ADVANCES I N
Immunology VOLUME 7
CONTRIBUTORS TO THIS VOLUME R. E. BILLINGHAM SYDNEYCOHEN ROSELIEBERMAN JOHN
P. MER...
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ADVANCES I N
Immunology VOLUME 7
CONTRIBUTORS TO THIS VOLUME R. E. BILLINGHAM SYDNEYCOHEN ROSELIEBERMAN JOHN
P. MERRILL
CESARMILSTEIN
MICHAELPOTTE~ DARCY B. WILSON JOHN
B. ZABRISKIE
ADVANCES I N
Immunology EDITED B Y
F. J. DIXON, JR.
HENRY G. KUNKEL
Division of Experimental Pathology
l h e Rockefeller University N e w York, New York
Scripps Clinic and Rerearch Foundotion
La 10110, Californio
VOLUME 7 1967
ACADEMIC PRESS A Subsidiary of Harcourt Brace Jovanovich, Publishers
New York
London Toronto Sydney San Francisco
COPYRIGHT 1967, HY ACADl;nfI<: I'RESS ALL RIGHTS RESERVED.
INC.
NO PART OF THIS BOOK MAY BE HEPRODUCED IN ANY FORM, BY PHOTOSTAT, MICROFILM, OR ANY OTHER MEANS, WITHOUT WHnTEN PERMISSION PROM THE PUBLISHERS.
ACADEMIC PRESS INC. 111 Fifth Avenue, New York, New York 10003
United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. 24/28 Oval Road, London NWl
PRINTED IN THE UNI'I'ED SI'A'TES O F AMEHLCA
808182
9 8 7 6 5 4 3 2
LIST OF CONTRIBUTORS Numbers in parentheses indicate the pages o n which the authors’ contributions hegin.
R. E . BILLINGHAM, Department of Medical Genetics, University of Pennsylziania School of Medicine, Philadelphia, Pennsylounia ( 189). SYDNEY COHEN,Department of Chemical Pathology, Guy’s Hospital Medical School, London, Englancl ( 1 ). ROSE LIEBERMAN, Laboratory of Clinical Investigations, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland ( 91 ). P. MERRILL,Department of Medicine, Peter Bent Brigham Hospital and Harvard hledical School, Boston, Massachusetts (275).
JOHN
CESARMILSTEIN, Medical Research Council Laboratory of Molecular Biology, Cambridge, England ( 1 ) .
MICHAEL POTTER, Laboratory of Biology, National Cancer Institute, National 1n.stitutes of Health, Bethescla, hlarylanrl (91 ) .
DARCY B. WiLsoN, Departnient of Afedical Genetics, [Tniziersity of Pennel~h~, ( 189). sgloania School of Medicine, P ~ i i l f f ~ ~ Pennsylvania R. ZABRISKIE,The Rockefeller University, New York, New York (147).
JOHN
V
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PREFACE With the retirement of Dr. John Humphrey, this serial publication is deprived of the last of the original editors. The development of the Advances in Immunology was a most successful venture; much of the credit for the high caliber of the first six volumes must be given to Dr. Humphrey. In the five years that this period encompassed the field of immunology expanded almost beyond belief. It no longer remains a province of the specialist alone since its challenging problems, which range from the mechanism of antibody synthesis to the immunopathogenesis of disease, have attracted scientists from many other disciplines. The elucidation of the structure of antibodies and the chemical basis for their variation represents the “new immunochemistry,” a far cry from that envisaged by Arrhenius when he first coined the word at the beginning of the century. Similarly, immunopathology has become virtually an independent discipline and one of the most exciting areas in clinical medicine. These developments herald an ever broadening range of subjects for coverage in the Advances, and such diversity is clearly evident in Volume 7. In the first chapter, Sidney Cohen has joined with Cesar Milstein to summarize the many developments concerning the chemistry of the immunoglobulins. Initially, this chapter was planned i s a supplement to that which appeared in Volume 4 on the same subject. However, it soon became apparent that this was virtually impossible in view of the rapid expansion of the field, and an entirely new review has emerged. Dr. Milstein, who has contributed greatly to the recent structural advances, has strongly supplemented Dr. Cohen’s wide knowledge of the immunology and genetics of the immunoglobulins. Numerous tables are presented which bring together many of the detailed chemical developments, particularly those concerning the light and heavy chains of y-globulin. Michael Potter and Rose Lieberman describe the many developments concerning the genetics of the immunoglobulins of mice in the second chapter. The discovery of myeloma proteins in mice by Dr. Potter some years ago immediately brought this species to the fore so that it rivals man in utility to the chemically oriented immunologist. Bence Jones proteins, myeloma proteins, and homogeneous macroglobulins have aided these workers in delineating five classes of immunoglobulins and a host of genetic antigens. It seems highly possible that an understanding of the genetic basis for antibody variability will eventually come from the mouse myeloma cell system. vii
viii
PREFACE
The third chapter, by John Zabriskie, reviews the role of structural similarities between parasite and host in initiating pathological events, primarily through immunological reactions to the parasite affecting the host. In many ways this is a new mechanism of disease and no one can predict just how broadly operative it is in man. Certainly, many so-called autoantibodies may well develop in this fashion. It is a particularly promising approach to the study of rheumatic fever and the various connective tissue disorders and is summarized well by a pioneer in this field. The fourth and fifth chapters deal with different aspects of transplantation immunity. Darcy Wilson and Rupert Billingham present a very complete review of the lymphocyte and its role in different types of immunological reactions in experimental animals and in in vitro systems. These cells which make up approximately one percent of the body weight are still mysterious, but progress has been extremely rapid recently and a large amount of information has been accumulated. john Merrill, on the other hand, presents a summary on the more practical side involving primarily renal transplantation in man. This chapter in some respects reads like a novel in recounting the fascinating recent developments that are revolutionizing the treatment of kidney disease. The new editorial team is particularly pleased with this volume, which despite its diversity of current topics integrated surprisingly well into an organized unit. It was a pleasure to deal with this stimulating group and to review their scholarly work. The complete cooperation of the publisher was present at all times and certainly facilitated our task, HENRYG . KUNKEL FRANKJ. DIXON
October, 1967
CONTENTS LIST
OF
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V
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vii
PREVIOUS VOLUMES .
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xi
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CONTRIBUTORS.
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PREFACE . CONTENTS
OF
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.
Structure and Biological Properties of lmmunoglobvlins
SYIINEY COHENA N I ) CESAHhflLbTvlN
I. 11. 111. IV. V. VI. VII. V 11I.
Introduction . . . . . . . . Ceneral Structure nnd Properties of IiiimunoFlol,uliiis Eiizymatic and Chemical Fragments . . . Striicture of 1mmiinoglol)uliii Chnins . . . Aiiti1)otly Coml,ining Site . . . . . . Synthesis m t l Assemlily of l'eptide Chains . . Genetic Iniplications of I n i m u n ~ ~ l o l ~ u Structure lin Coniments . . . . . . . . . References . . . . . . . . .
. . . .
.
. . .
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1
3
27 80 63 67 69 78 79
Genetics of Immunoglobulins in the Mouse MICHAEL POTTER A N D
ROSE 1 , I E B E H M A N
I. Introduction . . . . . . . . . . . . 11. Structural Characteristics of Immunoglobulins in Mice . . . 111. Preparation and Testing of Homologous Antisera . . . . . IV. Distribution and Localization of Heavy-Chain Determinants . . V. Comparison of the Results Obtained with the Inhihition of Prc. . . . . . . . . . . cipitation Method VI. Linkage of Genes Controlling Heavy-Chain Determinants . . . VII. Distribution of Heavy-Chain Determinants in Inbred and Wild M i e . . . . . . VIII. Myeloma-Specific Homologous Antisera . IX. Hemolytic Complement Component (Hc' or MuB') . . . . X. Concluding Remarks . . . . . . . . . . . References . . . . . . . . . . . . .
92 95 105 109 126 127 131 139 140 141 14.3
Mimetic Relationships between Group A Streptococci and Mammalian Tissues JOHN
B. ZABRISKIE
I. Introcluction . . . . . . . . . 11. Biological Mimicry and Enhancement of Pathogenicity ix
. .
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.
147 148
X
CONTENTS
111. Biological Mimicry in Relation to Pathogenesis of Disease
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IV Group A Streptococci and the Transplantation Antigens V . Summary and Conclusions . . . . . . References . . . . . . . . . .
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154 184 185 187
lymphocytes and Transplantation Immunity
DARCY B . WILSONA N D R. E . BILLINCHAM I . Introduction
.
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.
I1 . Identification of Immunologicolly Competent Cells III . The Process of Sensitization . . . . .
IV . V VI . VII . VIII .
. . . .
The Effector Side of the Imniunological Reflex . as Effectors of Immunity . . . . . . . . . . . . . . Macrophages and Antigen The Blastogenic Response of Lyniphocytes in Culture . . . Cooperative Interaction of Lyinphocytes a n d h4~~croplingc~s . . References . . . . . . . . . . . .
. Lymphocytes
. . . . .
189 194 196 205 225 236 238 260 265
Human Tissue Transplantation
JOHN P. MEHHILL
1. Introduction . . . . . . . . I1. Biology of Human Transplantation . . . . I11. Immunosuppressive Treatment . . . . IV . Complications . . . . . . . . V. Tissue Typing . . . . . . . . VI . Transplantation of the Human Kidney . . . . . VII Transplantation of Other Visceral Organs VIII . Endocrine Grafts . . . . . . . IX Corneal Grafts . . . . . . . . X Grafts of Bone and Blood Vessels . . . . XI Transplantation of Marrow . . . . . . . . . XI1 Transplantation of Human Skin XI11. Moral and Ethical Aspects of Human Transplantation References . . . . . . . . .
. . . . .
. . .
. . .
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. .
. .
. .
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. .
276 277 279 288 295 301 305 312 314 514 315 319 320 321
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AUTHOR INDEX.
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329
SUBJECTINDEX.
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346
Contents of Previous Volumes Volume 1 Transplantation Immunity and Tolerance
M. H A ~ E RA, LENGEROV.:, AND T. HRABA Immunological Tolerance of Nonliving Antigens
RICHARDT. ShfITH Functions o f the Complement System ABRAHAhl
G. OSLER
In Vifro Studies of the Antibody Response ABRAhX
B.
STAVITSKY
Duration of Immunity in Virus Diseases
J. H. HALE Fate and Biological Action of Antigen-Antibody Complexes
WILLIAM0. WEIGLE Delayed Hypersensitivity to Simple Protein Antigens
P. G. H. GELLAND B. BENACERRAF The Antigenic Structure o f Tumors
P. A. CORER AUTHORINDEX-SUBJECT INDEX Volume
2
Immunologic Specificity and Molecular Structure
FREDKARUSH Heterogeneity of y-Globulins JOHN
L. FAHEV
The Immunological Significance of the Thymus
J. F. A. P. MILLER, A. H. E. MARSHALL, AND R. G. WHITE Cellular Genetics of Immune Responses
G. J, V. NOSSAL Antibody Production by Transferred Cells
CHARLES G. COCHRANE AND FRANK J. D r x o ~ Phagocytosis
DERRICK ROWLEY xi
xii
CONTENTS O F PREVIOUS VOLUMES
Antigen-Antibody Reactions in Helminth Infections
E. J. L. SOULSBY Embryological Development of Antigens
REEDA. FLICKINGER AUTHOR INDEX-SUBJECT INDEX Volume 3 In Vitro Studies of the Mechanism of Anaphylaxis
K. FRANK AUSTEN AND JOHNH. HUMPHREY The Role of Humoral Antibody in the Homograft Reaction
CHANDLER A. STETSON Immune Adherence
D. S. NELSON Reaginic Antibodies
D. R. STANWORTH Nature of Retained Antigen and Its Role in Immune Mechanisms
DANH. CAMPBELL AND
JUSTINE
S. GARVEY
Blood Groups in Animals Other Than Man
W. H. STONEAND M. R. IRWIN Heterophile Antigens and Their Significance i n the Host-Parasite Relationship
C. R. JENKIN AUTHORINDEX-SUBJECT INDEX Volume 4 Ontogeny and Phylogeny of Adaptive Immunity
ROBERT A.
GOOD A N D
BEN w. PAPERhfASTER
Cellular Reactions in Infection
EMANUEL SUTERAND HANSHUEDY RA~ISEIER Ultrastructure of Immunologic Processes
JOSEPHD. FELDMAN Cell Wall Antigens of Gram-Positive Bacteria
MACLYNMCCARTY AND STEPHEN I. MORSE Structure and Biological Activity of Immunoglobulins
SYDNEY COHENAND RODNEYR. PORTER
CONTENTS OF PREVIOUS VOLUMES
Autoantibodies and Disease
H. G. KUNKELAND E. M. TAN Effect of Bacteria a n d Bacterial Products on Antibody Response
J. MUNOZ AUTHORINDEX-SUBJECT INDEX Volume 5 Natural Antibodies a n d the Immune Response
STEPHENV. BOYDEN Immunological Studies with Synthetic Polypeptides
MICHAEL SELA Experimental Allergic Encephalomyelitis a n d Autoimmune Disease
PHILIPY. PATERSON The Immunology of Insulin
C. G. POPE Tissue-Specific Antigens
D. C. DUMONDE AUTHORINDEX-SUBJECT INDEX Volume 6 Experimental Glomerulonephritis: Immunological Events a n d Pathogenetic Mechanisms
EMILR. WNANUE AND FRANK J. DIXON Chemical Suppression of Adaptive Immunity
ANN E. GABRIELSON AND ROBERTA. GOOD Nucleic Acids as Antigens
OTTOJ. PLESCIAAND WERNER BRAUN In Vifro Studies of Immunological Responses of Lymphoid Cells
RICHARD W. DWON
Developmental Aspects of Immunity
JAROSLAV STERZL AND ARTHURM. SILVERSTEIN Anti-antibodies
PHILIPG. H. GELLAND ANDREWS. KELUS Conglutinin a n d lmmunoconglutinins
P. J. LACHMANN AUTHOR INDEX-SUB JECT INDEX
Xiii
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ADVANCES I N
Immunology VOLUME 7
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Structure and Biological Properties of Immunoglobulins SYDNEY COHEN A N D CESAR MILSTEIN Department of Chemical Pathology, Guy’s Hospital Medical School, London, and Medical Rereorch Council Loboratory of Molecular Biology, Cambridge, hqland
I. Introduction . . . . . . . . . . 11. General Structure and Properties of Immunoglobulins . A. Antigenic Classification . . . . . . B. Isotypic Variants . . . . . . . . C. Allotypic Variants . . . . . . . . D. Icliotypic Variants . . . . . . . . . . . 111. Enzymatic and Chemical Fragments . Urinary Fragments of Immunoglobulins . . . . . . . . IV. Structure of Immunoglohulin Chains . A. Separation of Pcptide Chains . . . . . B. Heterogeneity of Peptide Chains . . . . C. Sequence Studies on Inimi~noglohulinChains . . . . . . . . V. Antibody Combining Site . VI. Synthesis and Assembly of Peptide Chains . . . . VII. Genetic Implications of Immunoglobulin Structure . A. Number of Genes Controlling C-Terminal Stretches B. Number of Genes Controlling N-Terminal Stretches VIII. Comments . . . . . . . . . . References . . . . . . . . . . I.
.
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1 2 4 5 17 26 27 29 30 30 32 34 63 67 69 70
70 78 79
Introduction
Since the multichain structure of immunoglobulins was established, many attempts have been made to define the chemical basis of their various biological activities and especially of that most characteristic property, combining specificity. This problem is greatly complicated by the size of antibody molecules and, more particularly, by their remarkable degree of chemical heterogeneity which is superimposed upon a constant basic structure. Recent studies have provided further evidence of this complexity (reviewed by Tomasi, 1965; Fudenberg, 1965; Gitlin, 1966; Fleischman, 1966). Types of immunoglobulins have been delineated within the main classes and the genetic variants, peptide chain structure, and general biological properties of these have been studied. Inevitably, however, comparatively little progress has been made in establishing the detailed chemical structure of these heterogeneous proteins. 1
2
S M N E Y COHEN AND CESAR MILSTEIN
The complexity of normal immunoglobulins and of specific antibodies has stimulated renewed interest in the monoclonal proteins found in multiple myelomatosis and related lymphoproliferative disorders. These proteins considered collectively show approximately the same distribution of antigenic and allotypic factors as normal immunoglobulin, thus suggesting that they can be regarded as species of the normal population having peptide chains sufficiently homogeneous to permit detailed chemical analysis. Data on the primary structure of these chains certainly comprises the most significant recent advance in the understanding of immunoglobulin structure and the genetic implications of this work have provoked much interest and speculation. II.
General Structure a n d Properties of Immunoglobulins
The 4-chain structure of antibody molecules consisting of 2 heavy and 2 light chains covalently linked by interchain disulfide bonds first proposed by Porter ( 1962) for rabbit immunoglobulin ( IgG) (Fig. 1) has been found to apply to all vertebrates having recognizable humoral antibody. The description and comparison of such immunoglobulins has been facilitated by adoption of a unified nomenclature summarized in Table I. The most primitive species investigated in detail is the elasmobranch, Mustelus canis (smooth dogfish ) . Immunoglobulins of this species are also made up of heavy and light chains (Fig. 2 ) separable by reduction and gel filtration in the presence of 6 M urea, and are present in proportions which suggest that the 7s molecule consists of 2 heavy and 2 light chains (Marchalonis and Edelman, 1965, 196613). Light
Heavy
.'.. 7cFc -
-Fob \
\
\
Papain
FIG. 1. Diagrammatic representation of the 4-chain structure of IgG linked by three interchain disulfide bridges. Broken lines show stretches in which heterogeneity occurs (or is presumedj within a given chain type. The undulating portion of the heavy chain indicates the area susceptible to proteolytic digestion giving rise to the fragments indicated.
STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS
3
TABLE I NOMENCLATURE OF IMMUNOGLOBULIKSAND THEIRSUBUNITS"
Huhtniice Inimriiioglol~uliii~
Recoinmeiided iionieiiclnturc
IgG o r .IG IgA or y h ~gni
-,~r
Previous noineiiclatrrre y:, y..,
T S y , 6.6 S y
m.1, P L . 1 y , M , PJI, 19 s 7 , ~-m:tcroglol) d i l l
1':~paiii fragmeiits
LgD or 71) F;tl,
Fc Peptic fragments Chains Classes of heavy chain Igc; IgA Ighl IgI> Types of light chain 'I
Fd Ip(ab')? Fab' Heavy chain Light chain y 01
p
I. 11, .I,c, s 111, €3, F
.Ipiece 5 S divaleiit fragment Uiiiv:dent frngnieiit H, A L, B
Chain Chain Chain
tl Challl I< or A cliaiiis I, or A chaiiih
1, 11, A
From B d l . World Health Otgan. 30, 447 ( 1964).
Sedimentation and viscosity measurements of rabbit immunoglobulin ( I g ) ( Noelken et al., 1965) have shown that the enzymatically separable fragments are more compact than the whole molecule and are presumably linked by a relatively extended area of the heavy chain (Fig. 1). This general molecular configuration is supported by the results of electron microscopy studies. Preparations of rabbit and human IgG examined by negative contrast appear as essentially symmetrical particles 80-120A. wide and about 34A. thick (Feinstein and Rowe, 1965). Pictures of ferritin and antiferritin systems taken at antigen excess show antibody molecules of similar appearance attached to antigen. At antigen antibody ratios nearer to equivalence, a number of Y-shaped strands are observed cross-linking antigen molecules and having a maximum length of 200A. which is twice the length of the intact antibody molecule ( Feinstein and Rowe, 1965); but see Fig. 3 (Valentine and Green, 1967). Examination of pepsin-treated antibody has shown that the base of the Y-shaped strand comprises the Fc fragment (Feinstein and Rowe, 1965; Rowe, 1966). These observations suggest that when antibody is crosslinked to antigen the Fab portion of the molecule can open to varying
4
SYDNEY COHEN AND CESAR MILSTEIN
FIG. 2. Comparison of reduced nlkylated humnn IgG ( l ) ,human IgM ( 2 ) , dogfish 17 S Ig ( S ) , and dogfish 7 S Ig ( 4 ) . Electrophoresis was performed on a starch gel in 8 M urea-formate buffer. (From Marchalonis and Edelman, 1966b.)
degrees and form a link twice as long as the original compact molecule (Fig. 3).Normal IgM examined by similar techniques shows a complex stellate structure made up of rods with a diameter of about 25A. and a maximum overall dimension of 230 to 250 A. (Feinstein and Munn, 1966, 1967). Anti-Salmonella IgM antibodies which visibly agglutinate were observed to crosslink flagella. At higher antibody concentrations, noncross-linked IgM antibodies appeared increasingly along the length of flagella and were seen in profile as staplelike structures with a maximum length of only 140 A. (Fig. 4).
A. ANTIGENICCLASSIFICATION The concept of immunoglobulins as a family of related but nevertheless heterogeneous molecules has arisen to a large extent from the study of their antigenic properties. Several degrees of antigenic difference between the immunoglobulins of a single species have been described (Oudin, 1960; Dray et al., 1962). These can be classified as ( 1 ) isotypic
STFtUcIzTRE AND A(;TIVITY OF IMMUNOGLOBULINS
5
speciiicities, common to all individuals of the same species, which differentiate classes and types of immunoglobulins; ( 2 ) allotypic specificities which distinguish polymorphic forms of immunoglobulin not present in all members of a given species; ( 3 ) idiotypic specificities which characterize individual antibodies (and myeloma proteins ) .
B. ISOTYPIC VARIANTS Different classes of immunoglobulins have been identified on the basis of physicochemical properties and the distinct antigenic specificities of their heavy chains; types within a single class of heavy chain show varying degrees of cross-reaction. This distinction is confused by the fact that heavy chains of different classes may, in fact, have common antigenic determinants. Thus rabbit antisera against human F( ab’) absorbed with light chains fail to react with Fc fragments, but show strong reactions with isolated y chains; such antisera, which are apparently specific for the Fd portion of the y chain, react with some IgA myeloma proteins (Kunkel et al., 1965) and also with some IgM macroglobulins (Kunkel et al., 1965; Harboe and Deverill, 1966). Similarly, antisera absorbed to show individual specificity for a particular IgG myeloma protein fail to react with many other proteins of the same class (Grey et al., 1965), but do react with certain IgM macroglobulins and also with a proportion of normal IgM molecules ( Seligman et al., 1965). The difficulty of antigenic classification is probably associated with the fact, discussed in detail below, that all classes of heavy chain seem to be derived from a common ancestor and have certain structural similarities. In addition, each has a portion which is relatively constant in structure and a part which is highly variable ( Fig. 1) . The variable portions considered collectively must contain a very large number of antigenic sites; their occurrence in different combinations probably accounts for some cross-reactions between heavy chains of different classes. The constant portions show a relatively restricted number of variants, and antisera which distinguish these will delineate clear-cut classes and types of immunoglobulins. The distinction between classes and types, as originally applied to whole immunoglobulin molecules, was based upon somewhat arbitrary differences. However, the terms will be retained here to indicate degrees of divergence in structural and biological properties, Classes of immunoglobulin are those having distinct antigenic properties which reflect major structural differences in C-terminal halves of heavy chains and are associated with discrete biological properties. Each class may contain several related types (or subclasses) which are not allelic products
STRUCTURE AND ACTIVITY OF IhihiUNOGLOBULINS
7
(C) FIG. 3. A. Electron microscopy of rabbit anti-DNP IgG saturated with a divalent DNP hapten (bisdinitrophenyl octamethylene diamine). Magnification: X500,OOO. Much of the antibody is linked to form closed rings with regular shapes (triangles, squares, pentagons). The hapten is at the mid-point of each side and the projections at each corner are at the middle of antibody molecules. Individual antibody molecules are therefore Y-shaped;- the angle between the- two arms may vary from 0" to 180". Each arm is about 60 A. long and about 35 A. wide. B. The antibody-hapten complex shown in (A) after peptic digestion. F c fragments a t each corner of the closed rings have been removed. C. The complex shown in (B) after reduction with dithiothreitol to convert F(ab')' to univalent Fab fragments. (From Valentine and Green, 1967.)
since they occur in all individuals. A given type of chain is defined by the amino acid sequence of its C-terminal half which is invariant except for polymorphic forms determined by ailelic genes.
1. Classes of Immunoglobulins The physical, chemical, and biological properties of the four main classes of human immunoglobulin have been established to a large extent by studying monoclonal proteins which characteristically belong to a
8
SYDNEY COHEN AND CESAR MILSTEIN
FIG. 4. Electron microscopy of flagellae of Salmonella paratyphi agglutinated in an IgM antibody at concentration 8 times the agglutination point. Negatively stained with sodium phosphotungstate p H 7. Magnification: X286,OOO. Bar = 0.1 p. (From Feinstein and Munn, 1966.)
STRUCTURE A N D ACTIVITY OF IMMUNOGLOBULINS
9
single antigenic class. The well-characterized human proteins are consequently regarded as prototypes for those in other species (Table 11). Differences between classes of heavy chains apart from those indicated in Table I1 include (1) molecular weights which appear to be characteristic for homologous chains in mammals as well as lower species (Table 111); ( 2 ) amino acid composition-comparative analyses have been recorded for human (Chaplin et al., 1965; Bernier et al., 1965), rabbit ( Lamm and Small, 1966), and bullfrog ( Marchalonis and Edelman, 1966a) heavy chains; ( 3 ) peptide maps which in human (Frangione and Franklin, 1965; Bernier et al., 1965) and rabbit (Lamm and Small, 1966; Cebra and Small, 1967) suggest dissimilarity in amino acid sequences of different heavy chain classes. The interspecies homology between chains of the same class is shown by their molecular weights (Table 111) and striking similarity of C-terminal sequences (see Table XX). The molecnlar weight of IgM is about 900,000 (Table 111). The molecule is split by reducing agents and reducing enzyme systems (Micheli and Isliker, 1966) into 4-chain nnits having molecular weights of 180,000 (Table I11 ) . IgM is, therefore, probably made up of five 7 S units linked by inter-p-chain disulfide bonds (Chaplin et al., 1965; Miller and Metzger, 1965) which may be formed between C-terminal cysteine residues (Doolittle et at., 1966). In some lower vertebrates, a protein which appears analogous to serum IgM occurs in the form of 7s monomers as well as larger polymeric forms (Marchalonis and Edelman, 1965; Clem and Small, 1966, 1967); a similar situation has been observed with certain horse (Sandor, 1962) and human antibodies (Rothfield el al., 1965; Stobo and Tomasi, 1966). The purification of IgA from normal human (Vaerman et al., 1965) and rabbit (Onoue et al., 1966) sera presents considerable difficulties. IgA can be more readily isolated from various seromucous secretions which contain a relatively high concentration of this immunoglobulin (see Schultze and Heremans, 1966). Exocrine and senim IgA show certain characteristic differences: 1. The sedimentation coefficient of IgA from saliva and colostrum (Tomasi et al., 1965) and bronchial secretions ( Masson and Heremans, 1966) is predominantly 11 S; over 90%of normal serum IgA, on the other hand, is 6.9 S (Tomasi et al., 1965). 2. The polymeric form of exocrine IgA is associated with an antigenically distinct fragment (transport or T piece) which is not present in 7s colostral IgA or in the polymeric IgA's of myeloma and normal sera (Tomasi et al., 1965). A similar fragment is excreted in the saliva
TABLE I1
PROPERTIES OF HUMAN IMMUKOGLOBULINS Properties
Biological Serum conc. (mg. %) (1)" 800-1680 Synthesis rate (mg./kg./d.) 20-40 (3) Catabolic rate (% I.V. pool/d.) 4-7 (3) Distrihution (% in I.V. pool) 48-62 (3) Ailtibody activity Complement fixation Placental passage (7) (14) Presence iri cerehrospinal fluid (8) Selective seromucnus secretionb 0 Skin sensitization heterologous species (9) homologous species 0 Immunological Light-chain types Kl Heavy-chain classes Y typesb 4 Allotypes, Gm IllV Ph ysicocheniical S?O.W 6 5-7.0 Ammonium sulfate precipitation (molar conc.) 1.49-1 64 (19) Total carbohydrate (%) (12) 2.9 Hexose (%) 1 10 Acetylhexosamine (%) 1 30 Sialic acid (%\ 0 30 Fucose (%j 0.20
+ + + + +
+ +
NOTE:
See footiiotes on faring page.
Igh
IgG
IgM
IgD
50-190 3.2-16.9 (5, 6 ) 14-25 (5, 6 ) 6 5 1 0 0 (5, 6 )
0.3-40 ( 2 ) 0.03-1.49 (6) 18-60 (6) 63-86 (6)
+
0 0
0
(b
t-
0
0 (10) ?
0 (8) 0 K,
x
140-420 2.7-55 (4) 14-34 (4) 40 (4)
+
0 0
+ +
Kt
K,
a
M
x
(2)"
d
2
2
0
+
0
+
0 (11)
7, 10, 13, 15, 17
18-20, >30 1.64-2.05 ( I S ) 11.8 5.4 4.4 1.3 0.7
6.2-6.8 (11)
7.5 3.2 2.3 1.8 0.2'2
11
STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS
of agammaglobulinemic patients lacking detectable IgA ( South et al., 1966b). The T piece dissociates from IgA of rabbit (Cebra and Small, 1967) and human colostrum (South et al., 1966a) in the presence of 5 M guanidine (Fig. 5B). The dissociated material has a molecular weight of about 50,000 and contains light-chain determinants which are presumably present either on the T piece (Hong et al., 1966) or on dissociated light chains (Cebra and Small, 1967). It has been postulated TABLE I11 MOLECULARWEIGHTSof IMMUNOGLOBULINS AND THEIRCHAINS
I3 at hit
Bullfrog (.5)
Dogfish (6)
y
Chain IgA (milk)
140,000 ( I , 2)" 53,000 (I, 2) 370,000 (4)
Chain IgM 7 S units of IgM p Chain Light chain
64,000 (4) 85&900,000 (3) 180,000 (3) 70,000 (3) 22-23,000 ( 1 , 2)
54,000 72,000 20-22,000
980,000 72,000 20,000
Immunoglot~ulin
IeG
01
-
' Key to references: ( 1 ) Pain (1963). ( 2 ) Small and Lamm (1966). ( 3 ) Lamm and Small (1966).
( 4 ) Cebra and Small (1967). ( 5 ) Marchalonis and Edelman ( 1966a ) ( 6 ) Marchalonis and Edelman ( 1966b).
.
that human colostral IgA (molecular weight 510,000) consists of three 4-chain IgA units noncovalently linked to the transport piece (Hong et al., 1966). The molecular weight of rabbit colostral IgA, on the other hand, is about 370,000 and the molecule is apparently made up of two 4-chain units and the transport piece (Cebra and Small, 1967). 3. After mild reduction, the IgA's from normal and myeloma sera are converted into 3.5 S units (Franklin, 1962; Fahey, 1963a; Deutsch, Key to references in Table 11: ( 1 ) Fahey and McKelvey ( 1965). ( 2 ) Rowe and Fahey ( 1965). ( 3) Cohen and Freeman ( 1960). ( 4 ) Solomon and Tomasi ( 1964). ( 5 ) Wochner et al. ( 1963) . ( 6 ) Rogentine et al. ( 1966). ( 7 ) Citlin et ol. (1963).
( 8 ) Schultze and Heremans ( 1966). ( 9 ) Ovary and Karush ( 1961). ( 10) Franklin and Ovary ( 1963). ( 11 ) Hansson et al. ( 1966). ( 12) Heimburger et al. ( 1964). ( 1 3 ) Olesen et al. ( 1905). ( 1 4 ) Kohler and Farr (1966).
See text. Myeloma IgDs are predominantly of Type L (Hobbs et al., 1966; Hansson el a/., 1966).
12
SYDNEY COHEN AND C E S A R MILSTEIN
1964), whereas exocrine IgA remains undissociated (Tomasi et al., 1965; Onoue et al., 1966). An additional class of immunoglobulin ( IgE ) has been postulated to account for the properties of human reaginic antibodies. The association of such antibodies with IgA was previously suggested by immune precipitation and by the observation that purified IgA (Ishizaka et al., 1963) or its isolated CY chain (Ishizaka et at., 1964) blocked the skin-sensitizing activity of reaginic sera. More recently, Ishizaka and colleagues have shown that serum reaginic activity may be retained after precipitation with antisera specific for IgG, IgA, IgM, or IgD, but was removed by coprecipitation with IgGanti-light-chain complexes ( Ishizaka and . activity was also Ishizaka, 1966; Ishizaka et al., 1 9 6 6 ~ )Skin-sensitizing removed with a rabbit antiglobulin which does not react with IgG, IgA, IgM, IgD, or with human light chains (Ishizaka et al., 1966a,b), but which precipitates with a protein having ?,-globulin mobility. This protein was designated IgE and was thought to contain light chains on the basis of radioimmunodiffusion tests using an anti-IgG antiserum absorbed with Fc. In several sera from patients sensitive to ragweed pollen extract, skin-sensitizing antibody was almost quantitatively removed by precipitation with anti-IgE. In addition, sensitizing activity and IgE were similarly distributed on diethylaminoethyl cellulose ( DEAE ) chromatography, gel filtration, and density gradient centrifugation of serum; the sedimentation coefficient of both was estimated to be 7.7 S. In some human subjects, therefore, reaginic activity is apparently associated with a fraction having the features of a distinct immunoglobulin class. It remains possible, however, that reaginic antibodies are heterogeneous and associated with different immunoglobulins in different patients Reid et al., 1966). Anaphylactic antibodies closely analogous to human reagins have been described in the rat (Mota, 1964; Binaghi and Benacerraf, 1964; Binaghi et al., 1964, 1966; Oettgen et al., 1966), the rabbit (Zvaifler and Becker, 1966), and the dog (Patterson et al., 1963, 1964; Rockey and Schwartzman, 1966). The sensitizing antibodies in these three species resemble human reagins in many respects; they are present in extremely low concentrations, have p mobility on electrophoresis, are destroyed by heating at %"C, have high affinities restricted to homologous tissues, their sedimentation coefficients are greater than 7 S and their skinsensitizing activity i destroyed by reduction and alkylation. 2. Types of Immunoglobulins a. Light-Chain Types. The occurrence of distinctive isotypic specificities within a single class of immunoglobulin led to the recognition of
STRU-
AND ACTJYITY OF IMMUNOGLOBULINS
13
two types of light chains in human (Mannik and Kunkel, 1963a; Fahey, 1963b) and mouse immunoglobulins (Potter et al., 1964; McIntire et al., 1965). Both human light chains occur on all classes of immunoglobulin and the ratio of Type K : L is about 2:l. Mouse light chains, on the other hand, occur with different frequencies on distinct immunoglobulins (see chapter by Potter and Lieberman); those designated K (previously A ) are found predominantly on yG-Bel, yG-Be2, y-A, and y-F myeloma proteins and a distinct type, h (previously K ) has been described in association with a yM-macroglobulin ( McIntire et al., 1965). Antigenic analysis of human light chains indicate that normal K and h chains each contain at least two and probably more subtypes, but it has not proved possible to separate these into clear-cut groups or to correlate observed antigenic differences with the nature of the N-terminal amino acid (see Table XV) or with InV specificity of K chains (Stein et al., 1963; Williams, 1964; Epstein and Gross, 1964; Solomon et a,?., 1965; Kunkel et al., 1965; Nachman et al., 1965; Putnam et al., 1966). Human monoclonal immunoglobulins that fail to react with antisera to K and h chains, may, nevertheless, contain one or other of the light chains in normal yield after reduction and alkylation (Rowe and Fahey, 1965; Osterland and Chaplin, 1966). These proteins apparently have light-chain antigenic determinants which are inaccessible in the intact molecule; partial screening of Iight-chain determinants has previously been described (Nachman and Engle, 1964; Epstein and Gross, 1964). When tested with certain antisera, light chains may behave as univalent antigens (Franklin, 1963) and for this reason Fab fragments of myeloma proteins may fail to precipitate with anti-light-chain antisera (Franklin et al., 1966). This may be of practical importance in typing samples which have undergone spontaneous fragmentation during storage. Two types of light chain have been demonstrated in the guinea pig ( Nussenzweig et al., 1966). Certain antibodies, notably, those against dinitrophenyl-bovine y-globulin ( DNP-BGG ) have light chains of a single antigenic type (designated Type K ) . After absorption with light chain from purified DNP-BGG, a rabbit antiserum against guinea pig light chains was specific for Type L immunoglobulins. Specific anti-K antisera were obtained directly by immunization with light chains from anti-DNP-BGG antibodies. About two-thirds of normal guinea pig y2globulin molecules have K chains and one-third have h chains; both have similar molecular weights as judged by gel filtration but differ somewhat in electrophoretic mobility so that the h-chain content of slow y,-globulin is only 10%and that of fast 7,-globulin, about 40%. b. 7-Chain Types. Types of the humar, y chain have been identified using rabbit antisera to purified human myeloma proteins (Grey and
TABLE IV TYPESOF HUMANIgG Nomenclature"
% Total (6) y2 yl
73
r4
"
(1)
Ne We Vi Ge
(2)
(5)
Y Z ~ 72%
yzC
C Z
T?d
Key to references: (1 ) Grey and Kunkel ( 1964 ). (2)Terry and Fahey (1964). , 3 ) Ballieux et al. ( 1964).
(4)
% Type K (4)
% Type L
(4)
11 77 9
5.8 54.5 4.7
4.2
3
2.6
0.5
100%
67.6%
-
Skin sensitization (5)
Location of type specificity
5.2 22.5
___ 32.470
( 4 ) Terry et al. (1965)-analysis of 191 IgC myeloma proteins. ( 5 ) Teny (1965).
( 6 ) Unified notation proposed by Kunkel et al. ( 1967).
STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS
15
Kunkel, 1964) or fragments of heavy-chain ( BalIieux et al., 1964; Takatsuki and Osserman, 1964) or monkey antisera to pooled human IgG (Terry and Fahey, 1964). These antisera made specific by absorption with selected myeloma proteins identified four types of IgG which have been variously designated (Table IV). IgG types are present in all normal human sera but techniques for their isolation have not been devised. There is evidence that the normal serum concentration of IgG types is influenced by the genotype of the individual (Yount, Kunkel, and Litwin, 1967). IgG myeloma proteins belong to one or other type, the commonest being yJl, ( W e ) (Table I V ) ; each may be associated with either K or chains, although available data suggest that Y chains may be commoner in y.,, than in yJ,, or -prproteins (Table IV). The typespecific determinants of y.,, and y?,, are located on the Fc part of the y chain (Terry and Fahey, 1964). Those of yZc appear on either Fc or F(ab')' depending on the antiserum used for testing; type specificity may, therefore, be distinguished in an analogous fashion by sera that react with determinants on different parts of tha chain. The Fc fragments from various types of human y chains have different peptide fingerprints ( Frangione and Franklin, 1965; Frangione et al., 1966b). The fragments of y..,, yl13, and y 2 . appear to be closely related, whereas y2,, has a greater degree of strnctural individuality. Differences observed in C-terminal amino acid sequences are only partially related to type specificity. Thus, y.n and y2,, have identical C-terminal peptides, and amino acid substitutions ( possibly related to allotypic specificity ) occur among individual yLLproteins (see Table XX). Guinea pig serum contains two immunoglobulins, referred to as yland yl-globulin (Yagi et al., 1962; Benacerraf et al., 1963; White et al., 1963). Whether these should be regarded as distinct Ig classes or types of IgG is uncertain. Both have sedimentation coefficients of 7 S and the same hexose content (0.74%)and both are transmitted from maternal to fetal circulations (Bloch et al., 1963b) so that y,-globulin is not regarded analagous to human IgA (Oettgen et al., 1965). The two guinea pig 7-globulins differ in their biological properties, only y l being capable of sensitizing guinea pigs to local and systemic anaphylaxis whereas y2 is involved in cytophilic activity for macrophages, complement fixation, and in complement-dependent phenomena such as cell lysis and the Arthus reaction (Ovary et al., 1963, Bloch et al., 1963a; Berken and Benacerraf, 1966). The specific determinants that distinguish y,- and yJglobulins are localized on the Fc portions of their heavy chains (Nussenzweig and Benacerraf, 1964; Thorbecke et al., 1963); the light chains and Fd fragments of both types are antigenically indistingurshable ( Nussenzweig and Benacerraf, 1966a ) .
16
SYDNEY COHEN AND CESAR MILSTEIN
Mouse serum also contains a y,-globulin which is capable of transferring passive cutaneous anaphylaxis in the mouse and does not fix complement (R. S. Nussenzweig et al., 1964; Ovary et al., 1965). The similarity of yl-globulins of mouse and guinea pig is also shown by the fact that their sensitizing activity is not inactivated by heating at 56°C. or by reduction and alkylation (R. S. Nussenzweig et al., 1964). It is of interest that reaginic antibody has not been detected in either species and that, conversely, distinct y ,-antibodies are not found in those species ( man, rabbit, rat) that have reaginic antibodies. In the bovine there appear to be two types of IgG with electrophoretic mobilities of 7,- and 7,-globulin. On serological testing, the light chains and Fab fragments of IgG, and IgG, are identical, but the Fc portions of their respective heavy chains give reactions of partial identity. IgG, sensitizes skin in the homologous species but in contrast to the y I of mouse and guinea pig, the bovine fraction fixes complement; IgG, is associated with precipitating antibody but fixes complement very poorly (Pierce and Feinstein, 1965, 1967). Horse serum contains what appear to be three variants of IgG having antigenically distinguishable heavy chains and common light chains [IgG(a), I g G ( b ) , and I g G ( c ) ] (Rockey et al., 1964; Klinman et al., 1965, 1966). An additional antigenically distinct immunoglobulin has a relatively high carbohydrate content, faster electrophoretic mobility, fails to fix complement and was designated IgA (Klinman et al., 1966). This may correspond to horse immunoglobulin which was recognized by van der Scheer et al. (1940) as being distinct from IgG and named T-globulin. This protein, on the basis of its yl electrophoretic mobility (Smith and Gerlough, 1947), carbohydrate content (Schultze, 1959), and distinct antigenic determinants (Jager et al., 1950) has been regarded as analogous to human IgA. More detailed comparisons of horse IgG and T-globulin have shown that the light chains and Fab fragments are antigenically indistinguishable and that the Fc fragments, although antigenically different, show some cross-reaction ( Weir and Porter, 1966) and have identical C-terminal sequences (Table XX). These chemical and antigenic similarities suggest that horse T-globulin is, in fact, an IgG type rather than a protein analogous to human IgA. The interspecies variation of immunoglobnlins and the difficulty of defining analogous classes or types on the basis of overall chemical and antigenic properties are emphasized by this work. c. chain Types. The occurrence of two antigenically distinguishable types of human IgA has been demonstrated using goat antisera to chains of a myeloma protein (Vaerman and Heremans, the isolated (Y
STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS
17
1966) and monkey or rabbit antisera to myeloma IgA’s (Kunkel and Prendergast, 1966; Terry and Roberts, 1966; Feinstein and Franklin, 1966). One type includes about 10 to 15%of myeloma proteins examined and is antigenically deficient when compared by gel diffusion with the major type. Absorption of antisera with antigenically deficient proteins leaves an antiserum which reacts only with the major group; attempts to produce a specific antiserum for the minor subgroup by direct immunization have been unsuccessful, nor has it been possible to demonstrate this subgroup in normal IgA, presumably because it constitutes a minor component. Subgroup determinants appear to be located on chains and are unrelated to the polymeric forms of IgA since both subgroups include these and since antigenic specificity is unaltered after conversion to 7 S units by reduction and alkylation. d. p-Chain Types. Antigenic heterogeneity has been observed with human IgM antibodies (Deutsch and MacKenzie, 1964; MacKenzie and Deutsch, 1965) and with monoclonal IgM’s. However, the first possible differentiation of two antigenic classes of IgM in patients with macroglobulinemia was made by Harboe et al. (1965a). A rabbit antiserum to a macroglobulin absorbed with normal serum gave a precipitin reaction with 8 out of 21 macroglobulinemic sera and failed to react with the remaining 13 as judged by direct precipitation or inhibition tests. The antiserum did not precipitate with either heavy or light chains but absorption tests indicated that its specificity was directed against determinants on the p chains. Since normal sera varied in their ability to inhibit the typing serum, it is possible that the observed specificity represents a genetic factor rather than a subtype of IgM present in all normal individuals. (Y
C. ALLOTYPIC VARIANTS Those antigenic specificities present on immunoglobulin molecules which differ between individuals of the same species, are referred to as allotypes. This phenomenon was first observed in rabbits (Oudin, 1956) and man (Grubb, 1956) and has since been described in the guinea pig (Oudin, 1958; Benacerraf and Cell, 1961) mouse (see chapter by Potter and Lieberman), baboon (Kelus and Moor-Jankowski, 1962), pig (Rasmusen, 1965), chicken (Skalba, 1964), and rat (Rarabas and Kelus, 1967) . 1 . Human Allotypes
The genetic polymorphism of human immunoglobulins detected on the basis of serological differences has been the subject of several recent
18
SYDNEY COHEN AND CESAR MILSTEIN
reviews (Steinberg and Polmar, 1965; Mhtensson, 1966; Steinberg, 1966; Oudin, 1966a,b). A t present over twenty allotypes have been identified on human immunoglobulins. A new terminology for these Gm and InV factors has been proposed (Table V ) but its general adoption is delayed TABLE V NOTATIONOF HUMAN ALLOTYPES' Specificities
Gm
Original a, X
[f"" and b2 b and b' c or "Gm-like" r e P
New 1 2 3 4 5 6
Original
New
tP
10 11 12 13 14 15 16 17 21 22 24
I? b? I-, 3
b4
7 8
si
9
g
t
Y n
InV
I a
b
1' 2 3
b
From Bull. World Health Organ. 33, 721 (1965). Does not produce a detectable antigen. InV( 1) is found whenever InV( a ) is present, but may also be found in the absence of InV ( a ).
by the expectation that recent work may lead to a more rational basis for a revised terminology. The distinct identity of certain Gm specificities is a matter of controversy and it has been claimed, for example, that G m ( b w ) and G m ( f ) are the same (Steinberg, 1965; Steinberg and Polmar, 1965) . Studies on the distribution of allotypic factors on immunoglobulins have shown that Gm groups occur only on IgG molecules; InV groups, on the other hand, are found on IgG, IgA, IgM, and Bence-Jones proteins (tests for InV activity of IgD do not appear to have been recorded). As would be expected from this distribution, Gm activity is associated with the y chain of IgG and InV with light chains (Polmar and Steinberg, 1964; Lawler and Cohen, 1965). The InV activity is, therefore, confined to the Fab fragment of the molecule which contains the light chain. The
STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS
19
distribution of Gm activity on enzymatic fragments of IgG varies with different specificities. Gm( a ) , Gm(x), Gm(b), Gm(b3), Gm(y), and Gm( n ) are located on the Fc portion of the y chain, whereas Gm(f) and Gm( z ) are associated with the Fd fragment and are, therefore, present on Fab (Steinberg and Polmar, 1965; Litwin and Kunkel, 1966). Gm(f) specificity is dependent on the quaternary structure of the molecule (Polmar and Steinberg, 1964). Thus, specificity cannot be detected on separated heavy or light chains, but is restored when chains are recombined provided that the y chain comes from a Gm(f+) IgG. On the other hand, light chains of either Type K or L and derived from IgGs of various Gm specificities are equally effective in restoring Gm(f) activity in the recombined molecule (Steinberg and Polmar, 1965). In a preliminary report, Litwin and Kunkel (1966) state that Gm( z ) specificity is partially dependent on quaternary structure. This may be true for many allotypic factors since the serological activity of isolated chains and fragments is frequently less than that of the parent molecule; this is especially the case for InV determinants on isolated light chains. Analyses of myeloma proteins have shown that each genetic factor is associated with a particular type of peptide chain. InV activity is present only on K chains and is not detectable on X chains (Terry et al., 1965); the reported association of InV(b) activity with a X chain was attributable to the use of an unreliable typing serum (Lawler and Cohen, 1965). The available data indicate that only those K chains associated with yZbor yzcheavy chains carry recognizable InV specificity; twelve yZRor yZa proteins of Type K were all negative for InV factors (Terry et al., 1965). The significance of this observation remains to be determined but it may be that InV determinants are masked by the quaternary structure in these types of molecules. The G m ( a ) , Gm(x), G m ( f ) , Gm(z), and Gm(y) occur only on yZh proteins, whereas Gm(b'), Gm(b3), and Gm(b4), Gm(c), Gm(s), and Gm(t) are found only on y2? (Kunkel et al., 1964b; Mirtensson and Kunkel, 1965; Terry et al., 1965; MArtensson, 1966) (Table VI). The recently described allotypic specificity, Gm ( n ) which is detectable in a precipitating system using primate antisera, is associated only with y?., proteins (Kunkel d al., 1966). Another allotypic specificity, Gm( g ) is probably the true allele of Gm( b ) (Natvig, 1966). Specificities of the Gm system are inherited in certain fixed combinations which differ from race to race (Table VII), and each set behaves as a unit of inheritance in family studies, i.e., no recombination of specificities is observed. It appears, therefore, that each set of specificities is determined by a cluster of sites in 1 chromosome. These sites
TABLE VI DISTRIBUTION OF ANTIGENIC AND ALLOTYPIC SPECIFIC~IES IN IgG MYELOMAPROTEINS
Gm Type
Caucasians
wa y2b
-
+ +
Y2c
Negroes 72s
Y2b
Y2C
YZd
Chinese Y2b
7%
+-
-
+-
-
-
-
+
-
a Gm( a ) and ( x ) are closely associated and occur together in about 50%of Caucasian (1965), Litwin and Kunkel (1966), and Kunkel et al. (1966).
Y2b
-
+
myelomas. Data from Terry et d.
STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS
21
have been regarded as constituting a single gene (defined as a unit of inheritance) with sets of specificities comprising a series of alleles (Steinberg, 1965). However, the fact that every Gm factor is confined to one type of heavy chain has suggested to several investigators that there are four closely linked genetic loci each directing the synthesis of one type of chain (Kunkel et al., 1964a,b; MHrtensson, 1964; Fudenberg et al., 1966). According to this theory, the y2b locus controls the synthesis of Gm(a), ( f ) , ( y ) , and ( z ) , the y Z c locus that of Gm(b) and Gm(g) (Natvig, 1966), and the palocus controls synthesis of Gm(n). TABLE VII SOMEGm SPECIFICITIES WHICHBEHAVEAs UNITSOF INHERITANCE’
‘I
Froiii Steinberg ( 1966 ).
It seems likely that the varying relationships of these genetic antigens in different races together with their molecular locations will provide a means of mapping the genes that control the synthesis of human heavy chains (see Mgrtensson, 1966). For example, the invariable association of Gm( n-) with Gm( a+z+) in Negroes and Caucasians suggests that genes controlling yZa and yZb are adjacent (Table VIII). The YZb immunoglobulins carry Gm specificities on both the Fd [Gm(z) and Gm(f)] and the Fc [Gm(a) and Gm(y)] portions of the heavy chain (see Table VIII). In Y2b myeloma proteins from Caucasians or Negroes these are invariably paired and such proteins are either Gma+, zf or Gmf+, y+ (Litwin and Kunkel, 1966). This pairing of specificities on the same myeloma protein implies that both are present on the same y chain. The two halves of the Y2b chain, therefore, appear to be inherited as a single genetic unit and this does not support the view
22
SYDNEY COHEN AND CESAR MILSTEIN
TABLE VIII TENTATIVE ARRANGEMENT FOR CHROMOSOMES CONTROLLING THE SYNTHESIS OF
COMMONLY OCCURRING y CHAINS“ Chromosomes
yZ b
(We)
Caucasians
Negroes
‘See data of Kunkel et al. ( 1966). The linear order of the genes is arbitrary.
frequently put forward (see Cohen and Porter, 1964) that Fc and Fd are, in fact, separate peptide chains. The Fd fragment probably contains a portion of the constant region and this is likely to include Gm(f) and Gm( z ) specificities. However, if these are localized in the “variable” N-terminal part of the y chain, then this must be a product of the same gene that codes for the relatively constant C-terminal part, and any mechanism involving gene fusion is excluded (see Section VI1,B).
STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS
23
In the Chinese a different relationship between the genetic factors of y r b myeloma proteins has been observed. Such proteins were shown by
Mkrtensson and Kunkel (1965) to be Gm( a+f+)-a combination never observed in Caucasians or Negroes-and more recently two such myeloma proteins were found to be Gm(a+f+y+) (Litwin and Kunkel, 1966). This indicates that Gm(a) and Gm(z) are carried on different molecules in Mongoloids, and this is in accordance with the observation that many Chinese sera are Gm(a+), Gm(z-). The fact that Gm(a) can remain associated with Gm(y) suggests that although both are on Fc, they do not occupy exactly homologous positions in the y chain (Table VIII). Considerable progress has been made in establishing the structural basis of InV specificity; InV(b+) and InV(a+) light chains have been shown to differ by the substitution of valine for leucine at residue 191 (see Table XII). Other substitutions which would account for InV( 1) specificity or for K chains without detectable allotypy, have not been reported. Peptide differences observed on Fc fragments of y chains were originally thought to correlate with the presence of Gm(a) and Gm(b) specificities (Meltzer et al., 1964; Fudenberg et al., 1964; Frangione and Franklin, 1965). The peptide regarded as characteristic of Gm(b) was later found in all Gm(a-) myeloma proteins of the pa,yZb,and y p c subtypes, but not in those of Xzd (Fudenberg et al., 1964; Thorpe and Deutsch, 196%; Frangione et al., 196%). It appears that these distinctive peptides characterize proteins which are Gm( a+ ) and those which are Gm( a - ) in three of the subclasses. Thorpe and Deutsch (1966b) obtained similar results and in addition found the following sequences for the peptides isolated from two Y.'b proteins: Gm(a +): Thr-Leu-Pro-Pro-Ser-Arg-Asp-Glu-Leu-Thr-Lys Gm(a - ) : Thr-Leu-Pro-Pro-Ser-Arg-Met-Glu-Glu-Thr-Lys
Multiple amino acid differences between what appear to be allelic products have previously been observed in sheep hemoglobins A and B (Huisman et al., 1965; Boyer et al., 1966). Although the Fc fragment produced by trypsin from y chains retains Gm(a) and Gm(b) activity (Lawrence and WiIliams, 1966), the tryptic peptide isolated from completely reduced Gm( a + ) proteins did not show allotypic activity (Thorpe and Deutsch, 1966b) so that the specific configuration may be dependent upon a longer sequence. A single amino acid substitution has been observed in the C-terminal peptides of ysr chains and may correlate with the allelic Gm( b ) and Gm(g) specificities (see Table XX).
24
SYDNEY COHEN AND CESAR MILSTEXN
2. Rabbit Allotypes Several rabbit allotypes have been described (Table IX); the molecular location and apparent genetic relationships of six of these have been studied in detail (reviewed by Oudin, 1966a,b; Kelus and Gell, 1967). Those designated A l , A2, and A3 appear to be controlled by alleles at one locus "a" and are located on the Fd portion of the heavy chain (Feinstein et al., 1963; Stemke, 1964). The specificities A4, AS, and A6 are controlled by alleles at a second locus "b" and are located only on TABLE IX ALLWIYPESOF RABBIT IMMUNOGLOBULIN
Ig Clitss
Specificity
IgG, IgM, IgA
Light, chain
IgG, ?IgM ?IgA
Light, chaiii Fc
IgG
"
Molecular locat ion
Key to references: ( 1 ) Hamers et al. ( 1964). ( 2 ) Oudin (1960). ( 3 ) Dray et al. (1963a).
Specificities occurring together
4: A;' AS A:' AS" A: A:
( 4 ) Kelus and Gell (1965). ( 5 ) Sell (1966). ( 6') Dubiski and Muller ( 1967).
light chains ( Wilheim and Lamm, 1966). An additional specificity (A9) appears to be related to the "b" locus, since in heterozygous rabbits A9 is associated with only one other specificity of the "b" group, whereas rabbits homozygous for A9 have no other allotypes of this group (Dubiski and Muller, 1967). Some antisera used for the detection of a specific allotype reveal several specificities systematically found together; these are designated with a prime or double prime (Table IX). The allotypic specificity designated A8 has been identified on rabbit IgG molecules some of which do not appear to carry A l , 2, or 3 (Hamers
STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS
25
et al., 1964). This specificity is present on the Fc portion of the heavy chain, has not been detected on IgM and is thought to identify a distinct subclass of rabbit IgG controlled at a locus closely linked to “a” (Hamers and Hamers-Casterman, 1965; Hamers et al., 1966). The specificity “e,” study of which was discontinued through lack of immune serum, is probably controlled by an allele closely linked to the “a” locus (Oudin, 1966a). The allotype P, on the other hand, appears to be determined at a locus distinct from “a” and “b,” whereas the genetic control of specificity T has not been established (Dray et al., 1963a). Individual molecules may carry specificities determined by different loci, but allelic specificities are found on separate molecules (Oudin, 1962; Dray and Nisonoff, 1963; Dray et al., 196313; Stemke, 1965). Up to 20% of molecules may have no allotypic specificity determined by the “a” locus (Dray and Nisonoff, 1963; Stemke, 1965) and a similar proportion have no “b” locus allotypes (Dray and Nisonoff, 1963; Oudin and Bornstein, 1964; Bornstein and Oudin, 1964; Stemke, 1964). Specificities controlled by both “a” and “ b loci have been detected on serum IgG and IgM (Todd, 1963; Stemke and Fischer, 1965) and have also been identified on both IgG and IgA in rabbit colostrum (Feinstein, 1963; Sell, 1967). However, Cebra and Robbins ( 1966) were unable to identify A1 or A2 factors on the IgA isolated from colostrum of five individual rabbits, although these specificities were readily identifiable on IgG from the same animals. Similarly, A 1 specificity present on serum IgG was not detectable on IgM (Lamm and Small, 1966). These discrepancies have not been resolved but may be due to differences in typing sera and reflect a complexity of rabbit allotypes which has not been adequately defined. An allotypic specificity confined to IgM (MS-1) was detected by Kelus and Gel1 (1965) who used a typing serum raised by immunization of an A3, A4, A5 recipient with anti-Proteus antibody from an A3, A4 donor. This antiserum reacted on gel diffusion with serum of the donor taken before immunization, with serum from 15 out of 40 of the donor’s offspring and 8% of unrelated rabbits. The specificity appeared to be localized on IgM as judged by immunodiffusion, ultracentrifugation, and gel filtration analyses. A second specificity present on rabbit IgM, but not on IgG and absent from a serum containing MS-1 has since been identified and designated MS-2 (Sell, 1966). The genetic relationship between MS-1 and MS-2 and between these and the specificities controlled by the “a” and “ b loci have not been determined. Several studies have been concerned with the structural basis of allotypic specificity in rabbit immunoglobulins. Data on the molar-
26
SYDNEY COHEN AND CESAR MILSTEIN
combining ratios of anti-A4 Fab fragments and A4 IgG from homozygous rabbits, suggest that light chains have three or four A4 determinants (Mage et al., 1966). Light chains isolated from the IgG of A4 and A5 homozygous donors have different amino acid compositions (Reisfeld et al., 1965) and show several distinct peptides on fingerprinting (Small et al., 1965). The heterogeneity of these preparations makes it difficult to assess the relationship of chemical differences to A4 and A5 specificities. Heterogeneity of the Fd fragment prevents its effective examination by the fingerprint method but, nevertheless, heavy chains with A2 specificity were distinguished from A 1 or A 3 chains by the presence of a yellow spot and absence of a brown spot (Small et al., 1966). Whether these are attributable to differences in amino acid composition or to distinct carbohydrate moieties in the heavy chains has not been determined.
D. IDIOTYPICVARIANTS Myeloma proteins and macroglobulins have been known for some time to possess individual antigenic specificity ( Kunkel, 1965). Antisera detecting such specificity do not react with normal immunoglobulin. Nevertheless, in the majority of instances, continued absorption with pooled immunoglobulin causes a progressive removal of the specific antibody; this suggests that normal immunoglobulin contains molecules analogous to most of those present in the spectrum of myeloma proteins. Determinants responsible for individual specificity are always associated with the Fab fragment and may be localized either on Fd or on light chain or be manifest only when these are recombined (Grey et al., 1965; Seligman et al., 1966). More recently, an apparently similar form of antigenic individuality, referred to by Oudin (1966a) as idiotypic specificity, has been demonstrated on a number of isolated antibodies (reviewed by Gel1 and Kelus, 1967). Antisera detecting a given idiotypic specificity react only with the individual antibody used for immunization and appear to have identical specificity when raised in different animals. Such antisera do not react with normal immunoglobulin, with preimmunization serum, with antibodies of other specificities from the donor, nor with antibodies having the same spectrum of specificities but raised in other animals. Idiotypic specificity, therefore, appears to be located in a variable region of the Fab fragment associated in some way with combining specificity. This variable stretch could be responsible for modulation of the combining site or for specific association of heavy and light chains (see Section V ) .
STRUCTURE AND ACTMTY OF IMMUNOGLOBULINS
Ill.
27
Enzymatic a n d Chemical Fragments
The IgG molecules can be split by a variety of enzymes which act at different sites, hut all within a limited and as yet incompletely defined area of the y chain (reviewed by Fleischman, 1966; Cohen, 1966). From molecular weights (Table X ) , the ability of univalent fragments to reform dimers, and sequence data (see Table XXI) it seems that papain splits the IgG molecule at the N-terminal side of the inter-heavychain disulfide bond (Fig. 1); pepsin, on the other hand, splits the y chain at a point nearer to the C-terminus. In accordance with this view, papain digestion of the peptic fragment F(ab’) 2, together with mild reduction and alkylation releases peptides which contain approximately TABLE X
MOLECULARWEIGHTSOF Enzlme ~
~~~~~
Papain Insoliible papaiii and S.D.S. Pepsin
THE
ENZYMATIC FRAGMENTS OF RABBIT IgG
Fragments
Molecular weight
Ref.
~
Fr Fab F(ab)? F(ab’)’
48,000 42,000 84,000 91,000
Soelken et al. (1965) Soelken et al. (1965) Jaquet and Cehra (1965) Jaquet and Cebra (1965)
one blocked sulfhydryl group per Fab fragment (Mage and Harrison, 1966). Analyses of such peptides have been carried out in rabbit IgG to define the structure of the 7-chain area which contains an inter-heavychain disulfide bond and is sensitive to proteolytic hydrolysis (see Section IV,C,3). Analysis of N-terminal peptides isolated from rabbit Fc has shown that the susceptible section of the chain has a high proline content ( 8 out of 18 residues) and that papain may act at several different peptide bonds in this region (Hill et al., 1966b). At acid pH, papain is able to hydrolyze the isolated rabbit Fc fragment releasing a C-terminal peptide of 113 residues; this is apparently devoid of certain biological activities associated with whole Fc (Prahl, 1967) and is similar to a fragment isolated after peptic digestion ( Utsumi and Karush, 1965). Another fragment of papain digestion (Fc’) has been isolated and may be part of Fc ( Poulik, 1966). The heavy chain is also susceptible to cleavage by cyanogen bromide. In the presence of 0.3 M HCl this reagent splits about half the methionyl residues of rabbit IgG and liberates a bivalent antibody fragment together with several smaller peptides ( Cahnmann et aE., 1965). Compari-
28
SYDNEY COHEN AND CESAR MILSTEIN
son of molecular weights and amino acid compositions indicates that the cyanogen bromide 5 S fragment is somewhat smaller than F(ab’)’. Reduction of the cyanogen bromide fragment releases two pieces resembling Fab and on reoxidation about 50% of the original bivalent fragment is reformed (Cahnmann et al., 1966). It seems likely that, in rabbit IgG, cyanogen bromide splits the y chain between the sites of cleavage by papain and pepsin perhaps at residue 221 from the Cterminus (see Table XXI) . Particular interest attaches to the isolation of the Fd fragment of the heavy chain in view of its likely association with the antibody-combining site. This fragment has been isolated from rabbit IgG, horse T-globulin, and a human myeloma IgG by gel filtration of reduced Fab under conditions that favor dimerization of Fd (Fleischman et al., 1963; Press et al., 196613; Weir and Porter, 1966). Isolation of normal human Fd’ from a fraction of F( ab’)2 soluble in 18%Na,SO, has been reported (Heimer, 1966). Preparations of IgG contain molecules which vary in their susceptibility to papain digestion; brief digestion of rabbit IgG releases Fab fragments (3.5 S ) and a 5 S fraction which appears to be an intermediate product containing an intact y chain linking one Fab and one Fc fragment (Nelson, 1964; Goodman, 1965). In addition, there remains a 6.9 S fraction with IgG specificity which is relatively resistant to redigestion with papain; its hexose content is almost twice that of the total IgG and this difference is confined to Fab (Goodman, 1965). Types of human IgG react differently to papain digestion. Human y Z Dappears to be unusually susceptible and its Fc fragment is hydrolyzed more rapidly than that of Y?b (Takatsuki and Osserman, 1964; Poulik, 1964; Frangione and Franklin, 1965; Thorpe and Deutsch, 1966a). Horse IgG and T-globulin (which appears to be a type of IgG-see Section II,B,2,b) differ markedly in their products of papain digestion in the presence of 0.01 M cysteine (Weir and Porter, 1966). IgG gives the expected 3.5 S fragment, whereas the T-globulin gives a divalent 5.6 S fragment (molecular weight 97,000) together with smaller peptides. This difference is associated with the presence of an additional disulfide bond linking the Fd fragments of T-globulin; the divalent fragment is converted to the univalent piece by reduction with 0.1 M mercaptoethanol. Guinea pig yl- and y2-globulins behave similarly on papain digestion; however, if both are dialyzed against phosphate buffer pH 7.6 in the cold, a crystalline fraction of the y,-Fc fragment is obtained while y2-Fc remains in solution (Nussenzweig and Benacerraf, 1964). Only limited information is available concerning the enzymatic
STRUCTURE AND ACTIVITY O F IMMUNOGLOBULINS
29
cleavage of IgA molecules (Deutsch, 1963, 1964; Bernier et al., 1965). As far as IgM is concerned, reports by Petermaim and Pappenheimer (1941) and by Deutsch et al. (1961) have suggested that fragments analogous to F(ab’)’ could be obtained by enzymatic digestion. In a more recent study Miller and Metzger (1966) observed that human macroglobulin or its 7 S units underwent a progressive cleavage when reacted with trypsin; after 18 hours incubation, about 52% of the IgM was recovered in the form of two fragments. One had a molecular weight of about 47,000, contained a single interchain disulfide bond, and both p - and light-chain ( K ) determinants which were separable by reduction and gel filtration in N-propionic acid. A second fragment with a molecular weight of 114,000 appeared to be a dimer of the first, linked by an inter-p-chain disuKde bond. These fragments apparently correspond to Fab and F(ab‘)?, respectively, and were designated F ( a b ) p and F(ab’)?p. Unlike the fragments of IgG, the divalent piece of IgM is converted to the univalent form by further tryptic digestion and without cleavage of a disulfide bond. URINARY FRAGMENTS OF IMMUNOGLOBULINS Human urine is known to contain low molecular weight proteins antigenically related to immunoglobulin and apparently identical to free light chains (reviewed by Cohen and Porter, 1964). More recently, fragments of light chain have been identified in the urine of several myeloma patients with Bence-Jones proteinuria ( Cioli and Baglioni, 1966; Williams et d., 1966; Baglioni and Cioli, 1966; Solomon et d., 1966). In some cases peptide fingerprints showed that the fragment consisted of the variable N-terminal portion of the corresponding Bence-Jones protein. Since the invariant C-terminal half was not detected in the same urine samples, this finding raised the possibility that the light chain is made up of two separately synthesized units (Cioli and Baglioni, 1966; Baglioni and Cioli, 1966). In other patients, however, immunological tests showed that low molecular weight, urinary fragments corresponded to either the variable or the constant portion of the light chain (Solomon et al., 1966). In one case the C-terminal peptide from such a urinary fragment was missing, but the common iiitrachain disulfide bridge peptides were detected in fingerprints (Milstein, 1966b). It now appears likely that these fragments arise through enzymatic splitting which occurs when light chain is incubated with serum (Baglioni, personal communication), but not readily on incubation with urine (Fagelman et al., 1966) . A similar explanation probably accounts for the presence in urine of
30
SYDNEY COHEN AND CESAR MILSTEIN
heavy-chain fragments antigenically related to Fc (Turner and Rowe, 1966). In the rare syndrome originally described by Franklin, on the other hand, fragments of heavy chain, having structural features of Fc and present in serum and urine, seem to be synthesized de nouo (Franklin, 1964). This has been thought to signify that the heavy chain consists of two separately synthesized units, but in fact there is no independent evidence for this ( see Section VI1,B). Antibody activity which has frequently been reported in urine is confined mainly to IgG (Turner and Rowe, 1964; Hanson and Tan, 1965). However, activity has also been found in fractions thought to have molecular weights of about 10 to 15,000 (Merler et al., 1963; Hanson and Tan, 1965). Such fragments have not been fully characterized; they appear to contain determinants of Fab, and their precipitating activity is lost on reduction (Merler, 1966). IV.
Structure of Immunoglobulin Chains
A. SEPARATION OF PEPTIDE CHAINS The separation of partially reduced immunoglobulin chains by the method of Fleischman et al. (1962) gives a yield of about 25% light chain and preparations of heavy chain partially contaminated by light chain (Porter, 1962; V. Nussenzweig et al., 1964; Nelson et al., 1965; Haber and Richards, 1966). The degree of dissociation of reduced chains in a given solvent may, therefore, vary among different immunoglobulin molecules. Human IgG and IgA molecules with h chains dissociate at higher p H than those with K chains; this difference, which was not observed with IgM, provides a means of partially separating normal human K and h chains (Cohen and Gordon, 1965). Heavy chains uncontaminated by immunological or chemical criteria have been isolated by repeated gel filtration of reduced Ig (Haber and Richards, 1966) or by reduction and fractionation in the presence of denaturing agents or detergents. Such complete separation of extensively reduced rabbit chains can be achieved by gel filtration in the presence of 0.03 to 0.05 M sodium decylsulfate ( Utsumi and Karush, 1964), 6 M urea (Frangk and Zikhn, 1964), or 5 M guanidine-HC1 (Small and Lamm, 1966) ; with these methods the yield of light chain is about 33%of the total IgG and 22%of IgM (Lamm and Small, 1966). Separation of mildly reduced guinea pig y,-globulin by gel fiItration in 4 M guanidine-HC1 gives biologically active chains uncontaminated by immunologically criteria and a yield of about 30%light chain (assuming equal extinction coefficients for both chains) (Lamm et al., 1966). Heavy chains which are soluble in neutral
STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS
31
Volume of e f f l u e n t h l )
(A)
I
40 I
Volume of effluent (rnl)
(B)
FIG.5. A. Gel filtration of rednced rabbit colostral IgA in the presence of 5.0 M guanidine HCl. The arrows show the elution volumes from the Sephadex G-200 column of the following reduced and alkylated materials: p = heavy chain of rabbit IgM; BSA = bovine s m u n albumin; y = heavy chain of rabbit IgC; L = light chain of rabbit IgM or IgG. B. Gel filtration of rabbit colostrnl IgA dialyzed against 5 M guanidine-HC1-0.01 M iodoacetamide, and passed through a column of Sephadex G-200 equilil~rateclwith 5 M guanidine-HC1. (From Cebra and Small, 1967.)
32
SYDNEY COHEN AND CESAR MILSTEIN
aqueous media, have been isolated by reduction of previously succinylated ( Lenard and Singer, 1966) or polyalanylated immunoglobulin (Fuchs and Sela, 1965). Isolation of the peptide chains of exocrine IgA is complicated by the association of these molecules with a fragment referred to as “transport piece” or T chain (Cebra and Small, 1967; Hong et al., 1966). After extensive reduction and gel filtration on Sephadex G-200 in the presence of 5 M guanidine-HC1, 29%of rabbit colostral IgA is eluted in the position of light chain (Fig. 5 A ) . This fraction contains a mixture of light and T chains, and these have not, as yet, been quantitatively separated. Unreduced IgA dissociates in the presence of 5 M guanidine and separates into three components on gel filtration (Fig. 5B). The first has a sedimentation coefficient of 7.2 S and after reduction can be separated into LY chains and light chains, the latter comprising 20.5%of the total fraction. The second component (molecular weight about 50,000) contains a mixture of T and light chains partly separable on DEAE chromatography; the third peak contains monomeric light chains. It appears, therefore, that the T chain is noncovalently bound to and light chains and, perhaps, stabilizes the IgA 4-chain dimer. The partial dissociation of light chains from the unreduced molecule in the presence of guanidine is difficult to explain in terms of the conventional, covalently linked, 4-chain structure unless disulfide interchange occurs under the experimental conditions used. (Y
B. HETEROGENEITY OF PEPTIDECHAINS Heterogeneity of peptide chains is the most characteristic feature of immunoglobulin molecules. The variability associated with differences in isotypic and allotypic specificities has been discussed above. Differences in peptide fingerprints of Fc fragments especially of y Z r molecules may be associated with unrecognized allotypes or technical artifacts (Frangione et al., 1966b). Definite heterogeneity within a single type of chain is shown by carbohydrate analyses (Thorpe and Deutsch, 1966a; Clamp et al., 1966) and more especially by distinct peptide patterns of Fd portions of heavy chains. Tryptic digests of rabbit (Small et al., 1965) and human (Frangione and Franklin, 1965; Frangione, Prelli and Franklin, 1966a, 1967) heavy chains characteristically show fewer peptides than the number expected from lysine and arginine contents of these chains. Although digests contain an unknown number of core peptides this discrepancy was attributed to variability of the Fd fragment as judged by comparing the peptide maps of iritact heavy chains and their corresponding Fc fragments. In the case of heavy chains
STRUCTURE AND ACT M T Y OF IMMUNOGLOBULINS
33
from whole rabbit IgG, only about 5 spots instead of the expected 13 could be associated with Fd, indicating that many peptides are either in a core or present in concentrations too low to be detected (Nelson et al., 1965; Small et al., 1965). Human myeloma proteins of a single type show 6-15 spots, apparently originating from Fd; about one-third of these peptides were common to several proteins, but the remainder had different distributions indicating a unique primary structure for the Nterminal part of each heavy chain (Frangione and Franklin, 1965; Fragione et al., 1967). Variations in the primary sequence of ,U and CY chains have also been suggested by differences of fingerprint patterns of monoclonal chains (Frangione and Franklin, 1965a). In accordance with these findings, the soluble tryptic peptides obtained in high yield from rabbit 7 chains are derived almost completely from the Fc fragment (Nelson et al., 1965; Hill et al., 1966a,b). Heterogeneity within a single heavy chain type has also been demonstrated by gel electrophoresis of y chains from horse IgG and T-globulin (Weir and Porter, 1966). Similarly human 7 chains isolated from monoclonal ySarp,,,y.,., or y>d proteins show multiple components on gel electrophoresis (Terry et al., 1966) but fewer than are present in pooled human y chain (Rejnek et al., 1966; Sjoquist, 1966; Sjoquist and Vaughan, 1966). Electrophoretic heterogeneity of the Fc fragment of the 7 chain arises, at least in part, from progressive degradation by papain; fractions of greater mobility are almost entirely absent from a 5-minute digest of rabbit IgG and increase progressively as hydrolysis proceeds ( Paraskevas and Goodman, 1865). The chemical heterogeneity of light chains is discussed in the following section. Such heterogeneity presumably accounts for the fact that on chains are reelectrophoresis in urea-glycine starch gels, both K and ,i solved into about ten bands (Cohen and Gordon, 1965), each differing by a unit net charge (Feinstein, 1966). On the basis of type specificity and allotypic and electrophoretic variation, there must be at least fifty different chemical forms of the human light chain. That the actual number is far greater (see Section VI1,B) is shown by the fact that distinct tryptic peptide fingerprints were obtained from human K chains of identical electrophoretic mobility and allotypic specificity ( Gordon and Cohen, 1966). Peptide chains isolated from antibodies of restricted specificity frequently show a degree of heterogeneity almost indistinguishable from that of the total immunoglobulin (Cohen and Dresser, 1965; Choules arid Singer, 1966; Reisfeld and Small, 1966; Lanckman, 1966). However, some antibodies from human, rabbit, and other species have relatively restricted heterogeneity (see Fleischman, 1966) especially when judged
34
SYDNEY COHEN AND CESAR MILSTEIN
by the distribution of antigenic and allotypic determinants and by the electrophoretic properties of heavy (Roholt and Pressman, 1966) and light chains (see Cohen and Dresser, 1965). A few antibodies have been reported which show an unusual degree of homogeneity at least in regard to certain properties of their constituent chains. The antibodies associated with chronic cases of cold agglutinin disease are always IgM molecules, and, in over ninety recorded cases, these have had Type K light chains (Mannik and Kunkel, 1963b; Franklin and Fudenberg, 1964; Harboe et al., 196%; Costea et al., 1966; Harboe and Lind, 1966). Some IgG erythrocyte autoantibodies are also associated with monospecific light chains, but these may be either Type K or L (Leddy and Bakemeier, 1965). Anti-DNP-BGG antibodies in guinea pigs may be either y,- or p-globulins, but in both types over 99%of molecules have Type-K light chains ( Nussenzweig and Benacerraf, 1966b). The proportion of h chains in anti-DNP antibodies is appreciably higher when the hapten is coupled to a different protein antigen; other evidence shows that properties of the carrier antigen used for immunization may influence the heterogeneity of antihapten antibodies (Sela and Mozes, 1966). The striking homogeneity of peptide chains from monoclonal immunoglobulins is widely recognized (see Fleischman, 1966) . Available data suggest, in fact, that the chains of such proteins have unique amino acid sequences (see below). It is possible, therefore, that the electrophoretic heterogeneity observed for both heavy and light chains of myeloma proteins (Terry et al., 1966; Melchers et al., 1966; Sjoquist and Vaughan, 1966) is not attributable to differences in amino acid structure. Heterogeneity of carbohydrate content has been demonstrated in a human myeloma IgA (Clamp et al., 1966) and in K chains isolated from a mouse myeloma protein. Three fractions of the latter were separable on starch-gel electrophoresis; all had the same amino acid composition, and charge differences resulted from variations in sialic acid content (Melchers et al., 1966). In the case of mouse myeloma proteins, electrophoretic heterogeneity has been shown to occiir after protein synthesis, either during the process of secretion or after exposure to serum (see Section VI).
C. SEQUENCESTUDIESON IMMUNOGLOBULIN CHAINS 1 . Light Chains
Heterogeneity has been a major difficulty in sequence studies of normal and antibody light chains. The use of homogeneous myeloma proteins Ied to a breakthrough in understanding the primary structure of
STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS
35
light chains and provided valuable information concerning the heterogeneity of normal immunoglobulins and their relationship to monoclonal proteins. Bence-Jones proteins are accepted as being free light chains (Edelman and Gally, 1962; Putnam, 1962; Schwartz and Edelman, 1963) which are of one or other antigenic type. Fingerprints of human K and h light chains show very few common peptides (Putnam et nl., 1963a,b; Schwartz and Edelman, 1963) and each type has a characteristic Cterminal sequence which seems to be common to all individual proteins of the same type ( Milstein, 1965). In fact the two types of chain have completely different sequences although an evolutionary relationship can be recognized (Milstein, 1966d; Hood et al., 1966; Milstein et al., 1967; Wikler et al., 1967). The most surprising finding emerging from sequence studies of Bence-Jones proteins has been that in both K and h chains the N-terminal halves are highly variable in structure whereas the C-terminal sequences are almost invariant for each type. Hilschmann and Craig (1965) first compared the partial sequences of two K chains and showed that, with the exception of a single residue, the C-terminal halves were identical. More detailed analysis of a third protein of the same type confirmed the invariant structure of the C-terminal half of the chain with the exception of the same single residue (Titani et al., 1965). These results were confirmed on large stretches at both extremes of the C-terminal half of several other proteins ( Milstein, 1966a,c). Results obtained with mouse myelomas on the corresponding chain type (Bennet et nl., 1965; Gray et al., 1967) and on human X chains (Milstein, 1966d) also indicated that the C-terminal half of the molecule remains essentially invariant. Differences in sequences of individual proteins were confined to N-terminal halves. a. The C-Terminal IZalf. Table XI shows the sequences of the C-terminal halves of human and mouse K chains and of human X chain. So far, the only well-established variation observed in this region among individual proteins of the same type is at residue 191 of human K chains. Valine is always present in that position in I n V ( b + ) , and leucine in InV(a+) proteins (Table XII). Eleven proteins have been analyzed so that the correlation is highly significant. Simple chemical techniques based on peptide patterns have been developed to distinguish the two allotypic forms (Baglioni et al., 1966; Milstein, 1966b,d). However, the chemical difference between the closely related InV(1) and InV( a ) antigens remains obscure. The important question of whether there are other differences in the
TABLE XI C-TERMINAL HALF OF LIGHTCHAINS“. *
I30
145
140
w
m
150
155
’4 Vol-L$-Asn-Alo‘///, , I l e -$$3y
////
‘/
A I a -
///
/
Leu-Gln -Ser-
160
1 65
/
/
/
’
- ~ ~ / L F l u - * r g - G l n - A ~ x ~ ~ ~ ’L~e u~G ,l<xi(<$
,V//
I70
m7nm’A7777 -~~5 5 Gly-Aso-Ser-Gln-6u -Ser/-vol -Thr-Glu-Gln - Asp-Ser-Lys - Asp-Ser7////,7// ///////////i://
////A
$;-(
- i l b P r o -Va 1 - Lys -A I a
/I/ ValGi
/////A
Asx
’/////. lu-Thr-Thr-Thr-
,&( Asx ,Trp ’I//
/’/’ii’/
) A
- Asp -S<<
////////,///,,,////
Pro- Ser- Lys-Gln
-
/ w
-1
<$G U < -IV (-;
///
20 5
200 / / G In-&{Leu -Ser-S
I95
/
210
I
//I///// '///:
////////
' ~ [ ~ L - A /I O
- ~ ~ ~ ~ -/L y s - - T h r - S e r - T h r - ~ / o l - I l-Val e
/////,
'/////
7//z
/C y ! i / d k - " ~ ~ - ~ h/ ~ /~ ~ ~ ~ - t l u - /, /~/ ~G Iu-dl-Thr~ S ~/ ~ T Valh r Alo-Pro-T - ~ ~ l - hr$u-&iSer
//y
L
////////
///
//A
75%
'//
/
/(//h
" Composite picture from several studies in man and mouse. Human K chains: Hilschmann and Craig ( 1965); Hilschmann ( 1966); Titani et al. ( 1965); Milstein ( 1966a,c). Mouse K chains: Gray et al. (1967). Human A chains: Milstein ( 1966d; Milstein et d.,1967; Wikler et al., 1967). Residues 140, 141 of human K chains are as indicated by more recent results (Putnam, personal communication). Residues 184, 185 of k chains are as determined by Milstein et al. (1967) but differ from those reported by Wikler et al. ( 1967 ). Common residues have been shaded.
38
SYDNEY W H E N AND CESAR MILSTEIN
C-terminal half of the molecule remains unanswered. An apparent difference in position 122 (an Asp for Asn) ( Milstein, 1966c) could result from technical reasons. However, the number of proteins fully investigated is not large. Studies based on amino acid composition and sequence of peptides are available for three human K chains and for large stretches in several others, and for two mouse K chains (see references, Table XI). Soluble tryptic peptides of the aminoethylated C-terminal half of K chains have been identified by their position and staining properties on peptide TABLE XI1 InV CHARACTERS AND RESIDUE 191 Protein
InV
Residue 191
Roy
-k
Leu VSl Leu Leu Val Val Val Leu Val
B-J4 B-J26
Val Leu
CU B-J
Ker Itad Day Man Fr 4
t
OF
HUMANti CHAINS Ref.
Hilschmanii arid Craig (1965)
Milstein (1966a,c)
t
Titaiii et al. (1966) Baglioni ct nl. (1966)
maps. By this method no further difference was detected in twenty-four Bence-Jones proteins, one macroglobulin light chain ( Baglioni and Cioli, 1966), and five other Bence-Jones proteins (Easley and Putnam, 1966), all of type K. The constancy of the C-terminal half of mouse K chains is indicated by almost complete sequence data of two proteins (Gray et al., 1967) supported by earlier fingerprint studies. The evidence on the human A chain is largely based on fingerprints and recent sequence studies on three human chains (Milstein et al., 1967; Wikler et al., 1967). Another important point concerns the position at which the invariant region starts. Variations have not been observed beyond residue 107, but residues 108 to 115 have been investigated in only three human and two mouse K chains and in three human X chains. However, it seems unlikely that variations will occur beyond residue 110 in view of the identity of residues 111-113 in the human K and A chains and in the mouse K chain. The C-terminal half of the molecule contains three half-cystine resi-
M o NOMER
"
'I
DlMER
IMMUNOGLOBULIN
----t--
-(NH~)
I
4
---7
H Chains
(-0Oc)
'----( N H ~ ) c ~ s ( c w - )
H Chains
4 (-0Oc) e--
f
t-i7
-4- - - -
FIG.6. The disulfide bridges of light chains in Bence-Jones proteins (monomer and dimers) and in immunoglobulins. The broken lines indicate the N-terminal half where multiple variations in amino acid sequences have been observed. (From Milstein, 196613.)
TABLE XI11
N-TERMINAL SEQUENCEAND SEQUENCE AROUND CYS 23 TYPE-K BENCE-JONESPROTEINS
'%
cu Rad Roy
Ag Ker
B-J Rad Day Mall Fr 4
Ale
SEVERAL
Sequence
Protein
Ker B-J 3 12
IN
Reference"
Asx-Ile-Gln-Met-Thr (Glx,Pro,Ser,Ser)Ser-Leu-Ser-lla-Ser-Val-Gly-hsp-Arg 10 Asp-Ile-G!n-Met-Thr-(:ll1-Pr~~-~~-Ser-Ser-~~i-Ser-.lla-Ser-~'a~-~!~-~~~p-Ar~ Asx(Ile,Glx)(Pulet,Thr,C;!ii,Pro,Ser,Ser,~r,LeujSer-.~la-Ser-T.'al-Gly-Asp-Arg Asx (Val,G lx)Met -Thr-G 111(Pro,Ser,Sex ,Ser,Leu) (Ser,Ala,Ser,Val,G 1y ,Asp)Arg ,lsp-Ile-Val-Leu-Thr-Gln Glu-Ile-Val-Val-Thr-Gln G Ix Glx Val(Thr,Ile,Thr,C?s,C;ls,~lla,Ser,(;lx,~l~~,Ile,Ser) Ile-Phe-Leu 20 30 Val-Thr-Ile- Th~-Cyi-C;ln-~I~tSer-C~l~i(.\.~x,Ile, Ser, dsu,Phe)Lea lsp-Ile- Lys(hw,Phe) Ile - Thr-Ile - Thr-Cys-Glii-.lIa-Ser-(:iliiVal-Thr-I!e - Thr-C) s-Glii-.~l~~-Ser-~lii-.l~p-Ilehsn-Lys (Tyrj Alla-Thr-Leu-Ser-Cys-Alrg-.lla-Ser-C;:li-Vd- Ser-Ser- .\sii-Ser-Tyr Val(Thr,Ile, Thr.Cys,Glx ,-lla,Ser,Glx. Aiu, Ile. Ser, -liu,Phe Leu Ile-Ser-Cys--Arg (Ala,Thr,Leu,Ser,Cys,Arg) Val(Thr,Leu,Thr,Cys,hrg)
Key to references: ( 1 ) Hilschmann and Craig (1965); Hilschmann ( 1966 ). ( 2 ) Putnain et al. (1966). ( 3 ) Milstein ( 1 9 6 6 ~ ) .
(4)Milstein (1966a). ( 5 ) H o o d e t d . (1966). ( 6 ) Milstein (unpublished). ( 7 ) Pink and Milstein (unpublished).
STRUCTURE A N D ACTIVITY OF II\.II\IUNOGLOBULINS
41
dues giving a pattern of disulfide bridges similar in both K and chains (Milstein, 1966b,d). The half cystines in positions 134 and 194 form an intrachain disulfide bridge which has been identified in several individual proteins. The C-terminal or next to C-terminal disulfide bridge is an interchain bridge, linked to a homologous sequence in the BenceJones dimer and to a lone hali-cystine in the monomer (Fig. 6 ) . The blocking of the free SH of the monomer could result from disulfide interchange during catabolism or represent a specific reaction occurring during assembly of the chains; in vitro labeling experiments should provide an indication of the mechanism involved. Direct evidence that light and heavy chains of IgG and IgM are joined through the same half-cystine has been obtained by isolating a cystine peptide made up of a half-cystine peptide from the C-terminus of either K or h chains and a half-cystine peptide from either or p chains (Pink and Milstein, 1967). The sequences shown in Table XI are very similar in some stretches, notably those between rcsidiies 110-140 and 160-180. In spite of this, only 60%of all the residues of the two K chains (mouse and human) are identical, as compared, for instance, with the 84% of identical residues in chains of human and rabbit hemoglobins. The similarity between the human h and K chains approximates to that observed in human and mouse K chains. This may indicate that the requirements for tertiary structure and association with heavy chains (as well as other biological functions) are localized in the conservative stretches, whereas the residues of other stretches alter in the course of evolution at a faster rate than occurs in the LY chain of hemoglobin (Zuckerkandl and Pauling, 1962), in cytochrome c (hfargoliash, 1963), or in pancreatic ribonuclease ( Epstein and Motulsky, 1966). I?. The N-Terminal Half. In contrast to the constant sequences of C-terminal halves, Rence-Jones proteins of the same type contain multiple variants in the N-terminal halves. This variation has been studied extensively only in the N-terminal stretch and around the cysteine residues of human K chains (Tables XI11 and XIV) and for this reason the overall degree of variation is still speculative. The results obtained (see also Table XV) suggest the presence of conservative residues either isolated (e.g., residues 2, 20, 23, 95) or occurring in stretches (e.g., residues 5-6, 25-27, 86-90, 97-98). Variation also occurs either in isolated positions (e.g., residue 96) or in stretches (e.g., residues 92-94). However, the distinction between conservative and variable residues is nncertain because of the restricted number of available sequences. Residues may be very conservative and yet variants appear when a suificient number of proteins are analyzed; for instance, rccidue I, was found to differ (Y
SEQUENCEAROUND CYS 88
Protein Roy
Ag Ker
B-J Cu Itad
TABLE XIV SEVERAL TYPE-KBENCE-JONESPROTEINS
IN
Sequence
Ref:
Leu-Gln-Pro-Glu-Asp-Ile-Ala- Thr-Tyr-Tyr-Cys-Gln-Gln- P he-Asp-Asn-Leu-Pro-Leu-Thr-Phe-Gly-Gly-Gly-Thr-Lys ( 1 ) 80 90 100 Leu-Gln-Pro-Glu-Asp-Ile-Aln-Thr-Tyr-Tyr-Cys-Gln-Gln- Tyr-Asp-Thr-Leu-Pro-Arg-Thr-Phe-Gly-Gln-Gly-Thr-Lys (8) Tyr-Tyr-Cys-Gln-Gln- Tyr-Asp-Asp-Leu-Pro-Pro-Thr-Phe-Gly-ProGly-Thr-Lys (3) Tyr-Tyr-CysGln-Gln- Ty r-Glu-Asn-Leu-Pro-Tyr (3) Val- Glx-Ala- Glx-AspVal- Gly-Val- Tyr-Tyr-CysGln-Met-Arg-Leu-Glu-IlePro-Tyr-Thr-Phe-Gly-Gln-Gly-Thr-Lys ( 1 ) Leu-Glu-Pro-Glu-Asp-Phe-Ala- Val- Tyr-Tyr-C ys-Gln-Gln- Tyr-Glu-Thr-Ser- Pro-Thr-Thr-P he (3)
* Key to references:
( 1 ) Hilschmann and Craig (1965); Hilschmann (1966). ( 2 ) Titani et al. (1966). ( 3 ) Milstein ( 1 9 6 6 ~ ) .
STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS
43
in only one out of thirteen proteins investigated. The large stretches without recorded variants may, therefore, reflect the insufficiency of data rather than real invariability. Some sequences may depart considerably from those included in Table XIII; for example, the N-terminal tryptic peptide present in a considerable number of proteins, as judged by fingerprints, is absent from others (Easley and Putnam, 1966; Raglioni and Cioli, 1966). Similarly, some cysteine peptides are so different from those more commonly found that they cannot a t present be related to any of the others studied (Milstein, 1966a), although observation of a larger number of sequences may disclose similarities. Some positions, on the other hand, seem to contain either one of two residues. This is well illustrated by the N-terminal residue (either Asp or Glu) and by the third residue (Gln or Val). Position 4 is either Met or Leu in ten out of eleven proteins investigated. The importance of all these observations lies in the possibility of using sequence data as a means of understanding genetic mechanisms (see Section VI1,B). Studies on the N-terminal stretch of mouse K chains shows a similar situation to that observed in the human (Hood et al., 1966; Perham et al., 1966). A blocked Glu has been reported in the N-terminal position of one protein (Perham et al., 1966). Almost complete amino acid sequences of two mouse K chains have been reported by Gray et al. (1967). Multiple differences were found localized in the N-terminal half of the chain (more precisely in the first 94 residues out of the 214 of the molecule, see Table X V ) . Differences between the two mouse N-terminal halves were larger than between a selected mouse and human pair. The two mouse proteins studied had a different total length, resulting from an insertion of four residues which could be confidently placed between residues 27 and 28 (see Table XV). In human K chains, residues 25/27 seem to be very conservative whereas a variation in residue 28 has been observed (see Table X V ) . Knowledge of the variable portion of h chains is more restricted. The N-terminal sequences of several h chains are presented in Table XVI. Sequences around the cysteine residue of human Bence-Jones proteins which appear to be related to the cysteine in position 22 are also shown. Again a difference in length seems to emerge from this comparison, probably related to a deletion at the N-terminal position. Table XV gives a summary of the variability observed in the N-terminal halves of human K and h chains and in mouse K chains. The comparison emphasizes the occurrence of conservative stretches within light chains. Homologous regions such as those occurring in the C-terminal halves of human K and h chains (Milstein, 1966d) are also present in the N-
VARIABILITY OF
THE
TABLE XV N-TERAIISALHALF OF HUMAN AND MOUSE LIGHTCHAISS"-'
Glu Val Val Leu 6 0-1 6 3-4 I(
Human
A la 1-2 Pro Ser Ser Ser Leu Ser Alo Ser Val Gly Asp Arg Val 1-4 1-4 1-4 2-4 2-4 3-4 3-4 3-4 3-4 3-4 3-4 4 5
-
_ 1
Val Leu
5 K
Mouse
Alo Val
Gin
A l a Thr
3
Asp I l e Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly Glu Arg V a l Ser 5 6 3 3 2 2 2 2 2 2 2
GlP
A Human
Alo
Val
K
Humon
25
Ile K
Mouse
Ser
Glu Ser Gly
Leu Thr
Cys Arg A l o Ser Gln Asx Ile
2
A Humon
30
2
2
2
Ile
35
Phe Met Asn
Phe?
Lys
Gly
Gly Ser Leu Ser Asx Trp Leu Glx Glx Gly Pro Asx Glx 1-2 1-2 2 ? 1-2 7 1-2 2
2m
46
F]e
C
5
k
I
n Pro
1
-
K
TY r
Gln
2
2
Leu Glu
2
Gly Gly Thr Lys Vol
Human
4
95
90
4
4
2
Ile Arg 2
Asp Phe Lys
4
2
100
I05
__ Glx K Mouse
Phe Cys Leu Glx
2
1-2
Ser Lys Glu
Val
Tyr Ala
Ser Pro Trp Thr Phe Gly
Ser
2
2
2
2
2
F l y Gly Thr L y s Leu Glu Ile
2
2
2
2
2
2
2
Lys 2
" Numbers outside the boxes are the numbering of residues from the N-terminus. Numbers within boxes show the number of ptoteins which have been described containing that particular residue; two numbers indicate that some results are inferred from composition of peptides: no number in the box means that it was found in only one protein. Glp = pyrrolidone-carboxylic acid. ' Shaded residues are those found in at least 5 out of 6 of the proteins investigated. ' In mouse the top row shows variants of protein 70 as compared with protein 41. Data from Perhani et al. ( 1966) are also included. In human K chain the bottom row i\ protein Roy. ' References-see Tables XI, XIII, XIV, and XVI. ' Many of the substitutions listed in the table are based on sequences of isolated peptides and the residue position is placed on comparative grounds. Aclditions or deletions may occur in the middle of the chain and this consideration should be borne in mind when making use of these results.
N-TERMINAL SEQUENCE AND SEQUENCE
TABLE XVI Fmsr CYSTEINE
AROUND THE
OF
SEVERAL HUMANk CHAINS ~~
Chain
Seqwnce
5
Sh
s
H BJ2 HBJ7 HBJR HBJ11 BJSS Sh
X IIil 3Ie BJ98
Reference.
10
15
Ser-C:lu-Leu-Thr-~~lii-.-Zsp-Pro-.-Zla-Val-Ser-Val-Ala-Leu-Gly-Gl~i-Thr-Val-ArgT y r-.-Zsp-Leu-Thr-Gln-Pro-Pro-Ser-Val-Ser-Val-SerPro-Gly-Gln-Thr-Ala-SerGlp-Ser-Ala-Leri-Thr-Gln-Pro-Pro-Ser-Al~-Ser-Gly-Ser- Pro-Gly-Cln-Ser- Val-Thr Glp-~er-V~~-Leri-Thr-Gln-Pro-Pro-Ser-.\ln-~r-Gly-~hr-Pro-Gly-Gln-Gl~-~a~-~hr Glp-,%r-a41a-Leii-.~a-Gln-Pro-Aln-Ser-Val-~r-Gly-Ser- Pro-Gly-Gln-Ser- Ile- ’I’hr Glp-Ser-Val-Leu (;lp(Ser,Val, Leu)Thr(Glx.Pro, Pro, Ser,\’al)Ser-Ala-~~la(,~s~,Gly) (C;lx,.ila)Val-Thr20 25 -Ile-Thr-Cys-C:Ii1-C;ly-Asp-Ser- Leu-;\rg-Cily-1le-Thr-Cys-Ser- Gly-.\sp-Lys-Leii-Gly-Asp -1le-Thr-Cy s-Gly-Gly- Asp-Glx -1le-Ser- Cys-Ser ( ~ l ySer) , Ser- Ser-.\sn-lIet Ser-Ile- Cys
J!
~
‘’ Key to references: ( 1 ) Milstein et al. (unpublished). ( 2 ) Hood et al. ( 1966). ( 3 ) Milstein (unpublished), ( 4 ) Wikler et al. (1967). ( 5 ) Baglioni ( 1967).
~~
STRUCTURE AND ACTIVITY O F IMMUNOGLOBULINS
49
terminal halves (Hood et al., 1966). This is indicated by residues at some positions (e.g., 5, 6, 7, 9, 12, 16) and by the position of Cys 23 when a deletion in the h chains is assumed (numbering is made with reference to K chains to give maximum homology). In spite of such striking similarities and of the variations observed in each chain, the available results strongly suggest that the variable stretch of h and K chains are recognizable as belonging to each type (Hood et al., 1966; Milstein, 1966d; Cioli and Baglioni, 1966). In other words, sequences shown in Table XI11 seem to be definable as belonging to K chains and the sequences of Table XVI as belonging to h chains. This distinction can be made, for example, on the basis of the N-terminal residue (Asp or Glu in K chains, Tyr, Ser, or a blocked Glu in h chains), residue 2, and the sequences around Cys residues. Thus, in K chains a recurrent sequence Cys-Gln ( o r Arg)-Ala-Ser-Glu has been found, while in h chains the equivalent position is the less conservative, Cys-Ser( or Gly ) -Gly-Asp (o r Ser)-Ser(or Lys or Glx). Similar observations apply to other residues in the N-terminal position (Table XV), to the sequences around other Cys residues (Milstein, unpublished), and to certain features of the fingerprint patterns ( Baglioni and Cioli, 1966). In all the proteins investigated so far, two cysteine sequences have been found in the N-terminal half of human K chains. This seems to be the case also in the mouse. Amino acid analyses indicating a lower number (Putnam et al., 1963b) ha\ e not been substantiated by consecutive work on the same protein. This is not surprising because of the known difficulties in accurate estimation of cysteine content. Evidence that the two cysteines present in the variable stretch are linked by an S-S bond has been presented in three proteins (Milstein, 1966b). Disulfide bridge peptide maps of several others suggest a similar arrangement. As in K chains, the N-terminal half of several h chains contains one disulfide bridge ( Milstein, unpublished). However, there are some proteins that contain three Cys residues in this stretch and one of them has been shown to occur as a free SH (Feinstein, 1966). 2. Bence-Jones Proteins and Normal Light Chains
If Bence-Jones proteins represent single species of the normal lightchain population, then peptides common to a large number of these myeloma light chains should be present in significant quantities in digests of normal pooled light chains. The peptides of K chains should be present in larger amounts than those of h chains. A quantitative estimate of the recovery of the C-terminal peptide of K and h chains (Table XVII)
50
SYDNEY COHEN AND CESAR MILSTEIN
showed that, within experimental error, normal pooled light chains contain one or the other in expected proportions (Milstein, 1965). Other soluble peptides from K chains, located by fingerprinting techniques (Putnam and Easley, 1965), may account for a large proportion of the C-terminal half of normal K chains starting at residue 146. Furthermore, the common intrachain disulfide bridge of Type-K Bence-Jones proteins has been found in good yields in normal light chains by the diagonal electrophoretic technique and by amino acid analysis of the peptides; these include the cysteines at positions 134 and 194 (Milstein, 1966b). YIELD OF
TABLE XVII C-TERMINAL TRYPTIC PEPTIDES FROM NORMALH U M A N LIGHTCHAINS" Yield of peptides Mole/mole
Light chaiii BeliceJones protein, Type I< (oxidized) Belice-Jones protein, Type L (oxidized) Oxidized riormsl light chairis "
K
O i
Corrected yield h
K
-
10 (nrbitmry)
-
0 5
0 45
0 20
-
0 63
A
-
10 (arbitrary) 0 40
Rec;ilculated from Milstein ( 1965).
Peptides of the A chain are more difficult to identify in unfractionated normal light chains because they occur in lower concentrations. Using a radioactive technique, two peptides that account for the last twenty-three residues of abnormal h chains have been identified in expected yields in fingerprints of normal pooled light chains ( Milstein, 1966d). These results indicate that C-terminal halves of normal light chains consist essentially of a mixed population of C-terminal halves of K and h chains. A similar study on peptides of the N-terminal half will be more difficult, because each variant sequence must be present in very small amount. However, sequences in the N-terminal half which occur in a high proportion of Bence-Jones proteins may be detectable. An Nterminal tryptic peptide has been observed in fingerprints of normal light chains (Putnam and Easley, 19651, although it is not possible to say whether it represented a mixture or a pure peptide. The study of the N-terminal sequence provides another approach to this problem. The N-terminal residues of human pooled light chains are Asp and Glu, which correspond to the two residues found in monoclonal K chains. The N-
51
STRUCTURE AND ACTIVITY O F IMMUNOGLOBULINS
terminal residue of monoclonal chains is either blocked or it is Tyr (see Table XVI), which is an elusive residue in N-terminal analysis. Stepwise degradation of normal human light chains gave Ile in the second position and either Val or Gln in third (Hood et al., 1966). This agrees very well with the residues more commonly found in the Nterminal sequence of the majority of K chains. In many cases a methionine is the fourth residue of Type K Bence-Jones proteins. The N-terminal peptide of cyanogen bromide cleavage has been prepared from such chains, and it should be possible to isolate the same peptide from normal pooled light chains. This has, in fact, been done ( Milstein, unpublished), but the amino acid composition of the peptide was not as straightforward as the one obtained from Bence-Jones proteins (Table XVIII ) ; however, TABLE XVIII THE N-TERMINAL PEPTIDEFHOM CNBr CI.EAVAC:E OF HUMANL m m CHAINS Light chain
Abnormal Ker ( I )"
B-tJ ( 1 ) Normal Poolctl 'ight chains (2)
'IS"
Ile
Val
Glri
HOSer
1 0 1 0
0 x5 -
10
1 1 1 1
0 75 0 5
1 1
o x
0 4
0.7
0.5
Minor components: Ser 0.2; Gly 0.2; Thr 0.1; Xla 0.1 'I
Key to references: ( I ) Milstein ( 1 9 6 6 ~ ) ;( 2 ) Milstein (unpublishecl).
this is not surprising because the isolation procedure may not have separated all the variants. The analysis nevertheless indicates that the most common N-terminal sequence shown in Table XI11 is present in normal light chains. The C-terminal residues of normal human IgG reported by Beiser et al. (1966) present a problem. The presence of Arg in very good yields in the C-terminal position, as shown by hydrozinolysis, is difficult to reconcile with analyses of myeloma proteins. The reason for the discrepancy remains obscure. An explanation for the pattern of electrophoretically separable bands of normal light chains can be given on the basis of differences in amino acid sequences. The electrophoretic mobility of a protein is a function of physical (size and shape) and chemical (charge) properties. If the former remains constant, the latter at appropriate pH values will vary stepwise by a minimum unit (one charge). Variation of charge may occur at several places in the N-terminal half, but mobility will be determined
52
SYDNEY COHEN AND CESAR MILSTEIN
by net charge and will be independent of the number and nature of the variations. The electrophoretic pattern of bands shows a characteristic random distribution, which suggests that the population is derived from a very large number of variants, which depart from a given or ancestral sequence. The same conclusion is suggested by the amino acid sequences of different Bence-Jones proteins. TABLE XIX PTH-AMINO ACIDS IDENTIFIED AT AMINO-TERMINALEND OF IhlMUNOGLOBULIN LIGHTCHAINS" Position in peptide chainb
Light chain source
1 Rabbit 7-globuliii
Ala
(Ile,Asp, Glu) Mouse and human
Asp Glu
2
Val (Leu)
Ile
3 Val Leu (Gln/Glu) Val Gln
4 Val (Glii/Glu) Val Leu Met
5
(;In
6 Glu (Thr,Ala)
Thr
Gln
From Doolittle ( 1966). numbered from amino-terminal end. PTH-amino acids shown in parentheses occurred in small amounts. a
' Positions
As regards other species, structural studies are more difficult because no preliminary information can be obtained from myeloma proteins. Doolittle ( 1966) has studied, by Edman degradation, the N-terminal sequence of rabbit y-globulin which can be compared with the corresponding sequence of mouse and human K chains (Table XIX). Since the y chains of rabbit have a blocked N-terminus, the results are considered to represent the N-terminal sequence of pooled light chains. 3. Sequence Studies on Heavy Chains
Elucidation of the primary sequences of light chains encouraged the more difficult task of studying the detailed structure of heavy chains; such studies are complicated by the size of the chain and by the number of classes and types. Nevertheless, progress in this area has been rapid and it seems IikeIy that sequences of myeloma and of large sections of normal heavy chains, will soon be available. Cyanogen bromide cleavage has been very useful in preliminary studies of heavy chains, and the arrangement of five fragments obtained from a human yzI, protein has been proposed (Press et al., 1966a)b; Piggot and Press, 1967) (Fig. 7 ) .The five fractions were separable by Sephadex filtration. The smallest was identified as the C-terminal peptide
STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS
53
and could be isolated and analyzed in several species and in types of human monoclonal IgG (Table XX). Normal IgG heavy chains from rabbit have also been split with cyanogen bromide and fractionated by Sephadex filtration (Press ct al., 1966a). A larger number of fractions was obtained, but a major fragment seemed to include practically thc whole of Fd (about 200 residues). However, there were indications that Fd was heterogeneous as far as methionine content was concerned and that a significant proportion ( 2 0 3 0 % ) of the population contained methionine in position 36 or 38 giving rise to an N-terminal peptide linked by a disulfide bridge (or bridges) to the rest of the Fd fragment. Papain
cleavage
Fd - f ragmen?
Fc - f ragrnent
FIG. 7. Cyanogen bromide fraginentc of a y:,, heavy chain ( D a w ) . The interfragment bridges and the moIecolar weights of fragments are indicated. The sequence of Fragment 2b is shown in the text. (From Piggot and Press, 1967.)
n. The C-Terminal Half. It has seemed likely that the C-terminal half of the heavy chain is invariant, since the pioneering work of Porter (1959) revealed that this part of the molecule readily crystallizes. This is being confirmed by finserprint studies of Fc fragments (Frangione and Franklin, 1965) and by sequence analysis of myeloma proteins and normal heavy chains. The similarity of C-terminal sequences of IgG in several species is remarkable (Table XX) , The C-terminal octadecapeptide of human yna.and yJb are identical but y Z c differs by either one or two residues. Larger differences between y types are to be expected in other sections of Fc fragments. The variation within y Z c may be related to Gm(b) and Gm(g) specificities, but more proteins are needed to establish a statistically significant correlation. Normal human IgG contains the C-terminal sequence of yna and Y.~, in very good yields as would be expected since these two types constitute the major part of normal chains ( see Table IV )
.
TABLE XX C-TERMINAL SEQUENCES OF HEAVYCHAINS ~
Chain Pooled human IgG Human yra myeloma Hiiman p b myeloma Human yzC H chain disease Gm (b+) Human yZe myeloma Gm(b+) Hiiman yZC myeloma Gm(b- g + ) Horse IgG(T) Horse IgG Rabbit IgG a
Sequence (.\let) His-GI u--lla-Leu-H is-.~sn-His-Tyr-Thr-Gln-Lys-Ser-Leu-Rr-Leii-Ser-Pro-~lS (AIet His-Gl~i-,Ua-Leu-His-;\sn-His-Tyr-Thr-Gln-Lys-~er-Leii-Ser-Le~i-Ser-Pr~-Gly (11et His-Glu-.~la-Leri-His-Asn-His-Tyr-Thr-Gln-Lys-Ser-Leu-Ser-Leri-Ser-Pro-Gly (Met ) H is-Gl~i-AIn-Leu-His-.~sn-d rg-Phe-Thr-Gln-Lys-Ser-Len-Ser-Leu-Ser-Pro-Gly (.\Iet ) His-GIu-Ala-Leii-His-.~sn-.-l,.g-Phe-Thr-Gln-L~s-Ser-Leu-Ser-~ii-Ser-Pro-Gly ( l l e t ~His-Glii-.~la-Leii-His-;\sn-.I rg-Tyr-Thr-Gln-Lys-Ser-Leli-Ser-Leu-Ser-Pro-Gly
(Net )His-G1ii-.lla-T‘al-Gl1c-~lsn-His-Tyr-Thr-Gln-Lys-il sn-T’al-Ser-His-Ser-Pro-Gly (Met)His-Glri-~~la-Leu-His-hsn-His-l’yr-Thr-Gln-I,~s-Ser-T’al-Ser-l,ys-Ser-Pro-Gl~~ (Met )His-Glu-Ala-Leri-His-.~sn-His-Tyr-Thr-Gln-Lys-Ser- Zle- Ser-.4 rg-Ser-Pro-Gly
Key to references: ( 1 ) Prahl ( 1966; and unpublished). ( 2 ) Press et d.(1966b). ( 3 ) Weir et al. ( 1966). ( 4 ) Givol and Porter (1965).
~~
Referenceo
STRUCTURE AND ACTIVITY O F IMMUNOGLOBULINS
55
Sequence stiidieq of rabbit Fc have been initiated by Hill ct al. ( 19666a,b). Twenty-seven tryptic peptides derived from the Fc fragment have been isolated; their total composition accounts, within experimental error, for the composition of the whole Fc. Chymatryptic digestion and cleavage with cyanogen bromide have provided evidence for the overlap of most of these peptides and a partial sequence of the Fc fragment of rabbit y chains has been proposed (Table XXI). Although two types of rabbit Fc have been suspected from studies of allotypes (see Section II,C,2), sequence studies suggest that this fragment is homogeneous. Rabbit IgG is known to be heterogeneous on papain digestion (Goodman, 1965), so that the seqiience may account only for a major fraction of Fc. However, when whole heavy chains were siibjected to tryptic digestion, no apparent discrepancy emerged. In fact only three extra peptides (one probably the N-terminus and two small peptides) were obtained in good yields. A number of unidentified peptides appeared in very low yields but their origin is unknown. A definite conclnyion about the homogeneity of rabbit Fc cannot be reached at present especially as the need to establish overlaps leaves considerable uncertainty about the partial sequence depicted in Table XXI. The results, nevertheless, suggest that more than haIf of the heavy chain (the C-terminal half) can be accounted for by a sequence which may be common to most or all of the rabbit y-chain population. A carbohydrate moiety is covalently bound to the F c fragments of IgG in several species (reviewed by Press and Porter, 19sS). It has been tentatively placed on residue 150 numbered from the C-terminus (see Table XXI) of rabbit y chain. However, in some IgG molecules the existence of more than one site of attachment of carbohydrate has been suspected (see Fleischman, 1966). In horse IgG( T ) , two-thirds of the carbohydrate is attached to the Fd fragment (Weir and Porter, 1966). The characteristics and distribution of the carbohydrate moiety might vary in different Ig molecules, even of the same type. Clamp et al. ( 1966) studied the composition of ten carbohydrate-containing peptides from a human IgA myeloma protein and found extensive heterogeneity. This very interesting observation raises the question of the specificity of structure and biological function of the carbohydrate in pure molecular species of immunoglobulin. 1). The N-Terminal Half. It is widely believed that the N-terminal half of the normal heavy chain of a given type is heterogeneous. This belief arises because there is little doubt that ( a ) antibody specificity resides at least in part in this half of the molecule and ( b ) antibody specificity is associated with variations in amino acid sequence (see
TABLE XXI A TENTATIVE SEQUENCE OF RABBIT FcaSb CXBr 2 40
u1
1
230
H,X-Cys-Pro-Pro(Pro,Glu) Leu(Pro,(;ly,(;ly,Vnl,Ser,Leu,Phe) Ile(Pro.Phe,Pro,Lys,Pro,Lys)~sp-Thr-Leu-?tIet-
0)
210
190
200
(.lsp?,Thr,,Ser3,C;lu3,Pro,,C~I~,i\lrt,C~s,Val~,Ile,,Leu,Phe,L~~) l(A~I)~,Thr~,Ser,C;l~i~. 180
160 (Ile-Asp-~~~p-C~lu-(C;I~i,Val~Arg) Thr-~lln-Arg-Pro-Pro-Le~i-~~rg-~l~~-
I
150
CHO
I 140 GIii-GIii-Phe-rlbl)-Ser-Thr-I le-~lrg-Val-Val-Ser-Thr-I,e~i-Pro-lle-.ila-Hiz-(;I~i-_ly,-Try-Leu-Ar~-Gly130
120
110
Lys-C;lu-Phe-Lps-Cys-L~z-Vrtl-Hiz-.l~p-Lys-Ala-Leu-Pro-Ala-Pro-Ile-C~lu-L~s-Thr-Ile-Ser-Lys-.llrtCSBr 100
1
90
Irg-Gly-G lu-Pro-Leu4 lu-Pro-Lys-Val-TS r-Thy-lkt-(: Iy-Pro-I’ru-Alrg-C;lu-C: lu-Leu-Ser-Ser-.irg-Ser-
CNBr
80 1 70 Val-Ser-Leu-Thr-Cys-Me~Ile-Bsp-Gly-Phe-Tyr-Pr~-Ser-.4sp-Ile-Ser-Gly-Val-Try-Glu-Lys60 50 hsp-Gly-Lys-Ala-Glu-Asp-Asp-Tyr-Lys-Thr-Thr-Pro-~4la-Val-Leu-Asp-Ser-Asp-Gly-Ser-Try-Phe-
40 30 20 Leu-Tyr-Ser-Lys-Leu-Ser-Val-Pro-Thr-Ser-Glu-Try-Gln-Arg-~ly-Asp-Val-Phe-Thr-Cys-Ser-ValCSBr
1 10 1 ~~et-EEis-Glu-Ala-Leu-His-Asn-His-Tyr-Thr-Glu-Lys-~r-Il~Ser-Arg-Ser-Pro-Gly-COOH a Vertical bars indicate positions where overlaps have not been established. The four peptide bonds cleaved by CNBr are indicated by the arrows. The numbering of residues is arbitrary and, in contrast to convention, residue 1 is at the COOH-terminal position. From Hill et a2. (1966a,b). There are differences between the sequence given by Hill et al. in 1966a and in 1966b. Where the discrepancies occur this table gives the data of the latter publication.
58
SYDNEY COHEN AND CESAR MILSTEIN
Section V ) . In fact, there is at present very little direct evidence for such variability. Heterogeneity in the N-terminal sequences of heavy chains of normal rabbits has been shown by Wilkinson et al. (1966) (see Table XXIII). Study of the N-terminal sequences has been greatly facilitated by the observation that the N-terminus of a myeloma protein (Porter and Press, 1965) was blocked by pyrolidone-carboxylic acid. This could arise under mild conditions from Gln residues in the N-terminal position; however, the authors found no evidence that the conversion was an artifact and favor the idea that it represents a more specific process (Press et nl., 1966b). The N-terminal sequences of rabbit heavy chains (Civol, unpublished) and of the cyanogen bromide fragment of a human pathological myeloma ( Daw ) heavy chain have been established and are compared TABLE XXII C O h l P A l ~ I S O N OF N - T E H M I N A L
CNHr FHACWENT 01%'A
* From Piggot
SEQUENCES O F RABBIT H E 4 V Y C H A I N S A N D OF TH1.:
H U h I A N PATIlOLOGlCAL
MYELOMA( b w ) HEAVYC H A I N " '
and Press ( 1967). residues, assuming a deletion in the second residue of labbit.
' It'ilics-identical
in Table XXII. As in the case of light chains of different species, there seems to be a significant degree of homology between the N-terminal sections of rabbit and human heavy chains. Apart from variations in the N-terminal peptide of rabbit (Table XXIII), very little is known about the variability of the N-terminal half of the heavy chain. Analysis of peptides isolated from antibody molecules by the use of hapten analogs (affinity labeling) indicates very extensive heterogeneity ( Singer and Doolittle, 1966); it is not clear, however, whether the tyrosines labeled by this method are in different parts of the molecule, in different types of chains, or, in fact, occupy equivalent positions in different sequences of a single chain type. Direct information
STRUCTURE AND ACTIVITY OF IhiMUNOCLOBULINS
TABLE XXIII THE ~ - ? ' E H b l I N A L
SEQUENCE OF I I E A V Y C H A I N S " '
59
'
Sormal mhbi t Glp-Ser-\.':tl-G In Glp-Ser-Leu-Glu Glp-Gln Longest sequence isolated :
Glp-Ser-Val-Glu-Crlu-Ser-C~ly-GI~-Arg " The numbers refer to approximatt. proportion of each sequence in the mixture. The pattern of rabbit peptitles \ w s essentially unchanged when a single hoinozygous r a l h i t and purified nntil,otly to hiiman serum allmmin were investigated. Glp = pyrralidone-carboxylic acid. " From Wilkinson et al. (1966).
will be obtained by sequence studies on Ftl fragments of myeloma proteins. The problem can also be approached by asking how much of the N-terminal half of the molecule is invariant in each type; two observations are of interest in this connection. First, some allotypic Gm characters are located in the F d fragment, and sequences determining their specificity should be essentially invariant (see Table VIII ). Second, light chains are attached to the Fd fragment and each class of human heavy chain appears to have characteristic sequences around the specific cysteine residue:
I
I& (ria) l'ro-I~eu-.~l,z-(Ser,Cys,I'ro)
I
(YLb)
Ser-Cys-A\sp-L>s
I IgM
Pro-Leu-Val-Ser-Cys-GIx-.2sx-Ser (Asp,Thr,Ser,Pro)
Those from IgG were obtained in expected yields from normal heavy chains (Pink and Milstein, 1967). Unfortunately the precise position of this particular cysteine is not yet known. It seems probahle, therefore, that the F d fragment resembles the light chain in having invariant sections and parts which vary within each type. This resemblance has been the subject of considerable speculation and some authors have suggested the possibility that the variable portions of light and heavy chains are controlled by a specific cistron (Burnet, 1966). The N-terminal sequence of the Y _ . ~heavy , chain of protein Daw can he compared with the N-terminal sequences of several human h chains (Table XV) and with a peptide isolated from the h chain of protein Daw ( Piggot and Press, 1967):
60 Heavy Daw
SYDNEY COHEN AND CESAR MILSTEW
Glp-Val-Thr-Le
ti- Arg-Glu-Ser
Humau
G1p-&--Ala Val-Leu-Thr-Gln-Pro
Daw
Asp Glp,Ser,Val
The N-terminal sequences of normal rabbit light (Doolittle, 1966) and heavy (Wilkinson et al., 1966) chains can also be compared: I:ahl)it~,light
Ilabbit, heavy
Ma (Ile, Asp Glu) 611)
Val (Leu)
Val (Leu Glu, Gln)
Ser (Gh)
Val (Leu)
Val Gln (Glii, Glu)
Glu
Glu
G 111 (Thr, Ala)
Ser
The results indicate that the light and heavy chain N-terminal sequences are quite different, This is in agreement with the fact that antibody specificity may be associated with isolated heavy chains but not usually with light chains (see Section V ) . The number of disulfide bridges between heavy chains is still a matter of controversy (see Fleischman, 1966). Smyth and Utsumi (unpublished) have isolated two carboxymethylated peptides from the rabbit Fc fragment after partial reduction under mild conditions and carboxymethylation with i ~ d o a c e t a t e - ~ These ~ c . peptides were rich in proline, carried 60%of the total radioactivity, and had identical sequences; one contained a blocked N-terminal carboxymethyl cysteine as an artifact of the isolation procedure. The carboxymethylated residue was believed to be the N-terminal cysteine of Fc (see Table XXI). However, a peptide also rich in Pro but containing two carboxymethyl cysteines has been isolated from mildly reduced and carboxymethylated myeloma proteins and human IgG proteins (Pink and Milstein, 1967). Utsumi and Karush (1964) have proposed that there are, in fact, two disulfide bridges joining the two chains in a nonsymmetrical bridge. Disulfide interchange is invoked to explain the low yield of carboxymethyI cysteine after mild reduction. The proximity of two cysteines in one peptide makes such a model attractive, but at the same time emphasizes the technical difficulties involved. Piggot and Press (1967) present evidence to indicate the position of some of the S-S bonds. They isolated a single, symmetrical disulfide-bridged peptide linking the two halves of Fc (interheavy-chain disulfide bridge ) and present in the N-terminal cyanogen bromide fragment of Fc (Fig. 7 ) . The authors point out that their results do not exclude the presence of other interchain bridges. The positions of intrachain bridges that seem to emerge from these studies also merit consideration. In light chains the “variable” and common sections are
61
STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS
bridged separately. If heavy chains followed the same pattern, Fragments 2b and 4 (Fig. 7 ) should be joined, but, in fact, both seem to form bridges with Fragment 2a. Of course, since no evidence about variable and common sections of heavy chains is available, it is still premature to draw further conclusions. The results do, nevertheless, emphasize the differences between the two chains. Elucidation of these problems requires more structural information and this may so011 be available.
4 . The Evolutionary Pattern of Immunoglobulin Chains There is convincing evidence from structural studies of hemoglobin chains and some proteolytic enzymes that related proteins in higher organisms may derive from a common ancestor gene (see Dixon, 1966; Epstein and Motulsky, 1966). The homology of myoglobin and hemoglobin chains is made even more striking by the close similarity of their tertiary structure. Similarities between the two types of light chains are sufficiently great to suggest that both are derived from a common ancestor. In the same way, similarities in the C-terminal peptides point to the common origin of heavy chains of different types. Although the evidence is tenuous for the p and y chains (and by extension perhaps to all classes of heavy chains), there are indications that these may also have evolved from a common ancestor; this has been the interpretation given by Singer and Doolittle (1966) for the common alIotypic specificities of the two chains of the rabbit ( Todd, 1963; Stemke and Fischer, 1965). Fingerprint similarities may also support this possibility (Lamm and Small, 1966; van Dalen et nl., 1967). In addition, IgM seems to precede IgG, phyIogenetically (Good and Papermaster, 1964; Marchalonis and Edelman, 196s) so that y and (1 chains may have evolved from a precursor p-type chain. Some authors have gone further in trying to understand evolutionary relationships between different chains. A similarity between C-terminal sequences of heavy and light chains has been suggested by comparing known stretches (Singer and Doolittle, 1966) and also the whole of the Fc fragment (Hill et al., 1966a,b) with light chains. The positions of the cysteine residues are also in agreement with this suggestion. If residues are numbered from C-terminal ends of human K chains and rabbit y chains, the cysteines occiir at the following positions:
I
p-l
K
Chain? (human)
191
134
. . . Cys
. . . Cys
195
Iinbhit Fc fragment
-
80 .
170 126(?)
. . . (Cys).
. Cys. .
..
Cys
.
Cvs
80
1
...
20 Cys
1
. . . Cyvl
2’2
. . . Cys .
.. .
..
C entl C end
62
SYDNEY COIIEN AND CESAR MILSTEIN
The similarity would be even more striking if it were found that heavyand light-chain disulfide bridges involve homologous residues. Doolittle et QZ. (1966) have reported the presence of cysteine as the carboxyterminal residue of a pathological, human, macroglobulin, heavy chain, which agrees very well with the hypothesis of a common origin of light and heavy chains. Another striking observation which points to a common origin of light and heavy chains is the position and sequence around the first Cys of a yZl,protein (Daw) and light chains. Daw heavy chain ( Piggot and Press, 1967), first Cys at position 22. Sequence: Thr-Leu-Thc-Cys-Thr
X chains (Table XVI), first Cys at position 22. Sequence:
K
chains (Table X V ) , first Cys at position 23. Sequence:
It seems that even in the N-terminal sections there may be significant similarities between heavy and light chains. The y chain (but not the p chain) is about twice the molecular weight of light chains, and the former could have arisen from a gene doubling of the latter (see Fig. 8 ) . However, the light chain includes a highly variable N-terminal half, whereas in heavy chains it is very unlikely that there are two “variable”
/,’
IS t Precursor gene
2nd ~
-~~~ + Precursor
gene
doubling
Ancestral heovy chain
gene
doubling
Frc. 8. A possible evolutionary pattern of I g chains.
K
STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS
63
stretches. Singer and Doolittle (1967) go even further and suggest that the light chain itself may have evolved from an ancestral half-molecule. This postulate is based mainly on the symmetry of the S-S bridges (see also Putnam et a?., 1966). This has also been suggested by studies of the Fc fragment. Hill et nl. (1966a,b) have observed that there is homology between the two halves of the Fc fragment. Comparison of residues 1-57 and 106-161 (numbering from C-terminus) shows 29%of the residues to be identical provided three gaps are left to obtain maximum alignment, This poses some interesting questions concerning the possible evolutionary pathway of the chains which may have important genetic implications (discussed by Singer and Doolittle, 1967). V.
Antibody Combining Site
The isolation of monovalent Fa11 fragments from IgG antibody molecules (Porter, 1959) proves directly that each 4-chain unit carries two combining sites. Nonprecipitating antibodies found in certain antisera are sometimes assumed to be univalent. However, Klinman et al. (1964) have shown by equilibrium dialysis that 7 S units of a nonprccipitating equine antibody each contained two combining sites of high affinity. The nonprecipitability of such divalent, high-affinity antibodies may be ascribed to structural features that prevent the opening up of Fab portions of the molecule to cross-link with antigen ( Fig. 3 ) . The available evidence indicates that IgM antibodies carry five or six combining sites per molecule. This has been shown by equilibrium dialysis experiments using purified rabbit anti-p-azobenzenearsonate IgM (Onoue et nl., 1965) and by precipitation tests with IgM antibody and isotopically labeled bovine serum albumin ( BSA) (Lindqvist and Bauer, 1966). In addition, the number of binding sites determined by equilibrium dialysis remained unchanged after dissociation of antihapten IgM to 7 S units by reduction and alkylation at pH 8 (Onoue et al., 1965). These results suggest that 7 S IgM subunits, which may retain full combining activity (Hill and Cebra, 1965) are, in fact, monovalent; whether this indicates the presence of only a single combining site or the availability for stereochemical reasons of only one of the two sites present on the 4-chain unit, has not been determined. Kaplan and Kabat (1966) obtained evidence that the combining sites of IgM antibodies are, in some instances, significantly smaller than those of IgG antibodies of similar specificity. The variable portions of heavy and light chains are present in the active Fab fragments which contain the antibody-combining sites; it is generally assumed that this variability in primary structure is related
64
SYDNEY COHEN AND CESAR MILSTEIN
directly to differences in combining specificity, The same conclusion is suggested by the finding that characteristic variations in total amino acid composition of specific antibodies are confined to active fragments and are located in the N-terminal portions of both heavy and light chains (Koshland, 1966), but whether such overall differences can be related directly to combining specificity is doubtful. The best evidence indicating that specificity is dependent on primary structure comes from experiments in which Fab fragments of various rabbit antibodies, completely reduced in 6 M guanidine-HC1, were shown to regain a significant degree of combining affinity after removal of denaturing and reducing agents (Buckley et al., 1963; Haber, 1964; Noelken and Tanford, 1964; Whitney and Tanford, 1965a,b). A similar recovery of activity has also been observed with whole IgG antibody (rabbit anti-BSA) which was reacted with polyalanyl residues before complete reduction in 8 M guanidine ( Freedman and Sela, 1966). The inference from these experiments, namely that amino acid sequence is the sole determinant of threedimensional structure and, hence, of specificity at the combining site, is supported by the fact that molecules denatured in the presence of guanidine have many properties of truly random coils (Tanford et al., 1966). Since available data suggest that the N-terminal regions of both heavy and light chains are highly variable, it might be anticipated that both polypeptide chains would be involved in forming the combining site. It has proved extremely difficult in practice to establish the relative importance of the two chains in this respect mainly because the conditions required for their separation lead to disruption of steric structure and considerable loss of combining affinity (see Porter, 1966). Nevertheless, isolated heavy chains have in several instances shown a degree of combining specificity which cannot be accounted for by contamination with light chain (Fleischman et al., 1963; Utsumi and Karush, 1964; Haber and Richards, 1966; Porter and Weir, 1966). In addition, catabolic studies have shown that Fd fragments, but not light chains isolated from specific antibody, bind to circulating antigen in vivo (Spiegelberg and Weigle, 1966). Antigen binding by the isolated light chain is uncommon but has been reported (Goodman and Donch, 1965; Mangalo et al., 1966). The light chain does, however, lead to enhancement of binding activity when recombined with specific heavy chain. Light chains derived from the original antibody are more effective than those from either nonspecific immunoglobulin, unrelated antibody, or even from antibody of the same specificity derived from a different pool or having different binding affinity (Metzger and Mannik, 1964; Roholt et al., 1965; Franek ct al., 1965; Hong and Nisonoff, 1966; Porter and Weir, 1966; Lamm et
STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS
65
nl., 1966). There is 110 evidence that the number of combining sites is altered by recombination, but combining affinity is greatly euhanced by association of homologous heavy and light chains (Haber and Richards, 1966). With the technique of affinity labeling, a specific hapten has been covalently bound to tyrosine residues in the vicinity of the combining site. The label is distributed in a constant molar ratio of about 2 : l between heavy and light chains of various specific antibodies; this suggests that both chains are involved in the combining site (reviewed by Singer and Doolittle, 1966). It is evident that no definite conclusion can be reached at present about the location of the combining site. Some heavy chains may carry all the information required to form the specific site. The remarkable heterogeneity of the light chain suggests its role in combining specificity but whether it has a relatively nonspecific modulating effect or participates directly at the site is a problem which remains unsettled. If combining specificity is determined by both heavy and light chains, then it is possible that a relatively restricted number of chains could generate a far larger number of distinct antibody specificities. The actual number generated might, however, be limited by the ability of various forms of heavy and light chains to associate. Studies on homogeneous chains from monoclonal proteins show that some recombination does occur between unrelated chain pairs and the ability to associate is apparently independent of the electrophoretic mobility and isotypic or allotypic specificity of the chains. However, there is a definite preference for autologous recombination which is revealed most strikingly when studied under conditions involving competition with homologous chains (Grey and Mannik, 1965; Gordon and Cohen, 1966; Mannik, 1967). Such specific interactions which have also been observed with the chains of antihapten antibodies (Roholt et nl., 1967) must be determined by regions within the variable portions of heavy and light chains which may not be the same as those areas which generate combining specificity. Studies on the chemical basis of combining specificity would be facilitated if monoclonal proteins with defined antibody activities were available for chemical analysis. Several apparently homogeneous IgG and IgM proteins with some form of combining affinity have been described (Table XXIV), but it has not as yet proved possible to stimulate the production of monoclonal immunoglobulins with well-defined combining specificity. Even if such proteins were available it seems likely that sequence data in the variable regions of heavy and light chains would have to be interpreted in terms of four properties which may or may not be related. namely ( 1 ) the Combining site, ( 2 ) regions modulating the
66
SYDNEY COHEN AND CESAR MILSTEIN
combining site, ( 3 ) the specific heavy-light chain association site, ( 4 ) idiotypic specificity of the antibody preparation. Preliminary attempts have been made to investigate the configuration of antibody-combining sites by means of physical methods. Antidinitrophenyl antibody can be differentiated from inert IgG by optical rotatory dispersion measurements in the ultraviolet region; this suggested the presence of a distinguishing configuration in the specific antibody apparently unrelated to allotypy and charge (Steiner and Lowey, 1966). TABLE XXIV MONOCIOV A L PHOI'EINSWITH SOME FORMOF COMBININGAFFINITY
Stored eryi Iirocyi es ICry Lhrocyt e " I-ant,igen' ' Htreptolysiii
niid p Ilpoproteiii Group A streptococcd carbohydrate (Y
Electron-spin resonance studies of the interaction between anti-DNP and a spin-labeled hapten (dinitrophenyl nitroxide), suggest that the combining sites have a high degree of structural rigidity (Stryer and Griffith, 1965). Chlorine-% nuclear, magnetic, resonance studies of the interaction between anti-DNP and a mercury-containing hapten [2,4dinitro-4- ( chloromercuri ) -diphenylamine] have shown that the Hg atom of the bound hapten is exposed to C1 ions of the solvent (Haugland et aE., 1967). Interpretation is complicated by the inany parameters which influence such physical measurements. However, in view of the inherent difficulty of interpreting sequence data in terms of biological activity, the use of physical methods and especially of X-ray crystallography (PoIiak and Dintzis, 1966) and electron microscopy, is of great potential interest in studies of antibody structure.
STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS
VI.
67
Synthesis and Assembly of Peptide Chains
Protein synthesis takes place on polysomes, and the size of the polysome appears to be related to the length of messenger and the polypeptide chain which is being synthesized (iVarner et ul., 1963; Staehelin ct al,, 1964; Kiho and Rich, 1965). Since the genetic markers of heavy and light chains are apparently unlinked, the chains should be synthesized an polysomes of different sizes. Several attempts to characterize the polysomes of lymphoid cells have been unsatisfactory (Stenzel et ul., 1964; Scharff and Uhr, 1965; Norton et al., 1965; Manner et al., 196s) because degradation to single ribosomes readily occiirs, probably through the action of nucleases. More recently, intact polysomes synthesizing immunoglobulin chains have been prepared by disrupting lymphoid cells in the presence of excess HeLa cell cytoplasm (Scharff and Uhr, 1965; Shapiro et al., 1966b) or under conditions of strict temperature control (Askonas and Williamson, 1966b; Williamson and Askonas, 1967). The study of mouse plasma cell tumors has shown that radioactively labeled, heavy and light chains, characterized either by electrophoresis (Shapiro et al., 1966b) or by immune precipitation (Askonas and Williamson, 1966b; Williamson and Askonas, 1967) are synthesized on polysomes of different sizes. Heavy chains are found only in association with larger polysomes (about 300 S ) and pulse-labeling experiments indicate that they are made within 60 seconds. Light chains appear to be synthesized within 30 seconds on smaller polysomes (about 180 S ) and can also be identified in free form in the cytoplasm (Askonas and Williamson, 1966;~;Shapiro et nl., 1966a; Nezlin and Kulpina, 1966). Labeled light chains can be demonstrated in association with heavy chains on the larger polysomes. However, in a chase experiment, labeled light chain was still present on the larger polysomes when all nascent heavy and light chains had heen removed (Shapiro et al., 1966b). Since free heavy chains are not detectable in the cytoplasm even after short labeling times, these findings suggest that assembly of the whole molecule occurs by attachment of free released light chains onto the polysome-bound heavy chains. Heavy and light chains also appear to be synthesized on separate polysomal sites in the normal lymphoid tissues of rabbits and rats (Becker and Rich, 1966) by processes essentially similar to those in other mammalian cells (Tawde et nl., 1966). Protein synthesis occurred on two sets of polysomes which, by analogy with those synthesizing hemoglobin in rabbit reticulocytes, were estimated to be of appropriate size for synthesizing peptide chains with molecular weights (25,000 and 55,000-60,000) corresponding to light and heavy chains, respectively.
68
SYDNEY COHEN AND CESAR MILSTEIN
In contrast to the remarkable heterogeneity of peptide chains isolated from the total immunoglobulin population, several observations indicate that individual cells synthesize only a restricted number of variants of each polypeptide chain. Immunofluorescent techniques using specific antisera have shown that heavy and light chains occur in the same cell. However, individual human or rabbit lymphoid cells produce only one type of light chain (Bernier and Cebra, 1964, 1965; Burtin, 1965), one class of heavy chain (Burtin and Buffe, 1963; Mellors and Korngold, 1963; Chiappino and Pernis, 1964; Bernier and Cebra, 1965; Cebra et al., 1966) including the d chain of IgD (Pernis et al., 1966), and probably only one type of the y chain. Moreover, in heterozygous animals, chains carrying allelic forms of allotypic specificity are always found in different cells (Pernis et al., 1965; Weiler, 1965; Cebra et al., 1966). This remarkable degree of specialization of immunoglobulin-producing cells is borne out by the properties of monoclonal proteins which characteristically are class- and type-specific and carry a single allelic form of alIotypic specificity (Table VI). In this connection it is of interest that the heterogeneity of myeloma proteins as judged by electrophoresis, arises at least in part from changes in charge properties which occur after secretion and can be induced in vitro by incubation of newly synthesized molecules with serum (Awdeh et al., 1966). Additional differences between the electrophoretic mobilities of intra- and extracellular Ig appear to arise during secretion and are attributable to changes located on the Fc portion of the heavy chain (Fleischman, 1963; Notani et al., 1966). Possible exceptions to the specialization of lymphoid cells have been recorded by Pernis and Chiappino (1964) who observed K and chains within individual cells of germinal centers, and Nossal et nl. (1964) who provided indirect evidence for the transient production of y and p chains by the same cell. It is possible, therefore, that at least in the differentiated cell, only two genes are active for the production of heavy and light chains and each chain represents predominantly the product of only one of two allelic cistrons. A similar degree of allelic exclusion has been observed in female animals with respect to genes located on the X chromosome, e.g., in heterozygous females only one allele controlling synthesis of glucose6-phosphate dehydrogenase is active ( Davidson et al., 1963; Beutler, 1964). However, in the case of autosomal genes controlling hemoglobin synthesis, both parental alleles are commonly expressed in each cell (Beutler, 1964). Immunoglobulin synthesis constitutes the only known example of complete or partial inactivation of one or other of a pair of
STRUClTJJX AND ACTIVITY OF IMMUNOGLOBULINS
69
autosomes. Whether this degree of differentiation precedes or follows antigenic stimulation is unknown. However, normal rabbit lymphocytes from A5, A6 heterozygotes show a summation of lymphoblast transformation with antisera to A5 and A6; this suggests that even primitive cells may be differentiated before antigenic stimulation to respond in terms of one or other chromosome of the pair (Gel1 and Sell, 1965). VII.
Genetic Implications of Immunoglobulin Structure
The combination of genetic experiments and protein sequence studies in bacteria and viruses has provided information having far-reaching consequences for understanding the molecular basis of protein synthesis. Unfortunately, what appear at first sight to be elementary genetic experiments on the nature of the immune response have sometimes proved to be of a complex nature (Green et al., 1966; McDevitt and Sela, 1965; Lennox, 1966). For this reason the increasing amount of protein sequence data has been used to the limit (and sometimes beyond it) in an attempt to understand some aspects of the genetic control of immunoglobulin synthesis. As in the case of mammalian hemoglobin and haptoglobin this approach has yielded some extremely informative results. Although many more sequences of the various chain types are needed, the available data indicate some puzzling contradictions. A good deal of space has been devoted to the evidence suggesting that both light and heavy chains are made up of a C-terminal section having a sequence defined by the type of chain, and an N-terminal portion specific to the clone from which the chain is derived. No two proteins from different clones (myelomas) have so far proved to be identical, and no two C-terminal sections have shown significant differences that could not be ascribed to an isotypic or allotypic distinction. This concept is derived mainly from studies on light chains, but recent results suggest that the same observations are applicable at least to the major types of y chains. In the following discussion we shall assume that regions of restricted and high variability are, in fact, characteristic of all types of immunoglobulin chains. Such an arrangement ideally fulfills the biological function of antibodies which must be unique in their specific recognition of an indefinite number of antigens and yet be able to maintain several properties characteristic of all antibodies of the same class or type. However, a structure which is in part repeated in all antibodies of a given type and, in part, is individually defined leads to an apparent contradiction regarding the number of genes controlling separate halves of the chains.
70
SYDNEY COHEN AND CESAR MILSTEIN
A. NUMBER OF GENESCONTROLLING C-TERMINAL STRETCHES There is convincing evidence that the minimum number of structural genes involved in the synthesis of immunoglobulins is of the same order as the number of classes and types of chains, i.e., in humans so far approximately twelve structural genes appear to be involved in the synthesis of C-terminal sections of immunoglobulins. That this is so is indicated by the following facts: 1. C-terminal halves of light chains of the same type and the known Fc fragments of heavy chains of a given type are remarkably homogeneous. It is difficult to imagine that an apparently identical sequence of over 100 residues (above 250 in Fc fragments) is under the control of many genes. 2. Population and family studies indicate that allotypes are segregated as Mendelian genes. It is difficult to visualize how homozygosity could be maintained for a factor controlled by, say, more than 100 different genes. One could postulate that, in the case of InV allotypes, mutation involves transfer ribonucleic acid ( RNA), and, therefore, affects the products of a triplet repeated in an equivalent position in many genes. But Gm(a) specificity, for example, appears to involve at least two different residues, and mutation in this case would have to involve at least two transfer RNA’s at the same time. These arguments taken together render most unlikely any hypothesis that necessitates a large number of genes coding for the C-terminal stretch of a given chain type,
B. NUMBEROF GENESCONTROLLING N-TERMINAL STRETCHES There is as yet no evidence that any two proteins derived from different clones are identical. All individual differences in chains of the same type and allotype are apparently located in the N-terminal sections. However, available data suggest that variability is restricted in several respects: ( a ) chain length is essentially constant, though variations in size due to insertions or deletions have been observed; ( b ) certain positions are highly variable and others highly conservative; ( c ) some positions involve one of two residues; and ( d ) the majority of the variations can be ascribed to a one-step mutation process involving both transitions and transversions (Table XXV; see also Gray et al., 1967). Even apparent exceptions may be ascribed to a one-step process when more data are collected; e.g., residue 91, Table XXV in which two steps would be required to go from Asp to Thr but single base substitutions would account for consecutive changes from Asp to Asn to Thr ( Milstein,
71
STRUCTURE AXD ACTIVITY OF IhlMUXOGLOBULINS
1966d). It must be emphasized that restrictions listed above are based on the study of a far from adequate number of proteins. Some proteins appear to have larger differences but these have not been studied in detail ( Milstein, 1966a; Baglioni and Cioli, 1966). From a statistical point of view it appears that, despite restrictions, the number of variants is theoretically very large. If variation can occur at 30 places (more than 30 have already been observed), and the number of variants at each place is restricted to only 2, then the total number of possible chains, assuming all theoretical combinations, is 2 or more than a million. If the average number of possible variants in each position is increased to 3 and the total number of variant sites to 50, then the theoretical number of variants becomes 3-"'-an astronomical figure. The chances of finding two identical proteins under these conditions is almost negligible. If, on the other hand, the number of variants is of a lower order of magnitude, one can ask how many proteins need to be studied before two are found to be identical. This can be calculated by assuming a population of infinite size containing n variants. The probability, Pr, of drawing r samples without observing a repeat is given by )'I,
Figure 9 shows a graphic representation of this equation for different values of n. If the number of variant sequences of a single type does not exceed 1000, then the probability of finding two identical sequences is large after studying 50 proteins (P,,, = 0.29). If after studying 77 proteins ( Pi, = 0.05), no two identical sequences were found it would be highly likely that the total number of variants is much larger than 1000. If the study is restricted to fingerprints rather than full sequences then the probability of finding complete identity is considerably increased because ( a ) core peptides are not detected and ( b ) variations such as Thr/Ser, Asp/Glu, Asn/Gln, and Leu/Ile are not likely to be detected. This approach has been initiated by Raglioni and Cioli (1966) who found no identical fingerprints after analyzing 25 K chains and 20 x chains, which indicates that the minimum number of variants is likely to be at least 250 K chains and above 200 chains (see Fig. 9 ) . Unfortunately no similar study has, to our knowledge, been carried out in inbred mice. In humans the possibility that some-or many-of the observed differences are due to allelic variation cannot be excluded. However, if this were not the case and each variant structure was under the genetic control of a separate gene, then a minimum approaching 500 genes for each type of light chain would be necessary.
TABLE XXV CODONSOF
THE
OBSERVED SUBSTITUTIONS IN HUMANK CHAINS"'~
1
(:APu; GAPy
39
GGX; AAPu
83
GUX; UUPy; AUPy
2
AUPu;GUX
46
SUPY
84
GCX; GGX
53
AAPu; AAPy
85
GUX; ACX
AUG; g:U;GUX
55
GCX; GAPu
90
GCX; GUX; AUPy
56
ACX; GCX; AGPy
60
lJCx. GAPy AGPy '
4
19
65 66
;E;
1
CAPu; AUG
I
ucx
92
GAPy; Gr\Pu;
=I
93
BCX; AAPy;
GAPy; GAPu
UUPU
CUPy
100
31
AUPy; AAPy; A1Pu
27
CAI’u; C U S
ccs;
C.1Pu;
I C;Gx
U
w
I]!,
” The numbers indicate the positions in the chain as shown in Table XV. In boxes are known variants that cannot be derived single base changes. Pu is either A or G; Py, U or C; X, any of the four. ‘’ From hlorgan et al. ( 1966).
74
SYDNEY COHEN AND CESAR MILSTEIN
I t is obvious that the minimum number of genes apparently required to code for N-terminaI stretches is of a different order of magnitude from that involved in coding for the C-terminal stretches. This contradiction involves a fundamental problem of immunology, namely, the origin of antibody variation ( and presumably of combining specificity ), and several mechanisms have been invoked to explain it. As discussed above,
r
FIG. 9. Probability ( P r ) curves indicating the number of proteins ( r ) which must be analyzed, out of a pool containing a total of n sequences, in order to obtain two identical sequences. (The collaboration of J. K. Moffat is gratefully acknowledged. )
conservation of invariant C-terminal sections and the Mendelian inheritance of allotypic specificity, indicate that the C-terminal end of each chain type must be controlled by a limited number of genes. The Nterminal sections must be controlled by a large number of genes which could have arisen during the course of evolution by a gene doubling process with selective preservation of variants in the germ line or be generated by somatic mutation of a limited number of genes. There are, therefore, two major problems in relation to the genetic control of immunoglobulin synthesis; namely: ( 1 ) Are the C- and N-
STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS
75
terminal sections of a given peptide chain controlled by two separate genes or by a single gene? ( 2 ) Is the variability of the genes (or specific parts of genes) controlling N-terminal sections of chains generated by mutation and selection during the course of evolution or by somatic mutation during the lifetime of the individual? No conclusive answer can be given to either of these questions at the present time, but it seems worth while discussing some of their implications in relation to the known structural features of immunoglobulin chains. The postulate that each variant chain is controlled by two structural genes visualizes that one is common to all proteins of the same type and the other is selected from a large set of genes which give rise to individual specificities. Fusion of the two halves could occur at the level of deoxyribonucleic acid (Dreyer and Bennett, 1965), messenger RNA, or at the protein level (Cioli and Baglion, 1966; Burch and Burwell, 1965) The general restrictions in chain variability, which were listed above, are qualitatively similar to those observed in several species for sequences of a given protein likely to have arisen by selection after random mutation [e.g., cytochrome c (Margoliash and Schejter, 1966) and insulin (Smith, 1966)l. Moreover, there does not appear to be a restriction of the type of base substitution since both transitions and transversions are observed. Proteins, Roy, Ag, B-J, and Ker are extremely similar in their sequences, but in 19 positions have 2 or more variants. Two of these cannot be accounted for by a one-step mutation; of the others, 8 are transitions and 15 are transversions. Such findings have suggested to several authors that genes controlling N-terminal sectiom have arisen by a mutational process followed by selection of suitable variants and are carried in the germ line. However, there are several difficulties in regard to such a theory involving two genes per chain. If the two polypeptides were synthesized separately and then joined, the two halves should behave as independent chains during amino acid incorporation; experiments with the pulse-labeling technique have, as yet, provided no direct evidence for this. In vitro studies of lymphoid cells have shown that polysomes involved in Ig synthesis are of appropriate size for heavy and light chains and not smaller units. Gin specificities located on different halves of monoclonal y.,, chains are invariably paired (see Section 11,C); if the specificities of the N-terminal half [Gm( z) and Gm( f ) ] are, in fact, localized in the “variable” region of the chain, then any mechanism involving gene fusion must be excluded. Finally, if the rabbit allotypes present on Fd and controlled by the “a” locus are associated with amino acid substitutions in the variable 9
76
SYDNEY W H E N AND (SESAR MILSTF.IN
region, then the explanation of their Mendelian inheritance becomes problematical. A major difficulty involving an independent set of genes for the Nterminal half stems from the evolutionary pattern of the system. If the N-terminal halves of the chains of different animal species are derived from a common ancestor set, then “conservative residues” present in the sncestors should have been selectively preserved. However, such residues are apparently not always common to digerent species (Table YIX) and may diger in the K and chains of a single species (Table XV ) despite evidence for their common ancestry. A further evolutionary difficulty concerns the specific attachment of a large set of N-terminal halves to a small group of C-terminal halves. The single evolutionary ancestor of K and C-termini must be capable of recognizing every N-terminal half. A doubling of the ancestor C-terminal half to produce the second type of chain would presumably have had the best chance of survival if it could still recognize the pre-existing set of N-terminal halves. The N-terminal halves should, therefore, be common to the two types of chains, and this is not the case (see Section IV,C,l,b). A more detailed discussion of evolutionary dif€iculties which complicate the two-gene model of immunoglobulin chain synthesis is given by Singer and Doolittle ( 1967). The available genetic and structural data, therefore, provide no positive support for the two gene model of chain synthesis, and certain structural features appear to be incompatible with this theory. An alternative postulate is that each variant chain is controlled by a single gene. If only a limited number of genes control C-terminal sections, then variability of the N-terminal stretch must arise by somatic hypermutation. Earlier hypotheses ( Burnet, 1959; Lederberg, 1959) did not restrict somatic mutation to a given part of the gene, but subsequent theories have attempted to account for variability confined to one-half of the chain. Sequence data can be used to test some of these hypothetical mechanisms. Because of their simplicity, crossing over mechanisms have been favored by some workers (see, e.g., Watson, 1965). One can ask, for instance, if the observed variants could have arisen from two different sets of nucleic acid sequences by multiple crossing over with strict preservation of phase. In other words, is it possible to construct two nucleotide sequences from which all amino acids in each position can be coded? This has been tested by Milstein (1966a). The more general test is to show that all substitutions involve only two bases in each position. In Table XV, however, there are 13 places in K chains which include more than two residues; among these at least three different bases are
STRUCl’URE AND ACTIVITY OF IMMUNOGLOBULINS
77
required either in the first codon position (residues 4, 83, and 96) or in the second codon position (residues 91, 96, and 100) (see ‘Table XXV). In h chains there are ten places (Table XV) with three or more variants, and five (3, 13, 15, 24, and 30) require more than two different bases in one of the codon positions. The possible occurrence of crossing over between more than two strands becomes very difficult to test in this way and is impossible if four or more strands are postulated since there are only four bases to code for any amino acid. As the number of postulated strands is increased, special assumptions must be made to explain the invariance of the C-terminal section and the Mendelian behavior of allotypes. Smithies (1965) has proposed that variations could arise by somatic rearrangements of genes controlling the polypeptide chains. Inverted duplications were suggested and these should give rise to variations clustered at the site of the inverted loop; in fact, residue differences occur along the length of the chain, cf. Roy, Ag, B-J, and Ker which have single residue differences in positions 2, 19, 30, 31, 46, 53, 56, 65, 67, 77, 91, 93, 96, and 100 (Tables XI11 to XV). Another hypothesis which can be tested by sequence data was proposed by Potter et al. (1965) and is based on a specialized messenger translation mechanism. Unusual triplets present in specific places in the gene could be translated differently depending upon small changes in the amino acid activating enzyme or in transfer RNA. This has been discussed by several authors (Milstein, 1966a; Titani et al., 1966; Gray et al., 1967; Singer and Doolittle, 1967). The fact that the chains showing a general restriction in size, may nevertheless, vary in length, is probably the strongest single argument against this hypothesis. However, it could be argued that there are, for instance, two genes of different sizes with identical C-terminal sections. Even then a variation such as that observed in residue 2 of human K chains, where an “invariant residue” (Ile) was eventually found to be replaced, seems difficult to reconcile with the prediction that “the final protein will vary by single amino acids at key points.” Other difficulties have been discussed by the authors mentioned above. Selective mutation could be achieved if a specific stretch of DNA at the beginning of the invariable part of the gene and common to all immunoglobulin genes acted as a recognition site for initiation of the mutation process. Brenner and Milstein (1966) have discussed a mutation mechanism which postulates a defective repair enzyme becoming operative after stretches of DNA coding for the N-terminal half of the chain have been split off. This model generates a far larger number of
78
SYDNF,Y COHEN AND CESAR MILSTEIN
variants than appears necessary (Dreyer and Bennett, 1965). One prediction of this hypothesis is that there should be an identical nucleotide sequence near the beginning of the C-terminal section of aZZ chains. In K and h chains, residues 110-114 are identical (Milstein, 1966e), suggesting an identical nucleotide sequence. A nucleotide sequence ( see Table XXV), Py, CCX, GCX, CCX, A, seems, in fact, to be common to K chains of human and mouse and h chains of human, starting at the end of residue 109 (Table XI). However, our knowledge of the nature and extent of the variable sections of chains is fragmentary and a stringent test of the above postulate requires further sequence data. It seems, therefore, that the available sequence data provide no positive proof for various hypothetical mechanisms whereby variability of certain sections of immunoglobbulin chains could be generated from a small number of genes. We are left with the fundamental question of whether or not each variable region is controlled by genes carried in the germ line. If this is so then a postulate that each variant chain is controlled by two genes seems unavoidable but, as outlined above, certain structural features seem to be incompatible with such a theory. A more critical assessment of its validity may come from an understanding of the evolntionary pattern of immunoglobulins and from studies on the inheritance of well-characterized variants. At present, the occurrence of somatic hypermutation seems more likely, but such a process may not necessarily be recognizable on the basis of sequence studies alone. VIII.
Comments
Since immunoglobulin structure was last reviewed in this series, the 4-chain model proposed by Porter has been amply confirmed and found to apply even to the most primitive vertebrates examined. Understanding of the general configuration of antibodies has been extended by electron microscopy which shows that, when cross-linked to antigen, the Fab portions of IgG antibodies are extended and the molecule appears as a Y-shaped strand. Although certain overall structural problems remainnotably in regard to the configuration of seromucous IgA and the apparent monovalency of the 7 S subunits of IgM, considerable progress has been made in defining the general chemical and biological properties of distinct immunoglobulin classes and types. Even more striking has been the accumulation of detailed sequence data which was obtained, in the first instance, from the study of monoclonal proteins and is now being derived increasingly from normal immunoglobulin chains; this work provides further evidence for monoclonal proteins being individnal species of normal immunoglobulin. It seems likely that these studies will
STRUCTURE AND ACITVITY OF IhIAfUiVOGLOBULIXS
79
before long lead to a detailed understanding of the chemical differences that distinguish classes, types, and allotypic variants of immunoglobulins and will also delineate evolutionary relationships between various chains. Amino acid sequence studies have revealed the surprising fact that light chains ( a n d probably also heavy chains) have C-terminal sections with sequences defined by isotypic and allotypic specificities alone, whereas N-terminal stretches appear to be specific for their clone of origin and no two chains have so far proved to have identical structures. The number of genes required to code for N-terminal stretches is, therefore, of a far greater magnitude than that involved in coding for C-terminal stretches. The fundamental problem of whether or not each variable region is controlled by a gene carried in the germ line or generated by somatic hypermutation cannot as yet be answered conclusively. It seems reasonable to assume that the remarkable degree of structural variation of immunoglobulin chains is related to combining specificity, especially as the successful refolding of antibody molecules in the absence of antigen has provided strong evidence that such specificity is dependent upon covalent structure. However, peptide chains also show individual antigenic ( idiotypic ) specificity related to, but not necessarily corresponding with combining specificity, and also considerable individual specificity in regard to interchain association. Even if monoclonal proteins with defined antibody activity become available for analysis, the relationship between combining specificity and primary structure of heavy and light chains may prove to be extremely complex. Understanding the structural basis of combining specificity may ultimately depend upon physical rather than chemical methods of analysis.
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Genetics of Immunoglobulins in the Mouse MICHAEL POTTER AND ROSE LIEBERMAN laboratory of Biology. National Cancer Institute. and the loboratory o f Clinical Investigations. National lnrfilute of Allergy and Infectious Diseases. Notional Institutes o f Health. Bethesda. Maryland
I . Introduction . . . . . . . . . . . . . . . . Structural Chanicteristics of Immnnoglol~ulinsin hlice . . . . . . . . . . . . A . General . B . Su1)unit Structure . . . . . . . . . . . C . Chemical Identification of Uiffercnt Im~iiuiioglol~ulin Chains . D . Localization of Allotypic Determinants on Different Imnirlno. . . . . . . . . . . . glohlins E . hlyeloma Proteins Ct)utiolled by Cenes Other than Those Found . . . . . . . . . . . in BALB/c . 111. Preparation and Testing of Homologous Antiscra . . . . . A . General . . . . . . . . . . . . . R . Preparation of Allotype Antisern with Normal Iiiimunoglol~ulins . C . Preparation of Allotype Antisera with Myeloma Imtiiunoglol~i~lins . D . Testing of Homologous Antisera . . . . . . . IV . Distribution and Localization of Ilcavy-Chain Determinants . . A. General . . . . . . . . . . . . . B . yG Heavy-Chain Determinants . . . . . . . . C. y H Heavy-Chain Determinants . . . . . . . . D . yA Heavy-Chain Determinants . . . . . . . . E . y F Heavy-Chain Determinants . . . . . . . . F . Unassigned Immunoglobulin Determinants . . . . . G . Failure to Produce Homologous Antisera in Some Donor-Recipient . . . . . . . . . . . Combinations V . Comparison of the Results 0l)tained with the Inhibition of Precipitation . . . . . . . . . . . . . hfethocl . VI . Linkage of &nes Cotitrolling IIcnvy-Chain Ileterminuits . . . A . Ceiwtic Studies . . . . . . . . . . . B . Structural Similarities I)rt\vwu (: ;inti f 1 1’;ipaiii Fc Fragrntwts of HALH/c hlicc . . . . . . . . . . . VII . Distrilxltion of IIcavy-Chain Dcterminants i n 1nl)rctl nntl Wild hlicc . A . Inbrcd Strains . . . . . . . . . . . B . Wild hlice . . . . . . . . . . . . C . Possible Evolution of Heavy-Chain Linkage Groups . . . VIII . hlyeloma-Specific Homologous Antisera . . . . . . . IX . Hemolytic Complement Component ( IIc’ or hluB‘) . . . . X . Concluding Rcmarks . . . . . . . . . . . References . . . . . . . . . . . . . 91 11.
92 95 95 97 9‘3
101 101 103 103 10:3
104 104 109 109 110 113 117 119 122 126
126 127 127 131 1S1 131
13’3 137 139 140 141 143
92
MICHAEL POTTER A N D ROSE L I E B E R M A N
I.
Introduction
The mouse is a highly advantageous species in which to study the qenes controlling immunoglobulin structure. The wild and domesticated forms constitute two different populations in which different selective factors affecting survival are at work. In wild mice, iminunoglobulin genes from individuals living under natural conditions can be examined. With domesticated, inbred strains developed by extensive consecutive brother-sister matings, it is possible to use classical genetic methods for establishing the segregation and linkage of genes controlling immunoglobulin structure. Hopefully, this will lead to a quantitative evaluation of the number of genes involved in immunoglobulin formation and ultimately provide insight into the mechanism of variation in immunoglobulin structure. Another important advantage in mice, very relevant to genetics, is that the polypeptide chain products of immunoglobulin genes may be recovered in sufficient quantity and purity to permit detailed chemical and amino acid sequence analysis (Hood et nl., 1966; Perham et al., 1966; Gray et al., 1967). It has become possible by the establishment of the colinear relationship between the gene and polypeptide chain ( Yanofsky, 1963; Yanofsky et nl., 1964) and the genetic code (Nirenberg and Leder, 1964) in other systems, to anticipate that immunoglobulin genetic structure may be established through amino acid sequence analysis of immunoglobulin polypeptide chains. However, normal serum immunoglobulins are a heterogeneous group of closely related proteins, and it is very difficult, if at all possible, to isolate homogeneous molecular forms which are espntial for this type of analysis. This problem is resolved by the homogeneous myeloma immunoglobnlins secreted by plasma cell tumors, and the mouse is one species in which plasma cell tumors can be readily obtained. In mice, plasma cell tumors very rarely occur spontaneously (RaskNieIsen and Gormsen, 1951; T. B. Dunn, 1954, 1957), but in one highly inbred strain, the BALBIc strain, they can be induced with high frequency by solid plastic materials such as Lucite disks or shavings ( Merwin and Algire, 1959; Merwin and Redmon, 1963), immunological adjuvants (Potter and Robertson, 1960; Lieberman et al., 1962), and mineral oils (Potter and Boyce, 1962). Plasma cell tumors can be propagated as transplant lines in syngeneic hosts where they grow to relatively enormous size in each new recipient. The cells of a tumor acti\lely synthesize and secrete specific homogeneous forms of immunoglobulins
GENEHCS OF IhfMUPiOGLOBULINS IN THE MOUSE
93
(Potter et al., 1957; Potter and Fahey, 1960). Each tumor consists of one type of plasma cell that is differentiated from others in the type of immunoglobulin synthesized. Further, tliese neoplastic cells are fixed, in a way that, when they continue to divide mitotically, the succeeding progeny produce the same type of immunoglobulin. For these reasons, one homogeneous farm of iminm~oglobulinappears in high concentration in the serum of tumor-bearing mice and permits both the separation of the immunoglobulin from the underlying normal immunoglobulins and its immunochemical characterization. There are five classes of immunoglobulins in mice: yM, yA, y F ( y l ) , 7G( y2a), and yH( 7211) (Faliey et a/., 1964; Potter et al., 1965). Each c l x s of immunoglobulin possibly has a specialized function in the immune response, although this has not been demonstrated for all forms as yet. For example, the yM-immunoglobulin is the most efficient immunoglobulin for binding complement. Eorsos and Rapp ( 1965) have shown that the presence of a single antisheep red blood cell yM molecule on a sheep red blood cell surface is sufficient to bind complement and lyse the red blood cell. YM-Immunoglobulins are 18 S molecules usually made in a pentameric form (Miller and Metzger, 1965). The large size and its multivalence endows the yM molecule with special physicochemical properties that are pertinent to some types of antigen-.antibody reactions. The yA-immunoglobulin class is of particular interest in the mouse, because the chief type of immunoglobulin-producing plasmocytoma induced by plastic ( Merwin and Redmon, 1963) or oil (Potter and Lieberman, 1967) produces yA myeloma protein. Further Mandel and Asofsky ( 1967) have shown that mesenteric node and intestinal tissue contain immunocytes that produce chiefly yA-immunoglobulins. These findings suggest that large numbers of the immunocyte population in the peritoneum (where the induced plasmocytomas arise ) are differentiated to produce yA-type immunoglobulin. Serum levels do not reflect the relative large numbers of yA cells in peritoneal tissues, as the yA leiiels in NIH-WS mice are low (0.4 mg./ml.) as compared to y F (2.5 mg./ml.) or yG plus yH (4.0 mg./ml.) (Fahey and Sell, 1965). One explanation for the low serum levels of yA is that these serum yA-immunoglobulins are concentrated in tissues or secretions. This is supported by the findings in the rabbit and in man that yA-immunoglobulins are transported across epithelial cells (e.g., salivary gland; mammary gland) and secreted into saliva, milk, nasal secretions, etc. (Tomasi et al., 1965; South et al., 1966). It is believed that during passage across the epithelial cell,
94
MICHAEL POTTER AND ROSE LIEBERMAN
the yA-immunoglobulin acquires an additional polypeptide called the “transport piece” (Cebra and Small, 1967). Thus far transport piece in the mouse has not yet been found. The 7 S mouse yF-immunoglobulins are able to bind skin (Barth and Fahey, 1965) and, thus, could be concentrated in specific tissue sites. The 7 S yG- and yH-immunoglobulins do not have this skin-binding property. The special function of these two immunoglobulin types in the immune response has not as yet been fully elucidated. In the humoral immune response in the mouse, five different classes of antibody molecules are produced (Fahey et al., 1964). The identification of genes that control the structure of these different immunoglobulin types are one of the primary genetic problems. As a direct outgrowth of the knowledge of genetic polymorphism of serum proteins and immunoglobulins in rabbits (Oudin, 1960; Dray and Young, 1959), it was discovered that genetic polymorphism of mouse immunoglobulins also existed. Kelus and Moor-Jankowski (1961) showed that mice of me inbred strain could be immunized with the immunoglobulins from a genetically different strain. The recipient mice formed precipitating antibodies that identified a specific, immunoglobulin, antigenic determinant in the donor strain. The genetic significance of this finding was immediately appreciated by observing that these determinants were present in some and absent in other inbred strains. Dubiski and Cinader (1963) identified a second immunoglobulin determinant, and Dray et al. ( 1963), utilizing reciprocal homologous antisera, demonstrated the allelism of two genes controlling different, immunoglobulin, antigenic determinants. When immunizations were extended to other donor-recipient inbred strain combinations, an elaborate system of antigenic determinants was identified (Lieberman and Dray, 1964). Based on the distribution of these determinants, five basic immunoglobulin allotypes were found among thirty-eight inbred strains. Herzenberg et al., (1965) extended this work and identified additional determinants. The assignment of immunoglobulin antigenic determinants to specific immunoglobulin chain types began with Mishell and Fahey’s (1964) observation that one of the homologous antisera identified a determinant on a yG ( y2a ) -immunoglobulin; Herzenberg ( 1964 ) found a second on a yA-immunoglobulin, and Lieberman et al. (1965) found a third on a yH-imrniunoglobulin. Herzenberg ( 1964 ) demonstrated that the genes controlling yG and yA were linked, and Lieberman et al. (1965) demonstrated the close linkage of the yG and yH genes. From these basic observations, a complex immunogenetic system in the mouse has evolved. The important general finding of genetic significance is that specific
GENETICS OF IMMUNOGLOBULINS I N THE MOUSE
95
homologous antisera can be used to identify the products of genes that control immunoglobulin structure in the mouse. Highly purified, myeloma immunoglobulins or their subunits can be used as the products of the immunoglobulin genes, and this constitutes a highly specific system. The identification reaction for this system can be an agar gel precipitin reaction which is easily accomplished with small quantities of material. It becomes possible with these antisera to test many different immunoglobulins for the presence of specific determinants. The potential of the mouse allotype system is far from being realized; for example, no polymorphisms of light chains have yet been found. Because of the rapid development of the mouse allotype system, we shall present the current data available pertaining to this system. We shall not make any attempt to describe all of the genetic aspects of the immune response; for example, discuss genes that regulate the recognition of antigenicity ( McDevitt and Sela, 1965). A brief mention of the distribution of the allotypes of Hcl (Herzenberg et al., 1963) or MuB' (Cinader and Dubiski, 1963) seriim protein among inbred strains is included. The Hcl component is not an immunoglobulin in the strict sense, but is related to the complement system and, therefore, participates in some immunological phenomena. Further, some inbred strains do not make any Hc'; these are the Hco strains. Some of these Hcn strains will form precipitating antibody when immunized with serum of mice that do produce Hc'. In preparing homologous antiserum to the immunoglobulin allotypes, antibody to the Hcl component is sometimes obtained and a brief description of the Hc' component is included here for purposes of distinguishing between the immunoglobulin allotypes and the Hc' component. 11.
'4.
Structural Characteristics of Immunoglobulins in Mice
GENERAL
The immunoglobulins of most vertebrate species, including the mouse, are constructed according to a common, 4-polypeptide-chain plan established by the noiv classic investigations of R. R. Porter and his associates (Porter, 1959, 1963; Fleischman et al., 1962, 1963; and Edelman and Poulik, 1961) . Essentially, the 4-polypeptide-chain unit consists of 2 identical light chains (25,000 mol. w. each) and 2 identical heavy chains (50,000 mol. w. each). The light chain is believed to interact with only about one-half of the heavy chain chiefly by hydrogen bonds, but also by a covalent disulfide bone ( L H disulfide). Amino acid sequence studies (Milstein, 1965; Perhani et nl., 1966) have shown the contributing cysteine from the light chain is actually the carboxyl
TABLE I IMMUNOGLOBULIN NOMENCLATURE IN MICE AND CLASSIFICATION OF
REPRESENTATIVE MYELOMAPROTEINS RZolecular forms of immunoglobulins in mice
Characteristics Classesa Chain types Light Heavy Forms0 Predominant sedimentation coefficient Representative myeloma proteins BALB/C
C3H
hL/N BL4LB/c-2(2/2 homozygotes)'
Y
Yz 4
YF
YG
IgM
IgA
IgGrl
IgGy2a
YH IgGy2b K
h
K
P
LY
(ma5 18 s
(K2a2)pb 7, 9, 11, 13 s
RIOPC 104
Adj PC-6.4 RIOPC 209 RlOPC 241 MOPC 153 X-5647 MOPC AL-3 lLIOPC 320
K
K
9 K292
Y
7
K2y2
x272
7s
7s
7s
MOPC 21 MOPC 31 MOPC 70A
Adj PC-5 RlOPC 173 LPC 1
RlOPC 141 MOPC 172 MOPC 195
X-5563 MOPC 300
MOPC 352
"Classes are distinguished by the differences in heavy chains. A class may contain two forms, i.e., two types of molecules possessing similar type heavy chains, but containing a different type of light chain ( K or h ) ; this prevails in man, but has not yet been demonstrated in mice. ' p = polymer. ' See Table 11; Section 1,E.
GENETICS OF IMMUNOGLOBULINS IN THE MOUSE
97
terminal residue. The remaining portions of the heavy chains are joined together again chiefly by hydrogen bonds and by a covalent bond, the H-H disulfide (Hong and Nisonoff, 1965). While most functional immunoglobulin molecules are constructed according to this 4-chain plan, some forms, notably the yM and yA classes, function as polymers of these 4-chain units. A second structural relationship between immunoglobulins within a class is based on the sharing of light-chain subunits, In the mouse, the yA-, yF-, YG-, and yH-immunoglobulins thus far appear to utilize the ronimon type of light-chain subunit, the K chain (Potter et al., 1965), whereas the yM-immunoglobulin contains the &-type ( McIntire et al., 1965) light-chain subunit (Table I ) . The mouse differs quite strikingly from man. In man, there are two light-chain types, the K and A. Either one of these light chains can combine with the same type of heavy chain. Six different heavy-chain types have been identified in man and taking into consideration the ability of either light-chain type to act as a subunit, there are at least twelve molecular forms of immunoglobulins in man (Kunkel et al., 1964; Terry et al., 1965).
R.
SUBUNIT STRUCTURE
There are two main types of subunits of immunoglobulins, the genetic subunits and the chemically derived proteolytic subunits. The light and heavy polypeptide chains are genetic subunits and are obtained by reducing the interchain disulfide bonds with reagents such as mercaptoethanol or dithiothreitol and dissociating the chains in urea, guanidine, propionic acid, etc. (Fleischman et a]., 1962; Small et al., 1963; Potter et al., 1965) by gel filtration. Dissociated heavy chains often become insoluble and lose their antigenicity and are not usable as antigens in agar-gel diffusion methods. The 4-chain immunoglobulin molecule is cleaved proteolytically by several enzymes (including papain which is chiefly used) into several types of subunits. The most common!y obtained are the papain F a b and Fc fragments (Porter, 1959). Papain splits the 4-chain unit in a vulnerable region about midway in the heavy-chain polypeptide into two similar Fab fragments and an F c fragment. Each Fab fragment contains an entire light chain and about one-half of a heavy chain. The Fc fragment ronsists of tbe two other halves of the heavy chains joined together by the H-H disulfide bond. The sedimentation and viscosity studies of Noelken et 01. (1965) show that the F c and Fab fragments each behave as compact globular proteins whereas the 4-chain protein is considerably extended. This has prompted Noelken et a]. to represent schematically
98
MICHAEL POTTER AND ROSE LIEBERMAK
the immunoglobulin molecule as consisting of three globular units, two Fab units, and one Fc unit. It is generally surmised that the Fab fragment is the region containing the interactive sites between the light and heavy chains. Further, Porter (1959) has shown the Fab fragments from rabbit antibody contain the antigen-combining sites.
4 Chain monomer
Papain
/
t
Reduction olkylotion
Llght chain
i
?l
COOH‘ICrOH
COOH Heavy chain
FIG. 1. A schematic iiiotlel of a 4-chain imnirinoglobulin molecule based upon the model described by Porter (1963), Fleischman et al. (1963), and Noelken et al. (1965) and the types of fragments or subunits obtained By papain digestion or reduction and alkylation. Coils not intended to connote a-helix.
The various fragments of the 4-chain immunoglobulin molecule are identified schematically in Fig. 1 which is a composite of the Porter (1963) and Noelken et aZ. (1965) schemes. In contrast to the isolated chains, the Fc and Fab fragments retain their solubility and antigenicity. Of particular relevance to the present study is the fact that most of the polymorphic, immunoglobulin, antigenic determinants are found on the papain Fc fragments. Thus, the structural basis for the antigenic poly-
GENETICS OF IMMUNOGLOBULINS IN THE MOUSE
99
morphism is probably accounted for by the amino acid sequence variations existing in the Fc region of the heavy chains. However, this is by no means the only possibility. The carbohydrate side chains are located on the Fc fragments in the rabbit (Fleischman et al., 1963), and presumably, also in the mouse and these could contribute to the antigenicity. The genes controlling carbohydrate structure would be independent of those controlling peptide structure. C. CHEMICAL IDENTIFICATION OF DIFFERENT IMMUNOGLOBULIN CHAINS The chemical identification of immunoglobulins is based upon the analysis of the polypeptide chains of a large variety of myeloma proteins (Table I ) . Initially, myeloma proteins of mice were classified by heterologous rabbit antisera (Fahey et at., 1964) and, then, further characterized by tryptic peptide maps of the heavy chains or the papain Fc fragments (Potter et al., 1965, 1966). When chains of the same type were compared by the tryptic peptide map technique, each chain derived from a different tumor source was found to differ from each other by several tryptic peptides while at the same time resembling one another by possessing a large number of common tryptic peptides. Although the genetic basis for the structural variation existing in polypeptide chains of the same type is not understood, it is chemically accounted for by changes in amino acid sequence (Hood et al., 1966; Perham et at., 1966, Gray et al., 1967). In the K-type light chains, it has been shown that amino acid variations are predominantly found scattered in the amino terminal half of the chain; the carboxyl terminal parts of K chains are similar in sequence. In the heavy chains, the same general picture probably prevails, and although this is not yet supported by amino acid sequence data, the tryptic peptide map analysis of Fc and Fab fragments indicate the heavy chain is divided into a common region (Fc) and a variable ( F d ) region (Fig. 1). Papain Fc fragments have been isolated from the yA-, yF-, yG- and yH-immunoglobulin classes in mice. Although it can be demonstrated that the heavy chain of each immunoglobulin class contains “distinguishing” tryptic peptides, the papain Fc fragments isolated from different myeloma proteins of the same class all produce the same tryptic peptide map. In the BALB/c mouse, we have found tryptic peptide maps of the Fc fragment for 2 yA myeloma proteins to be identical (Mushinski, unpublished operations) as have the maps for 6 yF, 5 yG, and 5 yH myeloma proteins. Further, the papain Fc fragments from myeloma proteins
FIG.2. Tryptic peptide maps of four types of BALB/c papain F c fragments upon which immunoglobulin determinants have been identified. Upper left and right are the G and H Fc fragments, arrows pointing right indicate common C and H tryptic peptides. Lower left is the y F F c fragment; arrow to left indicates common C and F tryptic peptides. Lower right is the S-carboxmethylated yA Fc fragment.
GENETICS O F IMMUNOGLOBULINS I N THE MOUSE
101
in the same class, have the same relative mobility in agar-gel electrophoresis which also helps in classifying the proteins (Potter, 196%). Tryptic p q t i d e maps of papain Fc fragments are, therefore, an effective means for classifying immunoglobulins (Fig. 2 ) .
I>. LOCALIZATION OF ALLOTYPICDETERMINANTS ON DIFFERENT IMMUNOGLOBULINS
Myeloma proteins from BALB/c inchding yM, yA, y F , YG, and yH types and free K- and A-type light chains (Bence-Jones proteins) were tested with a large number of homologous allotype antisera that precipitated with immunoglobulins in normal BALB/c serum. These specific antisera identified antigenic determinants on the yA, y F , yG, and yH myeloma proteins, specifically on their heavy chains in the Fc region (Dray ct al., 1965; Lieberman et a]., 1965, Lieberman and Potter 1966a; Potter et al., 1966). Allotypic antigenic determinants in BALR/c can now be assigned to specific heavy chains i.e., CY, 4, 7, and 7 (Table I ) . Thu$ far, no allotypes havc been found on light chains in the mouse. BY GENESOTHER E. MYELOMAPROTEINSCONTROLLED THAN THOSE FOUND IN BALB/c
To enable u s to compare immunoglobulin genomes in different strains of mice, it is important to assign any antigenic determinants found to a specific chain. We have only a few myeloma proteins from mice of other genotypes than BALB/c (Table I ) because plasma cell tumors are very rarely found in other strains or cannot be induced by the usual means (Merwin and Redmon, 1963; Potter, 1967a). However, it is possible to introduce genes controlling determinants not found in the BALB/c into the tumor-susceptible BALB/ c genotype by repeated back-crossing and by selecting progeny that carry the specific determinant. After several generations, it becomes possible to induce plasma cell tumors in the hybrids. For example, the unassigned immunoglobulin 2 determinant derivcd from the C57BL has been backcrossed into BALB/c for twelve consecutive generations (Tablr I1 ). At the sixth backcross generation, we mited backcross mice with each other and selected mice homozygous for the 2 determinant. Of forty such hybrids injected with mineral oil, sixteen have already developed plasma cell tumors. Tumors producing yA, ylF and yH myeloma proteins have been found. In other crosses, the genes controlling the 3 determinant from the DRA/2 and the 4 de-
102
MICHAEL POTTER AND ROSE LLEBERMAN
TABLE I1 SCHEMEFOR PRODUCING NEW PLASMACELL TUMOR-SUSCEPTIBLE TYPESOF MICE BY BAC~CROSSING DIFFERENT IMMUNOGLOBULIN GENESINTO BALB/c MICEORIGINOF BALB/c-2 Plasma cell tunior susceptible BALB/c
Plasma c e ll tumor resistant X
2/20
C57BLb
G1/G1 a C
G1/G1 C
(CX B)F,
I G1/G1 C
1
G1/GIC
G1/G1 C
tCbcl
X
G1/'+Cbc2
X
,.
Cbcnd
G1jz Cbcn
- - - - - .- .discard G1/l Cbcn
Continuous backc r o s s line, plasma cell tumor susceptible Cbcn parents of new plasma cell tumor susceptible s tra in 2/2
Defines antigenic determinant, see Table I V Symbols C = BALB/c and B = C57BL bc = backcross hc = number
terminant from AL have been backcrossed over six generations into BALB/c. Plasma cell tumors can be induced in some FI hybrids of BALB/c mice and other strains. Goldstein et al. (1966) have reported tumors in (NZB X BALB/c)F, hybrids, and we have observed them in several different hybrids (Potter, 1967a). Of particular interest, is the finding (Warner et al., 1966) that in neoplastic plasma cells originating in a host that is heterozygous for two different heavy-chain linkage groups, only one of the alleles appears to be used for immunoglobulin synthesis; the other is permanently repressed. It is possible, then, to recover the products of immunoglobulin genes not found in the BALB/c genome by backcrossing and hybridization to
GENETICS OF IMMUNOGLOBULINS I N THE MOUSE
103
BALB/c and inducing plasma cell tumors in the tumor-susceptible hybrids. Ill.
Preparation and Testing of Homologous Antisera
A. GENERAL In this section, the immunological methods used will be described. These differ from general immunological methods in only a few detailed respects but, nonetheless, it is essential to the discussion to describe the methods used as these reveal some characteristics of the allotype system, First, the immunization procedures with mice are of some interest. The mouse has not been as widely studied for its ability to produce precipitating antibody as have other species, and, especially, its ability to produce precipitating homologous antibody. Second, the methods for identifying polymorphic antigenic determinants vary in different laboratories, and this point requires some discussion. OF ALLOTYPEANTISERAWITH B. PREPARATION NORMAL IMMUNOGLOBULINS Washed “immune bacterial agglutinates” carrying the donor’s immunoglobulin are used uniformly by this laboratory to prepare homologous precipitins to immunoglobulin determinants. Whole serum has been used but usually evokes a poorer and delayed antibody response. Agglutinins to Proteris mirabilis are prepared in a selected inbred strain of mouse by immunization with 3 to 4, 0.5 ml. intraperitoneal injections, each containing approximately lo9 heat-killed organisms given at 3 to 4 day intervals. Immune agglutinates are prepared by mixing 0.1 ml. of anti- Proteus mirabilis antiserum with 0.1 ml. of packed P. mirabilis organisms. The mixture is allowed to stand at room temperature for 15 to 20 minutes, and the suspension containing the clumped organisms is centrifuged at 2000 R.P.M. for 10 minutes. The supernatant is discarded, and 0.85%saline is added to the packed organisms and mixed. The washing of the clumped organisms (agglutinates) is repeated three times. These immune agglutinates carry predominantly the 7 S (yF, YG, and yH ) immunoglolmlins of the immunized donor. Selected strains of mice are injected subcutaneously in several lymph node draining areas with a 0.1 ml. of the mixture of equal parts of complete Freund’s adjuvant and immune agglutinate (carrying the donor’s immunoglobulins ) . Three similar injections, one in incomplete Freund’s adjuvant and then two more without adjuvant are given at weekly intervals. Mice are bled f3-7 weeks after the first immunization injection, and the antisera are tested
104
MICHAEL POTTER AND ROSE LIEBERMAN
in Ouchterlony plates for precipitation with the donor’s serum. If antibody is not found, two additional subcutaneous injections of immune agglutinates without adjuvant are given. Thereafter, boosters are given whenever antibody begins to disappear. Whole-serum (Dubiski and Cinader, 1963; Herzenberg et at., 1965) precipitated 7-globulin ( Wunderlich and Herzenberg, 1963) , immune precipitates ( Gengozian and Doria, 1964), and allogeneic homologous anti-red-blood-cell ( RBC ) agglutinates ( Herzenberg 1964, Herzenberg et al., 1965) have also been used to produce homologous allotype antisera, but many of these fail to consistently evoke strong precipitating antisera. All of these antigen preparations contain mixed immunoglobulins in varying proportions. C. PREPARATION OF ALLOTYPE ANTISERAWITH MYELOMA IMMUNOGLOBULINS
Myeloma proteins have also been used to prepare homologous antisera and are especially useful since defined antigens can be used to immunize recipients. Recipient mice of selected strains are injected directly with myeloma . are proteins. Subcutaneous injections varying from 50 to 150 ~ g each given using the same regimen as is employed for bacterial agglutinates. Myeloma yG proteins evoke a strong antibody response in selected inbred strains and frequently require as little as 200 pg. for a total immunizing dose (Potter et al., 1966). This is also true of yA myeloma proteins but to a lesser degree. Myeloma yH proteins are poor immunogens and frequently require many injections, often 900 pg. of protein is injected before antibody is elicited. This is also true for y F myeloma proteins but with these, an even more intense immunization is rarely successful in inducing anti-yF antisera.
D. TESTING OF HOMOLOGOUS ANTISERA
1. Identification of Determinants Before describing the criteria for defining a determinant, it is necessary to describe some important characteristics of the homologous antisera that are produced by immunizations with immune agglutinates or myeloma proteins. Some antisera are highly specific and contain antibody to one determinant; the specificity of these antisera cannot be altered or partially changed by absorption. Further the specificity of the antiserum does not change during progressive immunization of the
GENETICS OF IMMUNOGLOBULINS IN THE MOUSE
105
mouse from which it is derived. Antisera with these characteristics can be used to define determinants. The majority of homologous antisera produced by immunization with immunoglobulins are polyspecific. These antisera may identify more than one determinant on a single immunoglobulin or may recognize determinants on two different immunoglobulins. Characteristically, the specificity of these antisera can be partially changed by absorption. In addition the specificity of this type of antiserum often changes with progressive immunization. A determinant is defined by the distribution of the antigenic determinant in question among a group of genetically different inbred homozygous strains. Monospecific antisera present no difficulties in assigning determinants, whereas polyspecific antisera that identify more than one determinant require absorption before the specificity can be determined and the determinants assigned.
2. Oitchterlonj Aiztilysis A diagram of a typical micro-Ouchterlony plate illustrating the antigens used in establishing the identity of an immunoglobulin determinant in this laboratory is given in Fig. 3. Wherever an antiserum identifies a determinant in the BALB/c and other strains presumably having thc same determinant, the antiserum is further tested in Ouchterlony plates to determine the specific immunoglobulin molecule as well as the region of that molecule bearing that determinant (Fig. 4 ) . Sevcml myeloma proteins of each class (Table I ) indicated in Fig. 4 iire roiitinely testcd for precipitation with the antiserum in question. Wherever a determinant has been identified that is not present in RALB/c or related strains and until a myeloma protein carrying the determinant can be obtained from the moiise strain in question, the determinant cannot be assigned to n specific immunoglobulin class. In those instances, immunoelectrophoresis can be used for identification. Characteristic precipitin arcs produced by the homologous antiserum reacting with its reference antigen are used to establish that the antiserum precipitates proteins in serum that are immunoglobulins. These arcs have the same shape and electrophoretic distribution as the arcs produced by similar systems where both the specificity of the antigen and the antibody are known (Lieberman and Dray, 1964; Potter and Lieberman, 1967). The specificity of some antisera can bc increased by iin absorption
19
24
31
36
FIG. 3. Model of a n Ouchterlony plate showing the procedure used to identify an immunoglobulin determinant. The homologous antiserum ( ) and the antigen (donors serum or myeloma protein) ( * ) are placed in alternate wells of the center row as indicated in each diagram. The precipitin bands between these alternate wells serve as reference bands. The top and bottom six wells of each diagram are filled with sera from the inbred strains of inice indicated. The inbred strain is considered to have the same determinant as the donor strain if the prcc:pitin hands produced by the donor strain coalesces with that produced by the strain in question. The distribution of the precipitin bands among the thirty-eight inbred strains is noted and identifies the specific determinant found in these strains and the donor strain.
+
GENETICS OF IMMUNOGLOBULINS IN THE MOUSE
107
PC-6A
@O PC-6A
000 MOW141
MOPC.21
FIG. 4. Model of an Ouchterlony plate showing the procedure used to identify the immunoglobulin carrying the specific determinant. Specific myeloma proteins from BALB/c are placed in the wells indicated. The homologous antiserum ( + ) and the antigen (donors serum or ntyelon>aprotein ) are placed in wells as indicated. Wherever a precipitin line occurs with a specific myeloma protein that coalesces with the precipitin line produced with the reference antigen, that immunoglobulin molecule is considered to carry the specific determinant that has been identified. The homologous antiseruni is further tested with the heavy-chain fragments of specific myeloma immunoglobulins to complete the identification of that determinant.
procedure. The absorbent is placed first in the antibody well and then the antibody is added; those antibody molecules that are not trapped in the immune precipitate diffuse toward the antigen well. In principle, the antiserum is absorbed with a specific antigen. This absorption procedure has been found to be particularly effective when the antigenic determinants are located on different immunoglobulin molecules. When two determinants are identified by an antiserum, one giving a strong reaction and the other a weak one; the weaker can often be removed by absorption. There are many advantages to using direct visual methods for
108
MICHAEL POTTER AND ROSE LIEBERMAN
studying antigen-antibody interactions in agar gels. One can often establish by the number of bands if there are antibodies to more than one antigenic determinant. 3. lnhibition of Precipitation hlethod
Immunoglobulin antigenic determinants have also been identified by the inhibition of precipitation method. In this highly sensitive procedure, antisera that fail to precipitate in agar gels can be used (Herzenberg et al., 1965). The sensitivity of the precipitin-inhibition method depends upon the ability of dilutions of antisera ranging from lo-' to to quantitatively precipitate small quantities of antigens of high specific activity. Iodine-125, 10,000 cpm, is introduced into 0.01 pg. quantities of r-globulins; the equivalence ratio of an homologous antiserum (or reference antibody Abr) is determined by reacting dilutions of the antiserum TABLE I11 SCHEMEOF PRINCIPLES OF PRECIPITIN-INHIBITION METHOD" lZ6IRadioactivity in 10,000 y Reaction
+ +
I *Agr Abr I1 *Agr 0 111 *Agr Agt Abr (a) Agt = Agr (b) Agt - Agr ( c ) Agt f Agr
+
+
Precipit2ate
1 /+ 4
l*Agr 0
- Abr]
Supernatant 0 *Agr
+
+ -9
+
[Agt - Abr] [Agt - A h ] [*Agr - Abr]
+ [*Agr + Abr]
*A@, 100% of I1 *Agr, partial of I1 Ayt, 0% of I1
Key to symbols and abbreviations: * = '"I substituted; Agr = reference antigen; Agt = test antigen; Ahr = antiserum that precipitates Agr; I / = run at approxiniateIy equivalence ratios; II/= same as I/ for Agr; III/ Agt is introduced at varying concentrations ( a ) is complete resemblance, ( b ) is partial resemhlance, and ( c ) is no resemblance to Agr.
with 0.01 pg. quantities of ""I reference antigen (Agr). After 3 hours at 37"C., the Agr-Abr suspension is centrifuged at 10,000 g for 10 minutes and the precipitate is removed; the amount of precipitation is determined by measuring the radioactivity remaining in the supernatant. An antiserum may identify a number of antigenic determinants in the reference antigen. The identification of specific determinants is established by introducing another unlabeled test antigen (Agt) into the Agr-Abr reaction at slightly below equivalence. These molecules may compete with the labeled lraIAgr molecules for the available Abr molecules to
GENETICS OF IMMIJNOGLOBULINS I N THE MOUSE
109
form precipitates. Three possible results are observed; no inhibition of precipitation, complete inhibition of precipitation, or partial inhibition of precipitation (Table 111). Antigenic determinants are identified in cases where the test antigen removes a part of the antibody molecules [Table I11 ( IIIa)]. Here it is postulated Agr and Agt share common determinants and that the Agr possess additional determinants not shared by Agt. IV.
Distribution and Localization of Heavy-Chain Determinants
A. GENERAL Immunoglobulin determinants have been identified by two kinds of homologous antisera in our laboratory. Homologous antisera were prepared with either mixtures of normal immunoglobulins (immune agglutinates-see Section III,B immunization methods) or with myeloma immunoglobulins derived chiefly from BALB/ c. Homologous antisera prepared with normal immunoglobulins have identified many determinants not present in BALB/c mice, and, hence, we are unable to assign them to any specific immunoglobulin class. TABLE IV IMMUNOGLOBULIN ANTIGENICDETERMINANTS OF MICE IDENTIFIED BY AGAR-GELPRECIPITINREACTIONS Strains
-
1 , 6, 7, 8
9, 1 1
12, 13, 14
1, 6, 7, 8
9, 11 9
12, 13, 14 -
9, 11
-
3
-
4, 10 4, 10 5
-
8
6, 7, 8 6, 7, 8 7, 8
-
13
-
9, 11
14
10 2
BALB/c, BDP, BRSUNT, CBA, C3H/He, C57BR, C57L, C58, MA, PL, ST, STR, 129 DD C57BL, C57BL/6, C57BL/10, NBL, HR, LP, SJL, Shl, STll/l DBA/l, DBAIL, I, RF, RIII, STOLI, SWIl, YBR A/He, A/d, AKIt, AL BL CE, UE, N H
At the present time, we have identified fourteen immunoglobulin heavy-chain determinants in inbred strains and their distribution among thirty-nine inbred strains is given in Table IV. It has been possible to assign nine of these to yG-, yH- or YA-, immunoglobulins; five have not been assigned. The yF heavy-chain determinants present a special
110
MICHAEL POTTER A M ) ROSE LIEBERMAN
problem and have not been included in TabIe IV but are described in Section IV,E. The notation system for immunoglobulin determinants used in our laboratory will be the one employed here. Essentially, when we have assigned a determinant to a specific chain, we have used a letter designation. For determinants assigned to the y heavy-chain, we have used G ; for the 7 chain; H; for the (Y chain, A (Table I ) ; and for the 4 chain, F. Consecutive numbers represent determinants as they are identified and are not speciated for G, H, A, or F. Thus a number may be assigned to two different letters, as is the case for the determinant 8 which is found on both the G and F immunoglobulins in BALB/c. (The symbols for the immunoglobulin genes G should be written Ig-G; for the sake of brevity, we have omitted the Ig prefix.)
B. YG HEAVY-CHAIN DETERMINANTS Four yG heavy-chain determinants, 1, 6, 7, and 8 have been identified among the inbred strains. As may be seen in Table IV, these are widely distributed among the thirty-nine inbred strains tested. The donor-recipient combinations used to prepare the homologous antisera that identify the four yG determinants are given in Table V. We shall TABLE V HOh~fOLOcoUs DONOR-RECIPIENT COMUINATIONS USED TO PREPARE ANTISERATHATIDENTIFY G DETERMINANTS Determinants on immunoglobulins of donor strain
Determinants on recipient strain
G Determinants 1, 6, 7 , 1, 6, 7, x I , 6, 7, X 1, 6, 7 , 1, 6, 7, 8
G Strain
Determinants
C58 DD BALB/C R.1
6, 7, X
ST
Y
7, 8
x
Determiiiaiits irlentified
Strsiii
.iL SM 3H DU.\/l
Lt F
7,8
NH
CRiBL
8
DBil/B
C57BL/10
1, 6,7 , 8
BALB/c $3"
LP
G1 (:
1
(:1
(;I GI
{ C:
I
+CX + Gti + (:7 +
~~8
G8 G1
+ G6 + G6 + '27 G I + (:6 + (;7 + C;8 G1
" Imiiirinogen was a BALB/c yG-type Inyeloma protein; in d l others, ilninune agglutinates were used.
GENETICS OF IMMUNOGLOBULINS I N TRE MOUSE
111
describe the determinants in some detail and show how the homologous antisera are prepared and how the determinants are identified. The G1 determinant is found in only fourteen of the thirty-nine inbred strains and has the narrowest distribution among the G determinants. Antisera specific for this determinant are very diacult to prepare since most recipients immunized with immunoglobulins carrying the G1 determinant make antisera simultaneously against the other G determinants that are more widely distributed. Antisera harvested in the early phases of some immunizations have proven to be the best source for this specific type of antiserum. For example, we have succeeded in preparing antisera specific for the G1 determinant in the early phases of some immunization of strain AL with C58, SM with DD, and NH with BALB/c immunoglobulins. These same combinations more frequently produce polyspecific antisera in later phases of immunization. Fw each donorrecipient combination, immune agglutinates carrying the donor’s immunoglobulins were used as immunogens. Anti-G1 antisera can also be prepared by absorption of specific isoantisera, e.g., AL anti-C58 antiserum absorbed with either a specific BALB/c myeloma protein ( yH MOPC 141 Fc fragment) or with a normal serum from a specific strain; namely, C57BL/6 (Fig. 5 ) . Homologous antisera identifying G6, G7, and G8 determinants are more easily prepared. The G6 determinant is identified by an antiserum prepared by immunizing strain DBA/1 with immune agglutinates from strain MA. This antiserum, like all the others described in this section, precipitated normal BALB/c serum and when tested with the different BALB/ c myeloma proteins, precipitated only the YG forms and the Fc fragments of the yG myeloma proteins. Normal sera from nineteen different strains were precipitated by this same antiserum (Table IV), and these strains are considered to have the same determinant, designated G6, found in BALB/c. I n another combination involving a recipient that lacks G6; namely, in RF immunized with immune agglutinates from a strain ST which has the G6 determinant, isoantisera are sometimes produced which precipitate the normal sera of the same nineteen inbred strains. More frequently, antisera from this same combination will precipitate the sera of the same nineteen strains and also the sera of three additional strains. Such an antiserum defines the G7 determinant and is found in twenty-two of thirty-nine strains (Table IV). It has not been possible to absorb an R F anti-ST antiserum (Table V ) with normal serum from a strain having only the G6 determinant to obtain an antiserum that identifies only the three additional strains. It, therefore, must be assumed that an isoantiserum such as the one described identifies both the G7 and the G6 determinants (Table V ) .
FIG. 5 . Identification of G1 and H9 determinants. Ouchterlony plates showing precipitin reactions of an homologous antiseruni 2684 ( + ) prepared in an AL mouse immunized with C58 immune agglutinates. The antigen wells contain the following: ( * ) C58 serum; ( - ) BALB/c myeloma yG Fc fragment; ( # ) BALB/c myeloma yH Fc; ( = ) BALB/c myeloma y F Fc (MOPC 21 ); and the sera from different inbred strains in wells numbered 1-38 (see Fig. 3 and Table I V ) . Precipitin reactions of antiserum 2584 after alxorption with BALB/c yH Fc (MOPC 141) and with C57BL/6 serum are also shown. The unalisorliet1 antiserrm identifies inimunoglobulins in all strains (except A/He, AKR, AL, and B L ) and on yC and yH Fc fragments. Thus this serum identifies G1, H9, and H11 determinants. Absorption with MOPC 141, yH Fc, fragment removes the antibodies identifying yH determinants and the resulting antiserum identify only the G1 determinants. C57BL/6 normal serum also removes the yH reactivity and the strong G1-specific antibodies remain.
GENETICS OF IMhfUNOC,LOBl.ILINS IN THE MOIISE
113
In another combination involving two strains, C57BL/6 and NH, neither of which carries the G6 determinant, immunization of C57BL/6 with NH immune agglutinates also produces an isoantiserum that identifies the G7 determinant in the same group of twenty-two mice (Tables IV and V ) . In more advanced phases of this immunization, the serum of eight additional strains are precipitated; this antiserum defines the G8 determinant (Table IV). Thus, R F anti-ST identifies both G6 and G7 determinants; C57BL/6 anti-NH identifies both G7 and G8 determinants (Tables IV and V ) . Antiserum identifying the G8 determinant may sometimes be shown to also identify the G7 determinant by absorption. For example SM antiBALB/c antiserum (Table V ) absorbed with DBA/2 carrying the 8 determinant will be specific for the G7 determinant; absorption with AL or CE serum will remove all the antibody reactions. On the other hand, SJL anti-DBA/2 antiserum (Table V ) only identifies the G8 determinant and not the G7 determinant but also identifies the unassigned 3 determinant (this can be demonstrated by absorption with BALB/c serum). When strain L P mice lacking G1, G6, G7, and G8 determinants are iminunizvd with BALB/c yG myc>loma immunoglobulins which carry these four determinants, antisera of different specificities are produced. Some antisera recognize only G1 G6, others G1, G6, and G7, and still others G1 G6, G?, and G8.
+
+
DETERMINANTS C. yH HEAVY-CHAIN
No homologous antisera prepared thus far with mixtures of normal immunoglobulins ( immune agglutinates ) recognize determinants exclusively on the yH BALB/c myeloma immunoglobulins. All these antisera have been prepared in either A/He or AL mice with immune agglutinates from inice with the G 1 determinant, and will identify both the heavy-chain H9 and/or H11 and G1 determinants (Tables IV and VI). Figure 5 shows an example of an antiserum identifying determinants on two different immunoglobulins. An AL mouse 2684 immunized with C58 immune agglutinates identified determinants H9 and G1. It is possible to remove the antibodies identifying G determinants from these antisera by absorption with BALB/ c yG myeloma proteins to make antisera specific for the H9 and/or H11 determinants alone (Dray et a?., 1965). The two determinants, H9 and H11, have a wide distribution among the inbred strains (Table IV). The H9 determinant was previously reported to be present in the RL strain and was based up011 precipitation of a BL pooled serum, obtained from another laboratory in 1963, with an anti-H9 antiserum. Subsequently, we found that sera from many
FIG. 6. Identification of determinants by nntisern collected at different stages of in~munizntionfrom an individual mouse. Ouchterlony plates of precipitin renctions of two homologous antisern placed in wells indicated ( ). Wells indicated ( a ) contain donor sera; wells numbered 1 3 8 contain sera of inbred strains (see Fig. 3 and Table I V ) . Antiserum N227 prepared in a C57BL/6 mouse immunized
+
115
GENETICS OF IhlhfUNOGLOBULINS IN THE MOUSE
TABLE VI L)ONOR-RECIPIENT COAIUINATIONS USEDFOR ANTISERA
THATIDENTlFY H, A,
BALB/r i.:L. BALB/c i.a. BALB/c. i.a. BAL13/[*i.a. lJAl,l3/c* ?l€ n1.p.”
A/He A/He A/He A/He A/He
BAIA13/c y L i ni.p.* BALB/c ?A n1.p. BALU/c yA m.p. HAIB/c yA 1n.p. C57BL/G i.n. DBA/2 Lit. AL i.n. NH i.a. BL La.
AND
PHEPAHATION OF €iohro~o::ous UNASSIGNED IXIhlUNOGLOUULlN DETERMINANTS
None h’oiie BALB/c. y U m p . <3iBL/6 Noiie
H9, H11, G1 H11, GI H l l and/or H9 1111 :iiid/or H9 HI), H1 1
AL A/He NH DE
Nonc None Noiie None
A19 or . i l 2 dl2 A12 or A12 A12 or A12
BALB/c BALB/c BALB/c CBFf or UALB/t! 129
None None Nolie
3
or AI, or AL o r AI, or AL or AL
Piolle
Nolie
+ .I14 + A13 + A13
2d 4 or 4 5 4 or 4
+ 10
+ 10
immune agglutinates; m.p. = niyeloina proteins. See Table I for examples. ‘ (BALB/c X C57BL/B)Fi. Determinants without letters = unassigned determinants.
I,
1.a. ‘ =
”
individual BL mice bred in our laboratory failed to precipitate with the anti-H9 antiserum. These results indicate that the H9 determinant is not present in BL mice in our stock. Recipient mice of one strain immunized with immunoglobulins from the same donor strain do not all recognize the same determinants. For example, an A/He mouse immunized with C58 immunoglobulins may identify the H9 or H11 determinants or both. In fact, an individrral mouse may identify different determinants at different stages of immunixation. For example, mouse 3177 of an A/HE anti-C58 combination identified H9, H11, and G1 determinants on day 330 following immunization, and on day 342, it only identified H11 and G 1 determinants (Fig. 6 ) . This type of response is also found in other immunizations involving the yG and yA determinants. with AKR iiiiniunoglobulins ( iminnne agglutinates ) identified the 4 determinant on days 48, antl on (lay 166 identified the 4 and G8 and possil>ly the G6 antl C7 tleterminmits; 3177 A/He inlti-C58 antiserum identified the H9, H11, and C;l rletenninants on day 330, and on day 342 identified the H11 and C1 determinants.
116
MICHAEL POTTER AND ROSE LIEBERMAN
FIG. 7. Identification of the H9 determinant by an antiserum 3146 preparcd in an AL mouse immunized with a BALB/c yH myeloma protein MOPC 195. Wells indicated ( ) have homologous antiserum, wells indicated ( * ) contain normal BALB/c serum, wells numbered 1-38 (except 4 ) contain serum of inbred strains (see Fig. 3), BALB/c myeloma proteins yG (MOPC 173), and yH (MOPC 195, MOPC 141, and MOPC 172) are in the wells indicated on the figure. This antiserum did not precipitate yF myeloma proteins (not shown),
+
GENETICS OF IMMUNOGLOBULINS IN THE MOUSE
117
Immunization of strain A/He or AL mice with BALB/c yH myeloma proteins ( MOPC-141, MOPC-195) produce antisera which are specific for heavy-chain yH determinants alone and eliminate the problem of having mixtures of antibodies to determinants on two different immunoglobulins. In general, it is difficult to prepare such antisera. The precipitin reactions of an antiserum prepared in an AL mouse immunized with MOPC-195 with the sera of thirty-eight inbred strains is shown (Fig. 7 ) . This antiserum 3146 identifies the H9 determinant; the reactions with other yH myeloma proteins and with a yG myeloma protein may also be seen. This antiserum did not precipitate any y F myeloma proteins. Homologous antisera that identify the H9 and H11 determinants, and absorbed with normal BALB/c serum, possess a curious, and as yet unexplained, capacity to precipitate specifically BALB/c yH myeloma immunoglobulins and normal ( y H ) immunoglobulins in the serum of 8 strains carrying the unassigned 3 determinant (Table IV). This finding suggests a similarity between BALB/c yH myeloma proteins and the normal yH proteins in genetically different strains that is not found in normal BALB/c yH proteins. D. yA HEAVY-CHAIN DETERMINANTS In assigning the y A-immunoglobulin determinants, several facts concerning the characteristics of yA-immunoglobulins must be known. First, in comparison to the other immunoglobulins, yA levels in the serum are apparently quite low ( Fahey and Sell, 1965); second, yA-immunoglobulins in some strains including BALB/c, AL, AKR, BRSUNT, CBA, BDP, and PL loose their antigenicity during storage and during repeated freezing and thawing. Reference samples must, therefore, be continually evaluated. Finally, the age of the mouse from which the reference sera are obtained is important. Adult mice of sufficient age, usually 2 months old or older must be used, since the concentration in a serum of young mice is so low as to often be undetectable. Particularly in strain AKR, detectable serum levels of 7A-immunoglobulins are only found in older mice. The immunizations used to produce specific yA antisera are given in Table VI. The assignment of the yA-immunoglobulin antigenic determinants is given in Table IV. In our previous assignment of yA determinants we separated strains PL, CBA, BRSUNT, and BL from strains BALB/c, BDP, C3H, C57BR, C57L, C58, MA, ST, STR, and 129. The PL, CBA, and BRSUNT strains differed from the others by lacking a determinant we called the A14
11s
MICHAEL POTTER AND ROSE LIEBERMAN
FIG, 8. Identification of the A12 determinant by an antiserum 2942 prepared in on AL mouse immunized with a BALB/c yA myeloma protein (Adj. PC-GA). Wells marked ( ) contain the antiserum, wells marked ( " ) contain normal BALB/c serum, and wells numbered 1-38 (except 4) contain sera from inbred strains (see Fig. 3); well 4 contains myeloma protein Adj. PC-GA. This antiserum (2942) did not precipitate yG, yH, or yM myeloma proteins of BALB/c (not shown).
+
GENETICS OF IMMUNOGLOBULINS I N THE MOUSE
119
determinant. This separation, however, does not exist when fresh serum is used. The sera of all these strains except BL are precipitated by an antiserum, e.g., 2942 prepared in an AL mouse immunized with myeloma Adj. PC-6A protein that identifies the A12 determinant (Fig. 8). The A14 determinant has been reassigned and is found in strains CE, DE, and NH and strains carrying the A12 determinant (Table IV). Fresh sera obtained on the day of testing from ten separate BL mice varying in ages from 4 to 12 months old failed to precipitate with antisera identifying the A12, A13, or A14 determinants. This was in contrast to previous results obtained with a pool of sera collected from several BL mice in 1963 and repeatedly frozen and thawed in the ensuing 3 years. This pool of sera continued to precipitate with the antisera identifying the A12 and A13 determinants. Until we can resolve this enigma we cannot assign the A12 or A13 determinants to the BL strain. The identification of any new yA determinants will be on the basis of reactions with fresh serum antigens. It is of considerable interest that the distribution of G1 and A12 determinants is the same among the inbred strains as is the G6 and A13 and the G8 and H9 determinants. Some plasma cell tumors of BALB/c mice produce a 3.9s yA protein that appears in the urine. This type of protein contains one K-type light chain and one chain (Potter and Kuff, 1964) and is called a ?A halfmer. We have prepared a number of homologous antisera to the halfmers Adj. PC-6C and MOPC 47A; none of these precipitate the normal sera from thirty-eight inbred strains but do precipitate the BALB/c halfmers MOPC 116, MOPC 88, MOPC 4G, MOPC 47A, and Adj. PC-6C irrespective of the halfmer immunogen used to prepare the homologous antisera. None of these halfmers are precipitated by antisera that identify the A19, A13, or A14 determinants. Only antisera prepared with Adj. PC-6A react with Adj. PC-6C halfmer and this may be attributed to a common myeloma protein specificity (see Section VIII). The two myelomas, Adj. PC-6A and Adj. PC-GC, were originally derived from the same mouse (Potter and Kuff, 1964). These results are described in detail ( Lieberman, Potter, and Mushinski, in preparation). Urine concentrates from hyperimmunized mice tested with the antihalfmer antisera give no precipitin reactions which may indicate that yA halfmers are proteins that are not liberated from normal cells. (Y
E. y F HEAVY-CHAIN DETERMINANTS The YF-immunoglobulins in mice have not as yet been shown to pos;ess unique polymorphisnis. Tryptic peptide maps of Fc fragments of
120
MICHAEL POTTER AND ROSE LIEBERMAN
y F myeloma proteins differ markedly from the YG and yH Fc pattern
(Fig. 2 ) (Potter, 1967). Composite maps show that a few peptides are shared, but for the most part, the peptides are nonoverlapping. This is in direct contrast to similarities in structure observed between the yG and yH Fc fragments (Potter et al., 1966). W e have previously reported that some antisera prepared with normal immunoglobulins ( immune agglutinates ) precipitated strongly with myeloma yG proteins and very weakly with myeloma y F proteins (Dray et al., 1965). At that time, we used the whole yF myeloma protein to test these antisera and did not attempt to determine if this precipitation would occur with the Fc fragments of the myeloma y F proteins. In agargel electrophoresis ( p H 8.2) the y F Fc fragments migrate far more TABLE VII DONOR-RECIPIENT COMBINATIONS USED IN PREPARATION OF HOhfOLOGOUS ANTISERATHATPHECIPITATE yF MOPC 21 Fc FRAGMENT A N D IDENTIFY F8 AND G8 DETERMINANTS Donor
net errniriants identified
Deterniiiiaiits
YG 1, 6, 7, 8 7, 8 1, 6, 7, 8 6, 798
8 8
I'
rF
Strain
8 BALB/c 8 NH 8 BDP 8 AKR 8 DBA/2 8 BALBlcyF m.p.n (MOPC 21)
Recipient *trail1
SM C57RL/6 SJL C57BLj6 SJL LP
YG
1, 6, 7 , 8 7,8 1, 6, 7, 8 6,7,8 8 8
yF
8 8 8 8 8 8
imp. = myeloina protein.
anodally than BALB/c yG or yH fragments (Potter, 1967), and when finally separated by this method, they can be considered to be essentially free of contamination. We have prepared papain Fc fragments from two y F myeloma proteins, MOPC 21 and MOPC 31C. These two fragments have similar electrophoretic distributions in agar gel and similar peptide maps. We have re-examined some of these antisera, and other antisera, and tested them against specific isolated y F Fc fragments (Table VII; Fig. 9 ) . Only the MOPC-21 Fc fragment was precipitated by these antisera. The data obtained indicate that the myeloma MOPC-21 y F and yG proteins have a common determinant. The determinants, designated G8 and F8, show the same distribution pattern among the thirty-
121
GENETICS OF IMMUNOGLOBULINS IN THE MOUSE
nine inbred strains (Table IV) and are identified on F c fragments of myeloma yG and y F proteins, respectively. Antisera to y F determinants are difficult to prepare by direct immunization with myeloma y F proteins. Thus far, we have only succeeded in immunizing one strain, LP, with a y F myeloma protein MOPC-21. As seen in Table VII, this antiserum behaves similarly to the antisera prepared with normal immunoglobulins and identifies determinant 8. Although the same distribution among the thirty-nine inbred strains is obtained for both G8 and F8, nevertheless, the great difficulty in obtaining
FIG.9. Precipitation of G8 and F8 determinants on Fc fragments of BALB/c yG (MOPC 173) and y F (MOPC 21) myeloma proteins, respectively. Ouchterlony plate showing the precipitin reactions of an homologous antiserum 313 ( ) (prepared in an SJL mouse immunized with BDP immune agglutinates) with the Fc fragments of several BALB/c myeloma proteins yG ( MOPC 173), yH (MOPC 141), y F (MOPC 21) and (MOPC 31C). Precipitin lines are formed with (MOPC 21) y F Fc and yG Fc and show partial identity; spurring may also be seen.
+
antibody to F8, and the ease in obtaining antibody to G8 determinant in the LP strain, suggests that some differences exist between these two determinants. (This is also shown by the spurring of the precipitin lines in Fig. 9.) An unexplained finding was that the antisera (Table VII) that identified the G8 determinant on MOPC 21 Fc fragment did not precipitate MOPC 31 Fc fragment. The tryptic peptides of Fc fragments of MOPC 21 and MOPC 31 are similar. This suggests genes controlling y F heavy chains are more complex and require further study. Possibly
122
MICHAEL POTTER AND ROSE LIEBERMAN
there are differences in MOPC 21 and MOPC 31 not revealed by tryptic peptide maps. Table VII lists the donor-recipient combinations in which the antisera were prepared that identified the G8 and F8 determinants. In general, these findings are supported by results obtained with heterologous rabbit, antimouse, immunoglobulin antisera ( Fahey et nl., 1964) where it has been observed that the yG and y F proteins share common antigenic determinants. Polymorphism in yF proteins in different inbred strains is demonstrable by a variation in electrophoretic mobility of y F papain Fc fragments derived from different strains (Herzenberg et al., 1967, Potter and Lieberman 1967). The BALB/c y F Fc fragments migrate more anodally at p H 8.2 than do those from C57BL. A y F producing tumor derived from the BALB/c-2 backcross (BALB/c mice in which the unassigned 2 determinant has been introduced) yields a y F Fc fragment of different mobility from any so far obtained from yF producing tumors of BALB/c origin. Herzenberg, using this electrophoretic characteristic, has evidence that'the y F gene is linked to the other genes in the heavy-chain linkage group. Preliminary studies have not shown that the genetic change which causes the electrophoretic variation also confers a special homologous antigenicity. This is another example of a genetic polymorphism to which we have been unable to produce an identifying homologous antiserum.
F. UNASSIGNED IMMUNOGLOBULIN DETERMINANTS Five determinants, 2, 3, 4, 5, and 10, have been found in sera of inbred strains that have not been assigned to specific immunoglobulins (Table 11). The donor-recipient combinations in which these antisera were prepared are given (Table VI ) . All of these five determinants are presumed to be 7 S immunoglobulin determinants since in immunoelectrophoretic plates the precipitin arcs have electrophoretic mobilities characteristics of 7 S immunoglobulins (YF, Y G or YH). Determinants G1, 2, 3, 4, and 5 have been shown to be controlled by five allelic chromosomal regions ( Lieberman and Dray, 1964). Homologous antisera to the 2, 3, and 4 determinants are the easiest to prepare in recipient strains having the G1 determinant. The 2 determinant differs from all other determinants inasmuch as antisera to this determinant alone can be prepared in mice having the 3, 4, and 5 determinants (Table IV). Antisera to the 3 determinant alone can also be made in mice having
GENETICS OF IMMUNOGLOBULINS IN THE MOUSE
123
the 5 determinant, e.g., DE anti-RF. However, in mice having the 4 determinant, all mice do not behave the same way when immunized with immunoglobulins carrying the 3 determinant. For example, AKR mice having the 4 determinant immunized with SWR carrying the 3 determinant will produce antisera that identify the 3 determinant alone. Another combination presumably of the same genotypes, namely, AL anti-DBA/ 2, will produce antisera that will identify the 3 and H9 determinants ( Table IV) . Recipient strains having the 2 determinants usually recognize the 3 and G8 determinants when immunized with immunoglobulins carrying the 3 determinant. Three types of antisera that identify the unassigned determinant 4 have been prepared. One type, e.g., BALB/c anti-BL (Table VI) identifies the 4 determinant. A second type identifies two unassigned determinants, 4 and 10. The 10 determinant is found in strains having the 4 determinant and also in one strain, DD, among the fourteen strains carrying the G1 determinant (Fig. 10; Table IV). The third type of antiserum in addition to the 4 determinant recognized other determinants that have been assigned to specific immunoglobulins. This is exemplified hy the N217 antiserum prepared in a C57BL mouse immunized with AL immunoglobulins ( immune agglutinates ) which identify the G8, G6, and 4 determinants. This can only be demonstrated by appropriate absorptions (Fig. 11). The unabsorbed antiserum identified determinant G8 which has a very wide distribution among the strains tested. When this antiserum was absorbed with normal serum from the DBA/2 strain, which contains determinants G8, H9, H11, and unassigned 3, the ability to recognize the G6 determinant remains. Hence the unabsorbed N217 antiserum identifies at least two determinants, G8 and G6. When this same antiserum is absorbed with normal CE serum, which contains G7, G8, H9, H11, and unassigned 5, only the ability to recognize 4 remains. Variation in recognition of antigenic determinants by an individual mouse at different stages of immunization is illustrated by mouse N227, a C57BL/6 immunized with AKR immunoglobulins, which recognized only the 4 determinant on day 48 following immunization and recognized several determinants, 4 and G8 and possibly G6 and G7 on day 166 (Fig. 6). An antiserum that identifies only the unassigned 5 determinant is very difficult to prepare. For example, BALB/c immunized with NH immunoglobulins rarely elicits precipitating antibody. The C57BL/6 antiNH combination, however, produces very strong precipitating antibody which identifies the 5 and G8 and G7 determinants. These antibodies appear to be equally strong for all the determinants, and it is difficult to
124
MICHAEL POTTER AND ROSE LIEBERMAN
FIG. 10. Identification of determinants 10 and 4. Onchterlony plate of precipitin reaction of antiserum 3484 ( + ) prepared in a BALB/c mouse immunized with BL immune agglutinates. Wells indicated ( " ) contain BL serum; wells numbered 1-38 contain sera from inbred strains (see Fig. 3 and Table IV).
GENETICS OF IMMUNOGLOBULINS IN THE hlOUSE
125
FIG.11. Identification of G8, and C6 and 4 determinants by a single antiserum. Ouchterlony plates showing precipitin reactions with homologous antiserum N217 ( + ) prepared in a C57BL/6 mouse immunized with AL immune agglutinates. The antigen wells contain the following: ( * ) AL serum; (-) BALB/c myeloma yG Fc (MOPC 173); ( # ) BALB/c myeloma yH Fc fragment (MOPC 141); ( = ) BALB/c myeloma y F Fc (MOPC 21); and the sera from different inbred strains in wells numbered 1-38 (Fig. 3 and Table IV). Precipitin reactions of antiserum N217 after absorption with DBA/2 show identification of G6 and 4 determinants; after absorption with CE serum, antiserum identifies 4 determinant alone.
126
MICHAEL POTTER AND ROSE LIEBERMAN
remove any by absorption without completely removing all determinants. Sometimes, however, specific antisera are obtainable from heterozygotes made from crosses of mice of two completely different genotypes (Table IV). For example, we were able to prepare an antiserum specific for the 5 determinant in a heterozygote of a cross of C57BL/6 and BALB/c immunized with NH immunoglobulin, The use of selected hybrids as recipients has considerable potential for the production of new types of homologous antisera.
G. FAILURE TO PRODUCE HOMOLOGOUS ANTISERAIN SOME DONOR-RECIPIENT COMBINATIONS Different donor-recipient combinations presumably of the same genotype do not necessarily recognize the same determinants, or for that matter, produce comparable strengths of antibody. For example, the BALB/c makes excellent antibody to the 2 determinant present in C57BL/6, whereas PL mice fail to recognize the 2 determinant in C57BL/6 mice. As far as we know, both these mice, BALB/c and PL, have the same immunoglobulin genotype. The MA mice presumably having the same genotype as BALB/c require much greater amounts of C57BL/6 immunogen to recognize the 2 determinant. Even then, only about half the immunized group will produce antibody. On the other hand, the MA mice are immunologically competent and have no trouble producing excellent antibody to the 4 determinant present in A L mice. These findings and others (Lieberman and Potter, 1966a) suggest genetic variations in the ability to recognize antigens and play an important role in the production of homologous antisera. V.
Comparison of the Results Obtained with the Inhibition of Precipitation
Herzenberg (1964) and Warner et al. (1966) have described sixteen immunoglobulin antigenic determinants in mice using both direct precipitation and the precipitin-inhibition methods (Table 111). The antigenic determinants described by Herzenberg and his group have been assigned to genetic loci designated Ig-1, the locus controlling the y heavy chain; Ig-2, the locus controlling the cr heavy chain; and Ig-3, the locus controlling the 7 heavy chain. The determinants are recorded as numbers, and for each locus, the first determinant identified is numbered 1 followed by consecutive numbers as determinants are identified. Thus Ig-1.1 denotes the determinant controlled by the y heavy chain gene, and the Ig-3.1 denotes the 1 determinant controlled by the heavy-chain gene. The 1 determinants of Ig-1.1 and Ig-3.1 are unrelated, In both
127
GENETICS OF IMMUNOGLOBULINS IN THE MOUSE
laboratories, the determinants identified and their distribution among tlic inbred strains, have been done for some of the same donor-recipient combinations, and it is possible to compare the findings. There appear to be seven determinants identified in both laboratories (Tab!e VIII ) . Thwe of the four yG determinants appear to be similar. Determinant G6 has only been identified b y our laboratory. Unessigned immunoglobulin CO&WAl3ISON OF
TABLE VIII ANTICENICDETERMINANTS DESCHIBED IN T H E
(:I)
(:I (;6 ( k i (;S ~
-.
-,A
YH
>(; Inimriiitrglol~riliiis~~
(1)) 1g-1 .10 -
Ig-1 . 1
hfOLJSE
lT~~:~srig~~etl
l n i n i i i i i : ) g l ~ ~ l ) ~ i i i i iIsn, i~iir ~ i r r o g I~ ~ I~ i i l iInimriiiogl~~l~uliiis~ i i s~ ~ (:I)
€19
HI 1 .-
Ig-1 . 2 Ig-1 9 1g-1 . (i
-
Ig-1 . 7 rg-1 .s
-~
-
. .-
(11)
(:t)
,112
~
-
-413
Ig-:il IK-3 2 lg-3 ::
,114
(1,)
(1))
2
Ig-1 4h Ig-1 :ih Ig-1 . 3 Ig-1 . I I h
~
-
)
4
.-
-
-
-
-
Ig-2. I Ig-2 .:<
-
-
-
~~
(3)
-
I
10 ~~
~
( b ) Herzenberg et al. (1966). Herzenberg assigns determinant to yG or Ig-1.
'* ( a ) Our laboratory;
determinants, 9, 3, 4, and 5, are similar to the Ig-1.4, Ig-1.3, Ig-1.5, and Ig-1.11 determinants. We do not propose to assign determinants 9, 3, 4, 5, and 10 to any specific immunoglobulin class until pure immunoglobulins are available from these strains. Thus far the yH and yA determinants of our group do not correspond to any of the Ig-3 and Ig-9 determinants. VI.
linkage of Genes Controlling Heavy-Chain Determinants
A. GENETICSTUDIES The linkage of genes controlling determinants on immunoglobulin heavy chains to other genes in the mouse has not been established. A few linkage groups have thus far been studied. Dray et al. (1963) reported that the genes controlling the immunoglobulin heavy-chain determinants were not sex linked and that there was no apparent close linkage to brown ( b ) on linkage Group VIII and dilution ( d ) on linkage Group 11. Herzenberg et al. (1965) also reported that the genes controlling the heavy-chain determinants are not on the X chromosome. Herzenberg et al. (1965) have not observed close linkage of various Ig-1 genes
TABLE IX CROSSES USEDTO TESTFOR LINKAGE OF UNASSIGNED 2 DETERMINANT TO VARIOUSMARKERS ON KNOWNLINKAGE GROUPS'
Marker and gene symbol Albino C Dilute d Hairless hr Piebald 8 Steel s1 Ragged Ra Danforth's short tail Sd Yellow AY Rex Re Black B Microptbalmia Miwb Pale ears ep Ruby eyes ru Looptail LP Satin sa Ataxia ax Varitint Va Oligosyndaotyly 0 s
No. of Mice'
No. of backcross Linkage genergroup ations I I1 111 111 IV V V V VII VIII XI XI1
XI1 XI11 XIV
xv
XVI XVIII
-
bc-66 bc-17 NIB. bc-43
NIB. bc-62 N-7 bc-17
Y-W N-5 F-5 bc-25
Cross
Albinos in 0 s cross used Dilute mice in piebald cross used (Hr inbred strain X BALB/c)Fz (NH(inbred) X C57BL/Ka)Fz (C57BL/6 SI X BALB/c)Fi X BALBjc (C57BL/6 Ra X BALB/c)Fi X BALB/c (Stock Sd X BALB/c)Fi X BALB/c (C57BLJG.J A Y X BALB/c)Fi X BALB/e (Stock Re x BALB/c)Fl X BALB/c (C57BL/6J 0 s X BALB/c)F1 X B.kLB/c (C57BL/6J Mi-ab X BALE/c)FI X BALB/c (C57BL/6J bep X BALB/c)Fz (C57BL/6J ru ru X BALB/c)Fz X BALB/c)Fi X BALB/c (LP Lp (C57BL/6J sa sa X BhLB/c)Fz (C57BL/6J ax X RALB/c)Fz (C57BL/6J \'a X BALB/c)F] X B.kLB/c (C57BLfGJ 0 s X BALB/c)Fi X RALB/c
+
Marker Marker 2+ 246 34 34 72 63 38 17 16 54
24 29 43 28 26 62 33 37 50
45 11
14 29 47 46 19 29 67 26 42 14 9 22 25 13
49 47
Wild 2+
Wild 2-
114 121 5s 34 31 72 37 23 90 111 104
45 263 113 59 38
Total progeny
Marker, 2 + mice obtained
tested
(9%)
-
91
55 42 67 45 51 101 20 22 128 44 39 39 113 40 59 45
217 264 225 163 118 218 178
50.5 75.5 71 71.3 57.2 45.2 47.4 35.6 44.7 48
45
95 289 212 180
132 463 202 204 185
41
75.6 76 54 71.5 72 43 51.5
SFor a description of phenotypic effects of markers see chapter "Mutant Genes and Linkages" by M. C. Green in E. L. Green, 1966, and Griineberg, 1952. b b c = marker was backcrossed to C57BL/6J by number of generations indicated. NIB = marker not on inbred background. Marker was backcrossed to 7 strain LP. The 2 is abbreviation for the unassigned immunoglobulin heavy-chain determinant 2.
GENETICS OF IMMUNOGLOBULINS IN THE MOUSE
129
to Agouti ( a ) on linkage Group V; brown ( b ) on linkage Group VIII, albino ( c ) and pink-eye dilution ( p ) on linkage Group I; dilute ( d ) and short ear (se) on linkage Group II; histocompatibility-2 on linkage Group IX; piebald ( s ) on linkage Group 111; caracul ( C a ) on linkage Group VI; and steel (Sl) on linkage Group IV. Approximately fifty segregating offspring were tested for each.
1. Linkage to Other Genes We have been pursuing this problem but have not as yet demonstrated linkage of genes controlling heavy-chain determinants to other genes in the mouse. (Mice carrying the various marker genes were obtained from the Jackson Memorial Laboratory, Bar Harbor, Maine. ) All of these markers were in mice that carried the gene controlling the unassigned 2 determinant. Some markers had been repeatedly backcrossed onto the C57BW6J background; for example, AY lethal yellow had been backcrossed 43 generations; varitint waddler ( V a ) for 25 generations; Miwh,white, for 62 generations, and steel (Sl) for 6 generations. Other marker genes were on stocks that carried the 2 determinant. In all cases we mated the dominant marker mice with BALB/c and tested the F, progeny for the presence of the 2 determinant and the marker, then these F, hybrids were mated to BALB/c and the progeny were classified. Linkage of recessive markers was tested in F, progeny of matings to BALB/c (Table IX). All mice were 3 months of age or older when tested. No evidence of linkage was obtained. The only suggestive results were seen with the markers S1 and oligosyndactyly ( 0 s ) . In view of the known sex differences in recombination frequency in mice (see Green, 1966), we examined both sexes in the S1 and 0 s crosses for recombination and found no further differences suggestive of recombination, Some data are presented here without further interpretation to illustrate the method of the linkage test. 2. Linkage within the Heavy-Chain LOCUS Herzenberg (1964) first demonstrated linkage in the genes controlling antigenic determinants located on yG- and YA-immunoglobulins. He studied the progeny of a cross between (C3H X DBA/2)F1 X C57BL with antisera that identified YA-immunoglobulins. No recombinants were found among the 149 progeny studied. Lieberman and Potter (1966a) have confirmed the linkage of genes controlling the structure of yA and yG immunoglobulins. Specific homologous antisera were prepared by immunizing NH mice with a BALB/c yA polymeric myeloma protein Adj. PC6A. The two antisera, NH anti-
130
MICHAEL POTTER AND ROSE LIEBERMAN
BALB/c yA myeloma protein (Adj. PC-6A) and C57BL/6 anti-BALB/c which identified the A12 and G7 determinants, respectively, in normal sera of BALB/c, were used to test the sera of 1054 progeny from a backcross between (BALB/c x C57BL)F, x C57BL. Both the A12 and G7 determinants were found in the same 539 mice and both determinants were absent in the same 515 progeny showing very close linkage and no crossovers. In view of the reports of the very low crossover frequency of some of the genes in the histocompatibility-2 locus in mice [0.05%or 2 recombinants in 3615 progeny ( Stimpfling and Richardson, 1965)1, we are continuing to test additional progeny for crossover. One of the reasons for pursuing these experiments bears upon the interesting question of the mechanism of structural variation. It is known from the tryptic peptide maps of the isolated y chains from five different myeloma proteins that each differs in structure from the others (Potter et d.,1965). The a chains isolated from six different yA myeloma proteins also show structural variations (unpublished observations). It is anticipated that as more samples are examined by this technique that more variants will be found. There may be a very large number of heavy-chain structural variants just as there are among the K-type light chains in the mouse (Potter et al., 1964), It is assumed that the Fd and Fc parts of the heavy chain are controlled by a single cistron. It is essential to determine if the locus for one of the chains contains a number of cistrons, one for each of the variants. If this is, indeed, true, the locus for the y and a chains should be quite large and the chances of crossing over will be greatly increased. Quantitative data on crossover frequency should provide some estimation of the size of this locus. Family study data obtained on the homologous locus in man has provided evidence that crossing over does occur rarely (Kunkel and Natvig, 1967). From the data obtained thus far in the mouse one might question if there was some natural interference for crossing over in this locus. One possibility is that the heavy-chain locus is so close to a centromere that crossing over is greatly diminished. Another might be the homologous chromosome regions in BALB/c and C57BL are structurally different and this inhibits crossing over (for further data see Potter and Lieberman, 1967). Lieberman et al. (1965) tested the linkage of genes controlling determinants on the BALB/c yG- and yH-immunoglobulins with an absorbed homologous antiserum ( AL anti-C58 absorbed with BALB/c myeloma yG protein) (Table VI) that was specific for the H11 and a second antiserum specific for the G1 determinants in 123 F, progeny of a cross between BALB/c and AL/N; no recombinants were found. This
GENETICS OF IMMUNOGLOBULINS I N THE MOUSE
131
finding indicated the genes controlling the G and H proteins in BALB/c were also linked. Because of the limited availability of the H11-specific antiserum at that time, only a limited number of progeny were tested. Clearly, it will be of interest to re-evaluate this result with a large number of backcross progeny. SIMILARITIES BETWEEN G AND B. STRUCTURAL H PAPAINFc FRAGMENTS OF BALB/c MICE Further chemical evidence points to the close relationship and possible linkage of the G and H genes. This developed from the study of the tryptic peptide maps of papain Fc fragments from the G and H myeloma proteins (Potter et al., 1966). Each map contained about 32 peptides and a composite map demonstrated that 12 of the tryptic peptides overlapped between the two types of Fc fragments. This result indicates that there is a large area of common amino acid sequence between the yG and yH polypeptides in the Fc region. This may have evolved when a gene controlling one of these chains arose from the other by a duplication error. The amino acid sequence analysis of these two peptides should reveal some clues as to the nature of this duplication process, VII.
Distribution of Heavy-Chain Determinants in Inbred and Wild Mice
A. INBRED STRAINS In 1964, over 200 inbred strains of mice were listed (Staats, 1964); many of these are close relatives of pre-existing strains. In our studies, only seven combinations of determinants have been found, and this is remarkable in view of the large number of possible combinations that one might expect to find with fourteen determinants. As already described, crossing over in the heavy-chain linkage groups has not been observed as yet in the genetic crosses thus far examined. It is probably safe to conclude that the heavy-chain linkage group is a small region on an autosomal chromosome and that because of the low incidence of recombination, only a few “chromosome types” or linkage group types are observed. An inbred strain may be characterized by the particular combination of antigenic determinants. There are five key determinants that serve as characteristic markers for the various heavy-chain linkage group types. We thought it was of interest to trace the origins of some of the inbred strains of mice using the five determinants, Gl, 2, 3, 4, and 5 (Table IV) as reference points (Fig. 12).
132
MICHAEL POTTER AND ROSE LIEBERMAN
Some background on Mus musculus is essential, The order Rodentia contains a very large number of species including Mus musculus; the taxonomic classification is not agreed upon (Wood, 1955). The Mus genus is in the Family Muridae, which from the fossil record is considered to be of southeastern Asian origin (Wood, 1955) in the late tertiary period. Mus musculus is not represented in the North American fossils, and commensals are believed to have gained access to this hemisphere by human migrations. The varieties of house mice in North and South America reflect some of the subspecies differences in feral and commensal European Mus musculus populations ( Schwarz and Schwarz, 1943). The wild mice in North America may be regarded as heterogeneous because of repeated new introductions and interminglings. The present domesticated laboratory stocks have been derived from a variety of sources. To illustrate this point it is pertinent to abstract the description of Mus musculus presented by Schwarz and Schwarz (1943). Four wild Mus musculus subspecies have been described: Mus musculus wagneri, spicilegus, manchu, and spretus. Wild forms inhabit dry areas, savannahs, steppes, or desertlike areas and feed on grasses, seeds, and grains. The commensals developed in association with human agriculture principally in three geographical centers, Russian Turkestan, Southern Russia, and Japan, from three wild subspecies, M . m. cagneri, spicilegus, and manchu, respectively. The most important of these locations was Russian Turkestan, for from there two main migrations occurred and from these several commensal subspecies originated. An Eastern group ( Mus musculus bactrianus, homomourus, castaneus, and urbanus) extended out to Persia, Northwest India, Indochina, and north to the Riu Kiu Islands. The Western group which includes Mus musculus praetextus, brevirostris, and domesticus was derived from migrants extending from Russian Turkestan to Persia, Iraq, Asia Minor, North Africa, the Nile Valley, Italy, Spain, and Western Europe. The house mice of the Northern US.and Canada are chiefly M . musculus domesticus, which is the chief commensal of Northern Europe. In the central states M . m. domesticus and brevirostris are found, whereas M . m. brevirostris is the chief subspecies in Southern U.S., in Latin America, and in Southern Europe. In appearance the wild forms are of medium size, the tail is always shorter than the length of the head and body. The lower side is white in all wild forms and the underfur may or may not be dark. A distinct line of demarcation between the upper and lower sides is observed. The commensals have several common characteristics, elongation of the tail
GENETICS OF IMMUNOGLOBULINS IN THE MOUSE
133
which is often much longer than the head and body; darker coat color; and a reduction in the bulk of the face and size of the molar teeth. Many of the strains today carry unusual mutations; some of these were collected from natural sources by both Oriental and European mouse fanciers (Keeler, 1931). The use of the mouse as an experimental animal in Europe and this country stimulated the development of laboratory colonies. Two important developments, the renaissance of Mendelian genetics in the early part of this century and the awareness of cancer in mice (particularly mammary cancer), greatly accelerated and enhanced the interest in this species. The selection of lines with high or low tumor incidence, is an example of one type of selective process involved in establishing inbred lines ( Heston, 1949). Laboratory and fancy mice were imported into this country by Miss Abby Lathrop and Leo Loeb from 1903 to 1915 (Lathrop and Loeb, 1915). These were bred both with mice obtained commercially from U. S. breeders and with wild mice. The European imports included mice from England and Germany ( Lathrop and Loeb, 1915). The Lathrop-Loeb collection was of special importance since mice from this heterogeneous colony were supplied to many investigators; notably, W. E. Castle at the Bussey Institute at Harvard, C. C. Little and L. C. Strong at Cold Spring Harbor. Little, working in Castle’s laboratory, began the genetic characterization of several coat color genes and in 1909 the now famous homozygous stock which contained three recessive characters, dilution, brown, and nonagouti was established. These were the ancestors of the DBA/2 strain of today (Strong, 1942). The present inbred strains of mice have emerged from many different sources, and contain a gene pool derived from many different geographical origins. By characterizing the inbred strains on the basis of immunoglobulin determinants ( G l , 2, 3, 4, and 5 ) , it may be seen that each has appeared several times from different sources (Table X ) . €3. WILDMICE
By using the chromosome types based on the G1, 2, 3, 4, and 5 determinants, we recently studied 123 serum samples obtained from wild mice from six different U. S. geographical locations (Lieberman and Potter, 1966h). The sera were collected by Dr. Robert Huebner of the National Institutes of Allergy and Infectious Disease in a field study of polyomavirus. The sera were diluted 1:10 or 1:s at the time of collection, which did not affect the testing of the G and H determinants. The low level of yA-immunoglobulin determinants in many strains precluded our testing
TABLE X GRIUINSAND ZELAIIONSHIPS OF THE VARIGUS INBRED STRAIWS OF KICE
?HE
[Thisw a s compiled fronr Staats (1964); Strong (1942, 1945), Heston (1949), Keeler (1931), Lathrop'and Loeb (19151, Hestun r l or. (1964), and several prsonal communications. (1) L . C. Durn mated a mouse of Little dilu+ebrvwn stock to a piebald stock at Sturrs. Connecticut: (2) dotted arrows between strains indlcate possible contamination and are'not sublines: (3) f u r details, s e e Strong (1942); (4) for details, s e e Strong (1945). 1 Isolate from wild population
Commercial breeder or fancier
% ;"'strain :
~~~~~
"'"
? Orient-Japanese
Mainland
fanciers circa 1850
Little 1921
;;-/r,
Littermates
/---";"
English A Lathrop-Loeb fanciers Collection anby.
52 x ~ 58-
5
r
__
-
~
~
~
Strow+
-~
~~
Gates
~~
~
MA --t
-
YBp,
BDP
Bussey Institute stocks .dd bb aa
Tk stock
1913
4
C57BL 6 C57BL 10
Dunn piebald
dealer,
3
NBL
Aygene-Little U.S. breeders Ohio, Mass.
2
7 C58 d
1903-1915
Michigan New England
Inbred strain immunoglobulin a n t i e m c determinant
G1
t
Strong D stock
__
STOLI DBA 2 DBA 1
-' stock N.Y. C.
Little
t t Snell
MacDowell 1922
Strong +Lynch
=
BALB/c
I
c BL
5
-
Strong A
-x
1928
I
A He A13--
Cold Spring Harbor albino
AL N
P
Princeton N.J. Rockefeller breeder v Institute 1922
J
K
-
PL
~
Swiss-WebsterJackson Lab I mixed stocks-MacArthur 1939 Pennsylvania breeder, S u p p l i e r t F u r t h bredto hrpont. 1928 f o r leukemia Swiss stock Lausanne RockefellerInstitute stock
: 2
STL SM
= AKR
AK -Lynch
-Lynch Furth circa 1930
-SWR : Rf
(low leukemia)
Engelbreth-Holm Denmark 1930
=
ST
Germany 1920
t
Japan
c Tsubura Heston
-(DD)
Paris -
1.C.R.S Mill Hi1 1930 -Bielschowsky
-
Hairless gene London 1926
Crew Carnochan
= -t
HR
NZB
NZD
DE (AA cCcr1
136
MICHAEL POTTER AND ROSE LIEBERMAN
for the A determinants (Fahey and Sell, 1965). Using only G and H determinants, we examined the various sera according to the known chromosome types that are found in inbred strains. Fifty-four samples behaved like the known homozygous inbred strains and among these we found mice having either the G1, 3, 4, or 5 determinants. The 2 determinant was not found in any of the 123 wild mice tested. There were two quite remarkable findings; first, 46 of the mice were judged to be probable heterozygotes (though not proven by genetic tests) of chromosomal types identified in inbred mice. Population samples were small and there were not enough to determine the preponderance of any particular chromosome type. The general conclusion was TABLE XI IMMUNOGLOBULIN DETERMINANTS OF INBREDMICE IN WILDMus musculus Determinants Relationship to inbred mice Resemble homozygous inbred strains Resemble hybrid of 2 inbred strains
Not found in inbred strains
No. of mice found
22 8 3 12
TG
-YH 9 9 9
7
3 4 5 3
4 22 2 8
2
Unassigned
4
5 31 4 31 5
9
3, 5
that all the populations from six different areas maintained several different heavy-chain chromosome types. This clearly suggests the ancestral original wild mice of the domesticated strains were potentially heterozygotes. Further, it may be conjectured that the G1, 3, 4, and 5 chromosome types are not deIeterious for their host and that a population may benefit from a balanced polymorphism of immunoglobulin chromosome types. A second interesting result from the study of the wild mice was the finding of a possible recombination type. These two mice from the same area each showed 3, 5, G1, G6, and H9 determinants (Table XI).
GENETICS OF IMMUNOGLOBULINS IN THE MOUSE
137
No heterozygous inbred combinations show these five determinants together. Explanations for the occurrence of these determinants in a single mouse are that they represent recombinations or that a new combination arose by independent mutation. C. POSSIBLE EVOLUTION OF HEAVY-CHAIN LINKAGE GROUPS The evolution of the immunoglobulin heavy-chain locus in the mouse is of considerable interest to the understanding of the genetic basis of antibody formation. First it has been shown that each of the four heavychain types, (Y,7 , 71, and (Potter et d,1965, Potter, 1967a,b), does not exist in a single form within the BALR/c strain. For the tryptic peptide maps of chains having the same common polypeptide component ( t h e papain Fc fragment), it was found that each chain had a unique structure located within the F d part of the heavy chain (Fig. 1; Potter et d., 1965, 1966). Further each variant thus far obtained contained the characteristic allotypic marker. It has been shown, too, that the ability to synthesize a particular variant, once established in a tumor cell, remains a stable heritable property of that cell during continuous transplantation ( Potter 1967b). These findings indicate that either individual genes within the heavy-chain linkage group, e.g., G, H, or A, have unusual properties or that the genes within the region are highly redundant and that individual cistrons have accumulated mutations. Genetic studies of the l o c ~ sare far too incomplete to provide much information as yet, but several facts now available suggest as improbable a highly redundant region that includes repeats of the part of the gene controlling the Fc fragment. The main basis for this is the uniformity of the heavy-chain types within the inbred strains. This reflects the constancy of all the polypeptide products deriving from that gene. Thus if there were from 50 to 100 copies of the Ig-G gene in each heavy-chain linkage group, all must possess the same structure €or at least half the gene, as each myeloma protein deriving from that gene in RALB/c carries the appropriate markers. Redundancy would create a number of genes each of which could accumulate mutations in the Fd segment-presumably these variants would be the basis of difference between antibody molecules. A highly repetitive locus might be expected to give evidence of recombination which as yet we have not observed except in wild mice. Further a mutation arising in the F c portion should affect one, but not all variants. This does not appear to be the case either. The structural relationship of the G and H genes in the mouse suggests one of these originated from a duplication that occurred in an im-
+
138
MICHAEL POTTER AND ROSE LIEBERMAN
mediate ancestor of Mus musculus. Such a duplication not only dupIicated the Fc part of the molecule but in all probability the Fd part containing the means for variation. There is no means yet available to explain by a duplication process, a mechanism for generating a set of variable genes all with a common segment unless the material duplicated were a single gene.' A basic nonhomologous crossover could generate a chromosome with one duplicate gene, and it is probable that limited gene duplication of heavy-chain genes has occurred. Recent work by Press et al. (1966) has shown that 17 of 19 amino acids in the COOH-terminal sequence of the yG heavy chains in three different mammals, man, horse and rabbit, are identical. Thus an ancestral, mammalian, G heavy-chain prototype gene has become an essential gene in mammalian genomes. The variants of this in man, guinea pig, mouse, etc., are possibly the result of mutations in independent duplications of this gene that have occurred outside the main evolutionary path. In this way the sequence studies will show chemical relationship of all the genes, but between species, they may not be comparably homologous for all types. Even more compelling evidence against a multigenic structure for each of the genes within the heavy-chain linkage group are the allotypes themselves. Mutations affecting amino acid sequence would not be expected to involve simultaneously all the members of such a hypothetical gene cluster in an identical way. Rather mutations would affect single genes independently. This would mean that individual heavy chains emanating from different units in the gene cluster might or might not carry the mutations. Thus far in our experience all the chains of like types uniformly carry the expected determinants. The allotypes thus behave as single Mendelian characters. Further direct evidence for this comes from the analysis of the heavy-chain types in two of the strains, BL and DD. Each differs from a more common type by only a single determinant, e.g., BL differs from strain A/He by lacking the determinant A13 and DD differs from BALB/c by containing the unassigned ten determinant. It can be postulated that these variants could have developed from single mutations. Thus strains BL ( Bagg Lynch ), BALB/c, and A/He (Fig. 12) may have had some common ancestors (Lynch, 1926, Staats, 1964) and the heavy-chain linkage group of A/He and BL became different via mutation in one or the other stock. Strain DD, though inbred only recently, has been genetically isoIated since
' It
would also be assumed hy this hypothesis that the variations were controlled could establish antibody activity. This may be a very unlikely assumption. fly random mutations and that these variants
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1926 providing ample opportunity to develop a unique mutation which is now distributed through the strain. In conclusion, the heavy-chain linkage group appears to have evolved by duplication of an ancestral mammalian gene. Individual genes in the group vary independently. The mutations assumed to affect polypeptide structure are distributed on all the variant polypeptides, apparently controlled by that gene. VIII.
Myeloma-Specific Homologous Antisera
When myeloma proteins are used as antigens in homologous immunizations, precipitins are often produced that identify determinants unique to the immunizing myeloma protein. The most striking example of this phenomenon has been the immunization of strain LP mice with different yG myeloma proteins from the BALB/c strain. Thus far we have immunized LP mice with five different myeloma proteins, Adj. PC5, LPC 1, MOPC 173, MOPC 74B, and MOPC 268, and in each case we have produced antibodies specific for each protein. In addition, each antiserum usually contains precipitins for the G1, G6, G7, and/or G8 determinants located on the Fc fragment, and these can be removed by absorption with normal BALB/c serum or another 7G myeloma protein. Each of the absorbed antisera are specific for the immunizing myeloma protein and do not react with each other. The determinants identified by these absorbed antisera are not found on the papain Fc fragment. The papain Fah fragments, however, are not precipitated by these absorbed antisera and this is thought to be due to the formation of soluble complexes by the identifying antiserum and the corresponding Fab fragment (Potter et nl., 1966). The genetic value of these specific antisera is limited by the fact that it has thus far been impossible to detect similar determinants in normal sera or in highly concentrated sera of hyperimmunized mice. This suggests that a population of antibody molecules consists of a large number of molecular farms and that no single form normally accumulates in sufficient quantity to be detected by a precipitation reaction in agar gel. These sera may be potentially very useful in identifying antibody molecules the structure of which, in the Fab portion resembles the myeloma proteins when more sensitive techniques are employed for detection. There appears to be considerable variation in ability to recognize myeloma-specific determinants depending upon the donor-recipient strain combinations used in the immunization procedure. The explanation for this is not readily apparent but suggests there are genetic differences in the ability to recognize the myeloma-specific determinants.
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(Hcl or MLIB~) Immunoglobulins are distinguished as a family of molecules having antibody activity or as a family of structurally similar molecules, e.g., the myeloma proteins that have not been shown to have antibody activity.' For example the myeloma proteins and antibody have the same genetically determined light and heavy polypeptide subunits. Although not considered in the strict sense to be immunoglobulins, the components of complement participate in a large number of immune responses. In genetic studies on inbred mice where immunological assays may reflect the participation of both antibody and complement, the genetics of complement is of obvious relative importance and hence is included briefly. Rosenberg and Tachibana (1962) developed a sensitive assay for hemolytic activity in mouse serum. They sensitized radioactive chromium-labeled sheep red blood cells with rabbit antibody and added fresh mouse serum or whole blood and tested the supernatants for radioactivity. Herzenberg et al. (1963) discovered that strain DBA/2J was completely deficient in "hemolytic complement" components ( the genotype of strain DBA/2J was Hc" Hc"), whereas strain C57BL/ lOSn contained "hemolytic" complement components ( genotype Hc' Hc' ). Appropriate crosses and backcrosses of Hc" Hen x Hc' Hc' demonstrated that Hc' behaved as a Mendelian dominant. The nature of the mutation is unknown. The Hc" mutation has not as yet been associated with manifestations of immunological incompetence affecting viability. This may be explained in part by the fact that Hc" mice have other components of complement, e.g., C'4,1,2,3 (Churchill et al., 1967), and these are sufficient to carry out essential immune responses. Recent studies by Nilsson and Miiller-Eberhard (1965) support this further as they have shown the Hc' complement factor is homologous with C'5 of the human complement system. In 1963, Cinader and Dubiski demonstrated an a-globulin allotype in the mouse (called MuB'). The inbred strains of mice were divided into two groups those with the MuB' component ( ML~B' positive) and those without MuB' ( MuB' negative). Homologous antisera were prepared by immunizing strain A/He J or DBA/BJ (MuB1 negative) with serum from strains C57L or DBA/lJ, respectively Cinader et al., 1964). The homologous antisera formed precipitin lines with serum from strains carrying MuB'; Cinader and Dubiski (1964) surveyed a large number of strains for this factor. IX.
Hemolytic Complement Component
Recently some myeloma proteins have been shown to have antibody activity (see Metzger and Stone, 1967; Eisen et al., 1967; and Cohn, 1967).
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In subsequent studies Cinader and Dubiski have shown that MuB’negative mice lack the hemolytic complement component, thus, correlating the absence of MuB’ with Hcn and indicating MuB’ and Hc’ are the same. The MuBl component is widely distributed in mammalian species ( Cinader et al., 1964). It is of considerable interest that the strains carrying the unidentified determinant ( 4 ) are all Hcn negative. Further, strain DD, which is related to mice carrying the 4 determinant by having the determinant 10, is also negative. Genetic studies have shown the MuB’ is not linked to an immunoglobulin determinant, specifically to the 2 determinant (Cinader et al., 1966). The association of the 4 determinant with Hcn or MuB’ negative mice may be a function of affinity-a genetic phenomenon which gives false evidence of linkage (Michie, 1953, Wallace, 1957 ) . Mutual affinity is believed to result from a general tendency of “like” centromeres to migrate to the same pole in meiosis. The phenomenon was originally observed in putative subspecies crosses between Mus musculus domesticus and Mus musculus bactrianus. At the time when many of the current inbred strains were evolving, chromosomes from different subspecies may have coexisted in certain stocks. X.
Concluding Remarks
In this chapter, we have been concerned with only one aspect of the genetics of immunoglobulins in mice, the polymorphism of genes controlling the structure of the yA, y F , yG, and yH heavy chains. Inbred strains of mice vary in their heavy-chain immunoglobuIin genotype, and it is possible in certain defined donor-recipient combinations to prepare precipitating antisera. These homologous antisera identify genetically controlled allotypic ( antigenic ) determinants that distinguish among mice with different heavy-chain immunoglobulin genotypes. Much of the chapter has been devoted to the means for preparing and evaluating these homologous antisera. Because there are a number of different classes of closely related immunoglobulins in the immunoglobulin family in mice, it is at present impossibIe to isoIate pure forms of normal immunoglobulins. However, pure forms of immunoglobulins can be obtained in the form of myeloma immunoglobulins that are produced by plasma cell tumors, and plasma cell tumors can be easily induced in the highly inbred BALB/c strain of mice. These tumors can be propagated by serial transplantation in syngeneic hosts. Polypeptide products of the BALB/c, heavy-chain, immunoglobulin genes have been obtained, specifically, the papain Fc fragments of the yA, yF, yG,
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and yH myeloma immunoglobulins. These four polypeptide fragments carry the allotypic markers found in the BALB/c strain. Tryptic peptide maps of fragments from different myeloma proteins of the same class are similar, indicating the papain Fc fragment represents a part of the heavychain molecule that is constant. The genes controlling the determinants on the BALB/c yA, yG, and yH papain Fc fragments are closely linked. This cluster of genes comprises the heavy-chain linkage group. Recombination of heavy-chain genes has not yet been observed in specific crosses of inbred mice in the laboratory. Evidence of recombination has been suggested, however, by the identification of unusual combinations of determinants in wild mice. The size and structure of the heavy-chain linkage group would shed a great deal of light on the mechanism of antibody synthesis as many structural variations of the a, +, 7,and 7 heavy chains have been identified. The tryptic peptide maps of heavy chains having the same Fc fragment differ from each other in the polypeptide structure in the Fd region. It is generally accepted that differences in primary structure distinguish among 4-chain antibody molecules composed of the same types of polypeptide chains. Thus, if each of these heavy chain variants was represented by a single heavy-chain cistron, it would be anticipated that the heavy-chain linkage group would be a long chromosomal segment and this should be reflected in a high rate of recombination. Further, if the heavy-chain linkage group was highly redundant for each of the heavy-chain gene types, then based on the current assignment of the fourteen determinants to specific strains, it would be difficult to imagine how the same combinations could have repeatedly appeared in mice of different origins. This apparent stability suggests the heavychain linkage group is small and contains only a few cistrons. If the polycistronic model is not the genetic basis for the variations of a single polypeptide chain type and only a few genes are present in the heavy-chain linkage group, it becomes necessary to explain how it is possible to derive more than one primary sequence from a single gene. Many possibilities are opened by a versatile system that translates a single genetic message into difi'erent polypeptides (Potter et nl., 1965). Thus far, the various gene products of heavy-chain genes other than those found in the BALB/c mouse have not been available because of the difficulties in isolating pure forms of normal immunoglobulins. It has become possible, however, to recover the polypeptide products of genes not found in BALB/c by backcrossing different heavy-chain linkage groups onto the BALB/c background. Mice carrying the non-BALB/c heavy-chain genes are selected, and after several backcrosses, these mice
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become susceptible to the induction of plasma cell tumors. Thus far, only a few types have been available, but because of the relevance of this approach to future work in this field, the means for obtaining these different immunoglobulin forms has been described. The chemical basis of the heavy-chain polymorphism has been assumed to be chiefly due to amino acid sequence variations. The possible role of carbohydrate side chains must still be considered as a contributing source of variation.
ACKNOWLEDGMENTS The authors gratefully acknowledge the skilled technical assistance of Miss Elizaheth P. Bridges in the linkage experiments; the technical contrilmtions of Messrs. William Humphrey, Jr., Cornelius B. Alexander, and Alvado F. Campbell; and the excellent photographs of Mr. Frank T. Caporael.
REFERENCES Barth, W. F., and Fahey, J. L. (1965). Nature 206, 730. Borsos, T., and Rapp, H. J. ( 1965). Science 150, 505. Cebra, J., and Small, P. A., Jr. (1967). Biochem. G, 503. Churchitl, W. H., Jr., Weintraub, R. M., Barsos, T., and Rapp, H. J. (1967). J. Erptl. Med. 125, 657. Cinader, B., and Dubiski, S. (1963). Nuture 200, 781. Cinader, B., Dubiski, S., and Wardlaw, A. C. (1964). I. Exptl. Med. 120, 897. Cinader, B., Dubiski, S., and Wardlaw, A. C. (1966). Genet. Res. Camh. 7, 32. Cohn, M. (1967). Cold Spring Harbor Symp. Quant. B i d . 32, In press. Dray, S., and Young, G. 0. (1959). Science 129, 1023. Dray, S., Lieberman, R., and Hoffman, H. A. (1963). Proc. Snc. Erptl. BioZ. Med. 113, 509. Dray, S., Potter, M., and Lieberman, R. (1965). J. Immunol. 95, 829. Dubiski, S., and Cinader, B. (1963). Nature 197, 705. Dunn, T. B. (1954). J. Natl. Cancer Inst. 14, 1281. Dunn, T. B. (1957). J. Nutl. Cancer Inst. 19, 371. Edelman, G. M., and Poulik, M. D. (1961). J. Erptl. Med. 113, 861. Eisen, H. N., Little, J. R., Osterland, K., and Simms, E. S. (1967). Cold Spring Harbor Symp. Quant. B id. 32, in press. Fahey, J. L., and Sell, S. (1965). J. Ezptl. Med. 122, 41. Fahey, J. L., Wunderlich, J., and Mishell, R. (1964). J. Erptl. Med. 120, 223, 243. Fleischman, J. B., Pain, R. H., and Porter, R. R. (1962). Arch. Biochem. Biophys. S ~ r ~ p 1, l . 174. Fleischman, J. B., Porter, R. R., and Press, E. M. ( 1 9 6 3 ) . Biochmn. 1. 88, 220. Gengozian, N., and Doria, G. ( 1964). J. Irnrnunol. 93, 426. Goldstein, G., Warner, N. L., and Holmes, N. C. (1966). J. Nafl. Cancer Inst. 37, 13s. Gray, W. R., Dreyer, W. J., and Hood, L. (1967). Science 155, 465. Green, E. L., Ed. ( 1966). “Biology of the Laboratory Mouse,” 2nd Ed. McCraw-Hill, New York.
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Griineherg, H. (1952). “Geneties of the Mouse,” 2nd Ed. Nijhoff, The Hague. Herzenberg, L. A. (1964). Cold Spring Harbor Symp. Quant. Biol. 29, 455. Herzenherg, L. A,, Tachihana, D. K., Herzenberg, L. A., and Rosenherg, L. T. (1963). Genetics 48, 711. Herzenherg, L. A., Warner, L. A., and Herzenberg, L. A. (1965). J. Exptl. Med. 121, 415. Herzenherg, L. A,, Minna, J. D., Warner, L. A., and Herzenberg, L. A. (1967). Cold Spring Harbor Symp. Quant. Biol. 32, in press. Heston, W. E. (1949). Roscoe B. Jackson Memorial Laboratory 20th Commenoration Lectures on Genetics, Cancer, Growth and Social Behavior. Bar Harbor Times, Bar Harbor, Maine, 1949. Heston, W. E., Vlahakis, G., and Tsubura, Y. (1964). J. Natl. Cancer Znst. 32, 237. Hong, R., and Nisonoff, A. (1965). J. Biol. Chem. 240, 3883. Hood, L. E., Gray, W. R., ancl Dreyer, W. J. ( 1966). Proc. N d . Acad. Sci. U.S. 55, 826. Keeler, C. E. ( 1931) , “The Laboratory Mouse.” Harvarcl Univ. Press, Cambridge, Massachusetts. Kelus, A., and Moor-Jankowski, J. K. (1961). Nature 191, 405. Knnkel, H. G., and Natvig, J. B. (1967). Cold Spring Harbor Symp. Qrrant. Biol. 32, in press. Kunkel, H. G., Allen, J. C., Gray, H. M., Martensson, L., and Grubb, €3. (1964). Nature 203, 413. Lathrop, A., and Loeb, L. (1915). 1. Exptl. Med. 22, 646, 713. Lieherman, R., and Dray, S. ( 1964). J. lmmunol. 93, 584. Lieberman, R., and Potter, M. (1966a). J . Mol. Biol. 18, 516. Lieberman, R., and Potter, M. (1966b). Science 154, 555. Lieherman, R., Mantel, N., Humphrey, W., Jr., and Blakely, J. G. (1962). Proc. Soc. Exptl. Biol. Med. 110, 897. Lieherman, R., Dray, S., and Potter, M. ( 1965). Science 148, 640. Lynch, C. J. (1926). J. Exptl. Med. 43, 339. Mandel, M. A., and Asofsky, R. (1967). In preparation. McDevitt, H. O., and Sela, M. (1965). J. Exptl. Med. 122, 517. McIntire, K. R., Asofsky, R., Potter, M., and Kuff, E. L., (1965). Scieiice 150, 361. Merwin, R. M., and Algire, G. H. (1959). Proc. SOC. Exptl. Biol. Med. 101, 437. Merwin, R. M., and Redmon, L. W. (1963). J . Natl. Cancer Inst. 31, 997. Metzger, H., and Stone, M. (1967). Cold Spring Harbor Symp. Qztant. Biol. 32, in press. Michie, D. (1955). Proc. Royal SOC. (London) B144, 241. Miller, F., and Metzger, H. (1965). J. Biol. Chem. 240, 4740. Milstein, C. (1965). Nature 215, 1171. Mishell, R., and Fahey, J. L. (1964). Science 143, 1440. Nilsson, H . R., and Miiller-Eberhard, H. J. (1965). Federation Proc. 24, 620. Nirenberg, M., and Leder, P. (1964). Science 145, 1399. Noelken, M. E., Nelson, C. A., Buckley, C. E., 111, and Tanford, S. (1965). J. Biol. Chem. 240, 218. Oudin, J. J. (1960). J. Exptl. Med. 112, 107, 125. Perham, R., Appella, E., and Potter, M. (1966’). Science 154, 391. Phillips, R. J. S. (1966). Genetics 54, 485. Porter, R. R. (1959). Biochem. J. 73,119.
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Porter, R. R. (1963). Brit. Med. Bull. 19, 197. Potter, M. (1967a). In “Methods of Cancer Research ( H . Busch, ed.), Vol. VII. Academic Press, New York. Potter, M. (1967b). In “Regulation of the Immune Response” (B. Cinader, ed.), C.C Thomas Co., Springfield. Potter, M., and Boyce, C. R. (1962). Nature 193, 1086. Potter, M., and Fahey, J. L. (1980). J. Natl. Cancer Inst. 24, 1153. Potter, M., and Kuff, E. L. (1964). J. MoE. Biol. 9, 537. Potter, M., and Lieberman, R. (1967). Cold Spring Harbor Symp. Quant. Biol. 32, in press. Potter, M., and Robertson, C. L. (1960). J. Natl. Cancer Inst., 25, 847. Potter, M., Fahey, J. L., and Pilgrim, H. I. (1957). Proc. SOC. Exptl. Biol. Med. 94, 327. Potter, M., Dreyer, W. J., Kuff, E. L., and McIntire, K. R. (1964). J. Mol. Biol. 8, 814. Potter, M., Appella, E., and Geisser, S. ( 1965). 1. Mol. Biol. 14, 361. Potter, M., Lieberman, R., and Dray, S. ( 1966). 1. Mol. Biol. 16, 335. Press, E. M., Givol, D., Piggot, P. J., Porter, R. R., and Wiikinson, J. M. (1966). Proc. Roy. SOC. B ( L o n d o n ) 166, 150. Rask-Nielsen, R., and Gormsen, H. ( 1951). Cancer 4, 387. Rosenberg, L. T., and Tachibana, D. K. (1962). J. Immunol. 89, 861. Schwarz, E., and Schwarz, H. K. (1943). I. Mamm. 24,59. Small, P. A., Jr., Kehn, J. E., and Lamm, M. E. (1963). Science 142, 393. South, M. A., Cooper, M. D., Wollheim, F. A., Hong, R., and Good, R. A. (1966). 1. Exptl. Med. 123, 615. Staats, J. (1964). Cancer Res. 24, 147. Stimpfling, J. H., and Richardson, A. (1965). Genetics 51, 831. Strong, L. C. (1936). J . Heredity 27, 21. Strong, L. C. (1942). Cancer Res. 2, 531. Strong, L. C. (1945). J. Natl. Cancer Inst. 5, 339. Terry, W. D., Fahey, J. L., and Steinberg, A. G. (1965). J. Exptl. Med. 122, 1087. Tomasi, T. B., Jr., Tan, E. M., Solomon, A., and Prendergast, R. A. (1965). 1. Exptl. Med. 121, 101. Wallace, M. E. ( 1957). Biometrics 13. 98. Warner, N. L., Herzenberg, L. A., and Goldstein, G. (1966). J. Exptl. Med. 123, 707. Wood, A. E. (1955). J. Mamm. 36, 165. Wunderlich, J. R., and Herzenberg, L. A. (1963). Proc. Natl. Acad. Sci. U.S. 49, 592. Yanofsky, C. (1963). Cold Spring Harbor Symp. Quant. Biol. 28, 296. Yanofsky, C., Carlton, B. C., Guest, J. R., IIelinski, D., and Henning, U. (1964). Proc. Natl. Acad. Sci. US.51, 266.
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Mimetic Relationships between Group A Streptococci and Mammalian Tissues JOHN B. ZABRISKIE The Rockefeller University, N e w York, N e w York
Introduction . . . . . . . . . . Biological Mimicry and Enhancement of Pathogenicity . A. Evidence from Streptococci . . . . . . B. Evidence from Other Biological Organisms . . . 111. Biological Mimicry in Relation to Pathogenesis of Disease . A. Rheumatic Fever . . . . . . . . B. Post-Streptococcal Nephritis . . . . . . IV. Croup A Streptococci and the Transplantation Antigens . V. Summary and Conclusion? . . . . . . . References . . . . . . . . . .
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Introduction
In a stimulating and provocative essay entitled Parasitism Zmmunity and Evolution, Sprent ( 1959) suggested that Darwin’s theory of biological evolution through natural selection was markedly influenced by the host-parasite interactions. In support of his thesis he cited Darwin’s descriptions of the various adaptations by parasites such as camouflage, mimicry or warning colorations, as evidence of the importance of protective adaptation in the biological world if the organism is to survive. Carrying his argument a step further, he proposed that host and parasites have undergone a series of “reciprocal adaptations” in which the invading organism survives by a series of adaptations to the host; the host, in turn, responding by variations in its mechanisms of defense. As the specificity of the immunological response on the part of the host has increased, the parasite has adapted by masking or altering its antigenic structures to more closely resemble those of the host. In its complete form this masking or “mimicry” might be identical to the host’s structures. In its less complete state, this adaptation might involve only a certain group of antigens or antigenic determinants on the part of either host or parasite. It is the concept of biological mimicry, i.e., the chemical and immunological similarities between organisms, that is to be the underlying theme of this review, and it is the present author’s intention to demonstrate how biological mimicry or cross-reactions between host and parasite may play a role in various disease states. Since the term cross-reac147
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tion is often used in the literature to denote various forms of structural identity, the words “cross-reaction’’ and “biological mimicry” will be used interchangeably in referring to these structural similarities. Partly because of the author’s interest but mainly because of the detailed immunological and chemical information that is available on the structural components of the hemolytic streptococci, the discussion will focus primarily on the antigenic similarities between the Group A streptococcus and its natural host, man. In this connection special emphasis will be placed on the nature of the role this biological mimicry may play in streptococcal infections and their nonsuppurative sequelae. Evidence for cross-reactions between host and parasite in other biological organisms will, in general, be discussed primarily as they relate to similar observations in the Group A streptococcus and its host. Orientation to specific details of biological mimicry between bacteria or helminths and the blood group substances, heterophile antigens and tissue-specific antigens, may be obtained from the excellent and extensive reviews of Kabat (1956), Damian ( 1964), Jenkin ( 1963), and Dumonde ( 1966), respectively. For the sake of convenience the discussion will be divided into three main sections. The first will be concerned with the influence biological mimicry has had on the enhancement of pathogenicity of hemolytic streptococci and other microorganisms. The second will focus on the possible role that cross-reactions between Group A streptococci and its host may pIay in the pathogenesis of various medical disorders. A final section will be devoted to the relationship of streptococcal components to the transplantation antigens. The long association of this organism to the pathogenic infections of man and its relationship to the nonsuppurative sequelae following a streptococcal infection make it an ideal model for the study of biological mimicry between host and parasite. It is hoped that the evidence to be presented of the structural similarities between Group A streptococci and the tissues of its natural host, man, will serve as a springboard for future investigations into the role biological mimicry may play in other host-parasite relationships. 11.
Biological Mimicry a n d Enhancement of Pathogenicity
A. EVIDENCE FROM STREPTOCOCCI Perhaps the most striking example of biological mimicry is to be found in the hyaluronic acid structures of streptococci and mammalian tissues. First extracted from bovine vitreous humor (Meyer and Palmer,
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( 1934), it was subscquently isolated from human umbilical cord, Groups A and C streptococci, bovine and human synovial fluid, and other mammalian tissues (Meyer, 1917). In its natural state it is a viscous mucopolysaccharide of high molecular weight and is composed of equimolar concentrations of N-acetyl glucosamine and glucuronic acid ( Meyer, 1958). Meyer has suggested that its function in animal tissues is threefold-namely, to bind water in interstitial spaces, to hold cells together in a jellylike matrix, and to serve as a lubricant and shock absorber in joints. A remarkable feature of this material is that, regardless of the source of isolation of the hyaluronate, the chemical properties of each preparation are identical. The only variations noted are attributable to differences in the molecular weights of different preparations. The question of whether hyaluronate is linked to a protein moiety has been the subject of some debate. The amount of protein present in these preparations has ranged from none (Laurent et al., 1960) to W O % (Ogsten and Stanier, 1952). The best evidence to date (Sandson and Hammerman, 1962) indicates that approximately 2% protein may be bound to the mucopolysaccharide. Although Meyer ( 1947) has suggested that this binding may be a result of weak secondary bonds and not a true proteinpolysaccharide complex, the work of Hammerman and Sandson appears to refute this concept. Taking care to avoid nonspecific absorption of tissue proteins to the viscous hyaluronate and using reducing agents and hyaluronidase digestion techniques, these authors feel that the protein moiety is an integral part of the hyaluronate complex. The wide distribution of hyaluronate in mammalian tissues coupled with the identical chemical configuration of hyaluronic acid obtained from either mammalian or streptococcal sources probably accounts for the lack of antigenicity of the substance. Although several groups of investigators ( Seastone, 1939a; Humphrey, 1943; Quinn and Singh, 1957) have employed a variety of immunizing procedures, all studies to date have been unsuccessful in producing antibodies to hyaluronic acid. More recently, Hammerman and colleagues ( 1965) have succeeded in producing antibodies to the protein moiety of hyaluronate but not to the polysaccharide itself. The evidence that hyaluronate is nonantigenic in animals is quite intriguing and suggests that in certain infections this material plays an important role in the virulence of the organisms. Indeed, Seastone (1939b) demonstrated that in Group C streptococcal infections in guinea pigs the presence of the mucopolysaccharide capsule played a major role in the infection of these animals. Furthermore, prior immunization with isolated hyaluronate was ineffective in protecting the animals
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against subsequent infection with encapsulated strains. Hirst ( 1941 ) subsequently confirmed Seastone’s original observations and demonstrated that the enzymatic removal of the encapsulated Group C strains by the enzyme, hyaluronidase, resulted in the loss of virulence of these bacteria. In Group A infections the influence of this polysaccharide on virulence is not as pronounced as that observed in Group C infections and it has been the subject of a difference of opinion among several investigators. Although Hirst (1941) was unable to demonstrate any significant effect of hyaluronidase in Group A infections in mice, Kass and Seastone (1944) found a definite, although minimal, protective effect if hyaluronidase injections were given in larger amounts and at more frequent intervals prior to inoculation with the Group A streptococcal strain. It was not, however, until the studies of Rothbard (1948) that the relative importance of M protein and hyaluronic acid capsules in the virulence of Group A streptococcal infections was firmly established. Using mouse protection, bacteriostatic and phagocytic techniques, he confirmed Seastone’s observations and demonstrated that the capsule plays a definite role in the resistance of organism to phagocytosis. Mice could be protected up to 10 MLD of Group A streptococci by prior treatment with hyaluronidase. The discrepancy between the results of Hirst and Seastone was apparently based on the timing and the amounts of hyaluronidase that had to be given during the experiments. However, the major role of M protein in the virulence of Group A streptococci was evidenced by the fact that M protein antisera protected mice up to 100,000 MLD of Group A streptococci. Rothbard concluded from these experiments that although the hyaluronic acid capsule does contribute to the virulence of Group A infections, M protein plays a far more important role in determining this property. Although M protein plays the major role in the virulence of Group A infections, the presence of a hyaluronic acid capsule is a significant factor in the phagocytosis of Group A streptococci. Hirsch and Church (1960), using an in tjitro rabbit leukocyte test system for the phagocytosis of Group A streptococci, found that encapsulated strains, with or without the presence of M protein, were not engulfed by the phagocytes. The importance of the capsule in the resistance of the organism to phagocytosis was demonstrated by the fact that addition of hyaluronidase resulted in efficient phagocytosis of the decapsulated strains, When similar experiments were performed using human leukocytes and human serum, the authors made a fascinating observation. Whereas neither rabbit nor human leukocytes were able effectively to kill encap-
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sulated streptococci in the presence of rabbit serum, human serum contained a factor that promoted phagocytosis and rapid killing of Croup A streptococci. In fact, if the concentration of serum was greater than 30%, the phagocytosis-promoting effect was even greater than that observed when hyaluronidase was used to decapsulate the strain. The effect did not appear to be based on the ability of human sera to decapsulate the strain since India ink preparations of the streptococci revealed that the capsule remained intact. The factor did not appear to be antibody, was thermolabile, and experiments with different human sera indicated that there was a quantitative difference in the amount of this factor present in human sera. Subsequently Stollerman and colleagues ( 1958, 1963) confirmed these observations and demonstrated that certain human sera were deficient in this factor. Attempts to isolate and further to identify this factor were equivocal. Although it did not appear to be any of the then known components of complement, the authors were not able to determine whether this factor could operate in the absence of complement. The fact that human sera differ in the amounts of this phagocytosispromoting factor may be of special significance in human streptococcal infections and obviously requires further investigation. In this connection, the presence of a capsule in hemolytic streptococci that is immunologically and chemically indistinguishable from a similar structure in mammalian tissues is also interesting. Capsule-containing strains would perhaps have an added advantage in entering the host during a streptococcal infection and more significantly in maintaining the infection. Seastone’s (1943) suggestion that the more virulent strains isolated from human infections were invariably mucoid would tend to support this concept. However, these studies were done before the importance of M protein in the virulence of Group A streptococcal infections was recognized and must, therefore, remain inconclusive. In any event, it provides an excellent example of biological mimicry on the part of the invading organism and an interesting and, as yet, unsolved reciprocal adaptation on the part of the host. FROM OTHERBIOLOGICAL ORGANISMS B. EVIDENCE
The evidence for structural similarities between host and parasite in other biological organisms has been recently reviewed by Jenkin (see Section I ) and will be summarized here mainly to give support to the concept of biological mimicry and enhancement of pathogenecity in diverse biological systems. Antigenic similarities between virulent Salmonellae typhimurium
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JOHN B. ZABRISKIE
organisms and mouse tissues have been described by Rowley and Jenkin (1962). By using an in vitro phagocytic test system they demonstrated that the inability of mouse macrophages to phagocytize the salmonellae was dependent on the absence of opsonizing factors in mice that were present in animals normally resistant to the organisms. Examination of sera from different species led to the observation that pig serum, which was particularly effective in promoting phagocytosis, contained hemagglutinating antibodies to mouse red blood cells. Immunization of rabbits with mouse tissues produced antiserum that was highly effective in promoting phagocytosis of the salmonellae. The concept of biological mimicry between the host and parasite was further strengthened by the fact that rats made tolerant to mouse tissues shortly before birth were quite susceptible to the infection. Control nontolerant rats remained resistant. In addition, mice who survived the infection and became “carriers” of the disease had positive Coombs test, indicating that antibodies to the organisms also bound to similar components on the mouse red blood cells (Rowley, 1966). Although these observations strongly support the concept that bioIogicaI mimicry plays a role in the susceptibility of the mouse to virulent S. typhimurium infection, the evidence is by no means conclusive. The isolation of the antigens involved in either the host or parasite and a demonstration that they are chemically or immunologically related would be an important step toward the confirmation of this attractive hypothesis. Furthermore, the observations of Mackaness and co-workers (1966) make it clear that cellular factors are also important in the resistance of the host to salmonellae infections. In a series of elegant experiments they demonstrated that, whereas the presence of opsonins is important for the original phagocytosis of these organisms, cellular immunity by the host’s macrophages plays a major role in the natural immunity of the host. These studies, however, in no way detract from the important observations of Rowley and Jenkin that biological mimicry between salmonellae and the host’s tissues may play an important role in the susceptibility of the host during the initial stages of the infection. A number of investigators have demonstrated cross-reactions between bacteria and the human blood group substances. With respect to gram-negative bacteria, Springer et al. ( 1961) has recently reviewed the pertinent literature and demonstrated that approximately 50% of 282 strains of gram-negative bacteria of different genera contained human blood group activity. Chemical analyses of these bacteria revealed that there was a close correlation between the components of the bacterial polysaccharides and the primary determinants of specificity of the
MIMETIC RELATIONSHIPS
153
blood group antigens. For example, blood group H (0)-active bacteria contained fucose, A-active bacteria contained N-acetyl glucosamine, and B-active bacteria contained glucose. What role, if any, these bacteria play in the production of naturally occurring anti-A and anti-B isoagglutinins in man at present is uncertain. Springer et al. (1959) has reported certain experiments in white Leghorn chickens suggesting that bacterial antigens may be important in their production. He noted that chicks raised under normal conditions develop anti-A blood group agglutinins by the age of 30 days whereas demonstrable activity was present in germfree chicks up to 60 days after hatching. By the sixty-sixth day, the titers were only 1:2 or 1:4, and, in fact, by the ninetieth day the titers were still only 10%of that observed in normally reared chicks. However, the ability of these chicks to respond to a specific antigenic stimulus was evidenced by the fact that easily demonstrable anti-B agglutinins were observed following the introduction of Escherichia coli 086 in the diet, a strain known to contain blood group B-active material. Furthermore, Springer ( 1956) and Muschel and Osawa (1959) have observed that hyperimmune human anti-B agglutinins will inhibit and even kill certain strains of E . coli. It is, therefore, conceivable that individuals with different blood groups might vary in susceptibility to infection with gram-negative strains on the basis of the cross-reactivity of these strains to specific blood groups. The importance of these observations in epidemiological studies of gramnegative infections and changing patterns of disease with these organisms is obvious and merits further investigation. Cross-reactions have also been noted between the Type XIV pneumococcal polysaccharide and the human blood group substances. Inadvertently discovered during the use of type-specific horse antisera for the treatment of human pneumococcal infections (Finland and Curnen, 1938, 1940), it is now clear that the partial reactivity is related to the presence of terminal galactose units in the Type XIV pneumococcal polysaccharide and the blood group substances ( Kabat and Mayer, 1961). An interesting observation of Finland and Curnen was the fact that rabbits, when immunized with Type XIV pneumococci, do not respond to antigenic determinants related to the blood group substances. This raises the intriguing question of whether biological mimicry in this particular instance may serve to protect rather than hinder the host. Since rabbits possess blood group factors that are closely related to the human blood group substances, the response might be only to those antigens that are not closely related to "self" antigens. This type of host response would thus prevent adverse reactions by the host to its own tissues. In
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JOHN B. ZABRISKIE
this connection, there is no evidence to suggest that adverse blood group reactions have occurred in patients with Type XIV pneumococcal infections. Nor is there support for the concept that individuals with anti-A or anti-B hemagglutinins may have a special advantage in resisting Type XIV pneumococcal infections. By now, it is obvious to the reader than many of the above-mentioned reports of biological mimicry and enhancement of pathogenicity are fragmentary and in many instances inconclusive. Yet the evidence does suggest that antigenic similarities between the invading organism and the host may play a role in the susceptibility or resistance of a particular host to infection. A combined immunological and biochemical approach will be needed to elucidate completely the nature of the many crossreactions in these reports, and the difficulties involved in separating contamination by exogenous sources from true adaptation by parasite or host are multiple. However, the concept of biological mimicry as an important factor in biological interactions between host and parasite appears to be established and, as will be reported in the next section, may have broad implications in medical disorders. Ill.
Biological Mimicry in Relation to Pathogenesis of Disease
A. RHEUMATIC FEVER 1. Heart-Reactiue Antigens Utilizing a variety of immunological techniques, numerous investigators (Brockmann et al., 1937; Cavelti, 1945; Osler et al., 1954; Kaplan et al., 1961a) have noted the presence of antibodies in the sera of patients with acute rheumatic fever and rheumatic heart disease which reacted against constituents of human heart tissue. In addition, Vasquez and Dixon ( 1957) and later Kaplan et al. (1961b) demonstrated that bound y-globulin was present at the site of histological damage in these patients. However, attempts to correlate the amount of circulating heart-reactive antibody in these patients with the amount of bound y-globulin in these tissues were unsuccessful (Kaplan et al., 1961a), and the nature and the origin of these antibodies remained obscure. Reasoning that the presence of the bound 7-globulin indicated an active pathological process involving the myocardium, Kaplan ( Kaplan and Meyeserian, 1962) hypothesized that an antecedent streptococcal infection might have played a direct or indirect role in the formation of these y-globulin deposits. As an outgrowth of studies that attempted to localize streptococcal products in these sites, he noted that a number of
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155
antisera prepared by immunizing rabbits with cell walls of certain Group A streptococci hound to heart muscle. Furth-rmore, protzin-containing HC1 extracts of these cell walls blocked the serological binding of this streptococcal antibody to heart muscle. The destruction of the streptococcal extract by various proteolytic enzymes, its association with M+ protein-containing strains, and its absence in M- variant strains suggested that the cross-reactive antigen was similar to but immunologically distinct from streptococcal type-specific M protein ( Kaplan, 1963 ) . Of the original 55 Group A streptococcal strains tested by Kaplan for the presence of the cross-reactive antigen, only 6 strains contained the antigen. Strains from other streptococcal groups were found to lack the antigen. The possibility that contaminating mammalian tissue products present in the growth medium was responsible for the cross-reaction was eliminated by obtaining identical results with strains grown in broth free of mammalian products. Experiments conducted along similar lines in our laboratory also provided evidence that antibodies to Group A streptococci reacted with cardiac muscle. However, this heart-reactive antigen appeared to be more widely distributed among Group A streptococci than the antigen described by Kaplan. When our preliminary experiments indicated that the heart-related antigen was present in the streptococcal membrane, a detailed investigation of the antigen-antibody reaction was begun ( Zabriskie and Freimer, 1966). Since much of what is to follow is based on an understanding of the chemical and immunological differences between the cellular fractions of the Group A streptococcus, it might be wise to digress for a moment and review some of the salient features of the cellular components of the Group A streptococcus. Largely through the efforts of Freimer et al. ( 1959) the morphological structures of the streptococcal cell can now be separated into immunologically and chemically distinct components called the “cell wall” and the “cell membrane.” Chemical analyses of these structures (Table I ) reveal that the sugars, rhamnose and hexosamine, account for approximately 40%of the weight of the cell wall. In contrast, only traces of these sugars are found in the cell membrane, a lipoprotein complex containing glucose. Whereas little or no rhamnose or hexosamine, the cell wall sugars, were found in membrane preparations, all cell wall preparations contained a varying but definite amount of glucose. As will be subsequently shown this was a highly significant finding since it suggested that the methods used for the isolation of streptococcal cell walls resulted in preparations contaminated with cell membranes.
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JOHN B. ZABRISKIE
TABLE I CHEMICAL COMPOSITION OF MEMBRANESAND CELL WALLS OF STREPTOCOCCAL STRAINT25/41P ~
~
~~~
Total lipid Structure Protoplast membranes Cell walls
~
_
Total protein
_
_
_
~
~
Glucose
Rhamnose
Hexosamine
(%I
%)
( %)
( %)
( %)
26 3
72 0
2 2
0.1
0.1
-
0.3
28.4
16 2
2.0
Immunological studies lent strong support to the evidence that the streptococcal membrane was a distinct entity. Using antisera prepared against each of the cellular fractions, Freimer (1963) found that membranes of Group A streptococci possessed a common antigen which was unrelated to antigens of the other fractions (Table 11). It also was observed that cell wall extracts consistently reacted with antisera to highly purified membranes, which again suggested that the cell wall preparations were contaminated with membranes. TABLE I1 SEROLOGICAL SPECIFICITY OF STREPTOCOCCAL CELL FRACTIONS Rabbit antisera to
-
Soluble antigens from Cell wall Protoplast membrane Cytoplasmic material
Killed Group A streptococci 4+
+ +
Protoplast membranes
+
4+ 0
Cytoplasmic material 0 0 4 t
Furthermore, agar diffusion &dies demonstrated that membranes prepared from different Group A streptococcal strains all contained a common antigen. Among the other streptococcal groups only membranes from streptococcal Groups C and G showed a partial reaction of identity with Group A membranes. Thus it was possible by chemical and immunological techniques to separate at least two of the major components of the streptococcal cell ( Freimer, 1963). a. Immunofluorescent Staining of Cardiac Muscle with Group A Streptococcal Antibody. With this brief review in mind, the recent evidence for an antigenic relationship between streptococcal antigens and mammalian tissue will now be discussed. The use of a Group C streptococcal phage-associated muralytic enzyme made it possible to
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157
prepare streptococcal protoplast membranes free of cell wall and cytoplasmic contamination. When fluorescent-labeled antisera prepared against these membranes are appIied to human cardiac tissue, the streptococcal antibody binds to the cardiac muscle cells in the manner illustrated in Plate I, Figs. 1and 2. The most intense staining was present in the region of the sarcolemmal sheath, and the nature and distribution of this immunoflourescent staining was quite similar to that observed by Kaplan ( 1963). Although these observations suggested that the sarcolemma complex was the site at which streptococcal antibody was bound, precise definition could not be obtained at the limit of resolution of fluorescence microscopy. Both rheumatic and nonrheumatic heart tissue stained with equal intensity. In addition, the fluorescent staining was not limited to cardiac muscle, and specimens of skeletal muscle obtained from a number of different voluntary muscles also bound the streptococcal antibody. In general, smooth muscle cells did not react but the layer of smooth muscle in the subendocardium of heart tissue and smooth muscle cells in the media of mediumsized arterioles were quite reactive (Plate I, Fig. 3 ) . Although arterioles always stained in sections of tissues such as the synovial lining of joint spaces, the other cellular elements of synovium as well as those of liver, spleen, and portions of the genitourinary tract did not react. In contrast to this tissue specificity, the reaction of streptococcal antibody with muscle cells was not species-specific. For example, heart sections from normal rabbits or guinea pigs exhibited the same staining patterns as those observed with human heart sections. Since the distribution of this antigen among the various groups of hemolytic streptococci was of special significance, rabbit antisera to a wide variety of Group A streptococcal types as well as those representing most of the other streptococcal groups were prepared and tested for the presence of the cross-reactive antigen. The presence of heart-reactive antibody in each serum was determined by both the direct and indirect fluorescent staining technique and the intensity of each staining graded from 0 to 4f. The results are summarized in Table I11 and demonstrate that 47 out of 48 Group A and A-variant strains contain the antigen. The antigen did not appear to be related to the presence or absence of typespecific M protein, since a variety of serological types as well as Group A strains that contained little or no protein, produced identical reactions. Furthermore, six antisera prepared by Dr. Eugene Fox to highly purified M protein antigen failed to react with the heart sections (Fox et al., 1966). In contrast to the Group A antisera, only a few of the other streptococcal group-specific antisera showed positive reactions, and the
PLATE I FIG.1. Ilemonstration of immunofluorescent staining of human heart muscle by antisera to membranes of Group A streptococci. Magnification: x 1250. FIG. 2. Cross section of muscle cells from a biopsy of the left auricle of a rheumatic patient. The immunofluorescent staining is primarily localized to the sarcolemmal region of the myofiber. Magnification: x 1250. FIG. 3. Immunofluorescent staining of the smooth muscle layer of an arteriole as well as striated muscle in a section from a biopsy of the left auricle of a nonrheumatic human heart. hlagnification: x 250. FIG.4. Staining of cardiac muscle cells with serum obtained from a patient with acute rheumatic fever using the indirect staining technique. The section of heart muscle was obtained from an auricular biopsy of a patient with rheumatic heart disease.
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MIMETIC RELATIONSHIPS
TABLE I11 DEMONSTRATION OF THE HEART-STAINING ANTIBODY IN ANTISERA TO GROUP A STREPTOCOCCI Immunofluorescerit reactinus
Ttabbit antisera t o whole cells of Hemolytic streptorocci Group h .4 var.
B C D I!! F G H L M N Staphylococcus aweus Staphylococcus albtts Diplococcus pneitmoniae Preimmrine serum coiitrols
so.( I f sera
48h :3 4
So. positive
4i 3
n
6
4
4
0 0
3 6 6 2 1 1
1r 0
0
n 0
1
0
1 1 2
n n
15
0
0
So. negative
Id
n 4 2 4 3 .i 6 2 1 1 1 1 1 2 15
Intensity o f staiiiingfL
2+/3+ 2+/3+
n
I+P+
n n
+ 0
0 0
0
n 0 0
n 0
These results represent the average staining reaction of each antiserum after absorption with rabbit liver powder. The 48 group antisera were unabsorbed, and represented 21 different serological types as well as two strains, J17A4 and S43 glossy, that had no type-specific M protein. This serum was prepared with strain H127 isolated from a human throat infection. This serum was prepared with strain, B514, originally isolated from a mouse infection.
reactions with Group C antisera were in general weaker in intensity than those of Group A. When antisera to purified membranes were examined for the presence of heart-staining antibodies, the results were even more striking (Table IV). All of the antisera to Group A streptococcal membranes produced extremely bright (4+) staining, whereas none of the antisera to membranes of other streptococcal groups or other gram-positive cocci were reactive. b. Localization of Heart-Reactive Streptococcal Antigen to the Cell Membrane. Absorption studies lent further support to the concept that
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JOHN B. ZABRISKIE
the heart reactive streptococcal antigens resided in the membrane fraction. These tests were based on the assumption that the heart-reactive antibody would be specifically absorbed by the streptococcal cell fraction which contained the heart-reactive antigen. The reference point in each experiment was a section stained with an unabsorbed fluoresceinlabeled heart-reactive antibody. The degree of absorption of heartreactive antibody by a given weight of the antigen was evaluated by comparing the intensity of the fluorescent reaction produced by the absorbed antibody with that of the unabsorbed antibody. As can be seen TABLE IV REACTIONSWHICH DEMONSTRATE THE HEART-STAINING ANTIBODY IN ANTISERA TO MEMBRANES OF Gnow A STREPTOCOCCI
IMMUNOFLUORESCENT
Immunofluorescent reactions Rabbit antisera to membranes of
No. of sera
No. positive
No. negative
Intensity of shining
Hemolytic streptococci Group A
15
15
0 0 0 0
0 2 3 2 1
4+
2 3 2 1
15
0
15
0
C D Streptococcus viridans Staphylococcus aureus Preimmune serum controls
0 0 0 0
in Table V as little as 1 mg. of Group A streptococcal membranes completely abolished the staining reaction. Membranes from 36 strains of Group A and A variant streptococci representing fourteen different serological types were all equally effective in inhibiting the serological reactivity of the antibody. The only exception was a membrane preparation from a Group A streptococcal strain, A 236, originally isolated from a mouse infection. Although membranes from some strains of Groups C and G streptococci partially absorbed the immunofluorescence, at least 5 mg. of these membranes were necessary to produce 50% inhibition. Membranes from other streptococcal groups as well as membranes from other gram-positive cocci were nonreactive. Whereas cytoplasmic material obtained from a number of Groups A and C strains did not decrease the fluorescent staining of cardiac tissue, absorption with either 5 mg. of cell walls or 10 mg. of whole cells significantly reduced the intensity of staining. As the cell membrane represents 10%of the dry weight of the
161
MIMETIC RELATIONSHIPS
streptococcus, it seemed likely that the absorption of the heart-staining antibody by whole cells was a reflection of the presence of the cell membrane. This concept was supported by the serological evidence that almost all sera prepared against whole cells of Group A streptococci contained membrane antibodies. TABLE V ABSORPTIOXOF HEART-REACTIVE ANTIBODYBY MEMBRANESOF GROUPA STREPTOCOCCI dbsorptioii with ~~
IVhole cell Aiitigetis prepared from Hemolytic streptococci Group -4* X var.
c
D E F G Streptococcus t2iridan.s Staphgloroccus aureiis rnabsorbed globulin co ti trol
SO. of
n-t.
straiiia (mg.)
33 3 4 3 1 1 2 2 1
10 10 I0
1iit.n
+
2 2+
4+
Cell wall nt . (mg.)
5 5 10 10 10 10
10 10 10
4+
Cell memhraiie
liit.'l
+/z
+/s
+
+
4+ 4+ 4+ 4+ 4+ 4+ 4+
4+
wt . (mg.)
1 1 5 10 10 10 5 10 10
cytoplasmit. material wt.
Iiit."
0 0 2+ 4+
(mg.) Irit."
10 10 10
4+ 4+ 4+
4+ 4+ 2
+
4+ 4+ 4+
4+
Int = intensity of the immunofluorescent staining reaction after absorption. Group A membranes used were prepared from the following types: 1, 3, 4, 5, 6, 12, 14, 19, 22, 24, 26, 30, and 50 and several nontypeable strains. Of the 33 strains tested, only one, A236, a strain isolated from mice, failed to absorb the heart-reactive antibody. "
"
c. Presence of Streptococcal Membranes in Cell Wall Preparations. The absorption of the heart-reactive antibody by cell walls appeared at first paradoxical since the evidence up to this point indicated that the cross-reactive antigen resided in the cell membrane fraction. However, upon review of previous studies by Freimer (1963) and Schmidt ( 1965a) of mechanically disrupted cell walls, it was noted that a number of cell wall preparations examined by both immunological and chemical techniques contained a considerable amount of cell membrane contamination.
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JOHN B. ZABRISKIE
Re-examination of a number of cell wall preparations in our laboratory ( Zabriskie and Freimer, 1966) demonstrated that all preparations contained a significant amount of glucose, a sugar found in the cell membrane complex but not in the cell wall. Indeed, at times, membrane contamination accounted for almost 40%of the weight of the cell wall preparation. Double-diffusion studies again confirmed the fact that membranes were present in cell wall preparations since extracts of these cell wall preparations formed lines of identity with antigens extracted lroin membranes. Experiments were, therefore, designed in an attempt to reduce the amount of membrane contamination of these preparations. However, extensive rewashings of these preparations in saline solutions failed to remove the membrane material from the cell wall preparations, and sucrose density gradients experiments were unsuccessful in separating the two cellular components. It was obvious from these experiments that mechanical separation per se was not the method of choice in the removal of contaminating membrane material. However, the idea was entertained of using the Group C, streptococcal, phage-associated enzyme to dissolve the cell wall fraction and leave the insoluble membrane residue behind. In this we were aided by the fact that the enzyme’s action was primarily muralytic (Barkulis et al., 1964) and had either little or no effect on the membrane fraction. Unfortunately, there was one drawback to the use of the lysin in its vs-.7al form. Thc Group C streptococcal phage-associated enzyme contsiced small amomts of Group C streptococcal membrane material and, as mentioned previously (see Table V ) , Group C membranes were able to absorb partially the heart-reactive antibody. This obstacle was overcome by centrifuging the phage-associated lysin at high speeds to sediment the membranes leaving the active muralytic enzyme still in solution. Control absorption studies with the supernatant fluid demonstrated that the lysin per se did not react with the heart-reactive antibody. d . The Absorption of Heart-Reactive Streptococcal Antibody b y Membrane Material in Cell Wall Preparations. Following treatment of a number of cell wall preparations known to contain membrane contamination with this enzyme, the soluble and insoluble fractions were examined chemically and immunologically for their ability to absorb the hpart-reactive antibody. As a control, protoplast membranes were also subjected to the lytic action of the phage-associated enzyme. The soluble fraction contained 96%of the cell wall sugars, rhamnose and glucosamine, whereas the insoluble residue had a chemical composition similar to that of protoplast membranes. Both the membrane material and the insoluble residue were found to be free of rhamnose and glucosamine.
163
MIMETIC RELATIONSHIPS
Table VI records the results of the absorption studies which compared the relative effectiveness of equal weights of the original wall preparations, the two fractions of enzyme-treated walls, and the protoplast membranes in removing the heart-staining antibody. The data show that as little as 1 mg. of either the insoluble residue or protoplast membranes essentially abolished all immunofluorescent reactivity and was as effective as 6 mg. of the original cell wall preparation. Indeed, it can be ABSOHPTIONOF
TABLE VI FRACTIONS OBTAINED STREPTOCOCCAL CELL WALLS
IhlM UNOFLUORESCENCE BY
LYSISOF
BY E N z n I A n c
Intensity of st n i i I i ngfl
(:ell walls
In.;olrilrle residue ( i f cell wdls
Poliil)le materid of (sell walls
IVeight (mg.)
Lot I
Lot I1
1 L’
3+ 2+
4 G
*+
3+ 2+ +/2+
1 2 4 0 1 2 4
l’rotoplast rneml)r:wes 1roliil)le residue of protopl:wt rnemkiraneb
G 1
+
0 0 0 3+ 3+ 3+ 3+
Lot 111
+/+
+/+ 0 0 0 3-k 3+ 3+ 2+/3+
+/+ 0
L‘ 1 L‘
f/4 0
3+
3+
Complete absorption rend ns 0.
seen that the insoluble residue representing only one-fifth of the weight of the cell wall was 5 times as active in quenching the fluorescence. Since the soluble material did not contain membrane material by chemical analyses and did not absorb any heart-reactive antibody, it appears that the absorptive capacity of the cell wall preparations was in reality due to that portion of the cell membrane that remained associated with the wall during the isolation process. The establishment of the membrane of the Group A streptococcus as the locus of the heart-reactive antigen led to attempts to isolate the
164
JOHN B. ZABRISKIE
specific antigen involved in this reaction. Work still in progress indicates the antigen may be extracted by treating the membranes with either dilute HC1 or digestion with pepsin, whereas treatment with trypsin or chymotrypsin is without effect. The portion of the HC1-extracted antigen that absorbed the heart-staining antibody appears to be primarily a protein moiety. This was suggested by the fact that rapid destruction of the antigen occurred following digestion with proteolytic enzymes such as trypsin and by the fact that defatting the membranes did not affect the activity of the extract. e. The Sarcolemmal Fraction from Cardiac Muscle Which Reacts with Streptococcal Antibody. As the sarcolemma seemed to be the part of the cardiac muscle cell involved in the cross-reaction, this fraction was isolated by a modification of a method used to isolate sarcolemma of skeletal muscle (Zabriskie and Freimer, 1966). These structures did not contain cytoplasmic material, and chemical analyses of this fraction revealed that it contained less than 1%nucleic acid, 70%protein, and 151 lipid. The cross-reaction between streptococci and heart tissue was supported by the fact that as little as 1 mg. of these sarcolemmal sheaths absorbed the heart-staining antibody from streptococcal antisera. Hydrochloric acid extracts of sacrolemma also were active in blocking this reaction. Treatment of the sarcolemmal sheaths with collagenase or defatting did not alter the ability of the residues to absorb the heartstaining antibody. Furthermore, antisera prepared by Rothbard and Watson (1965) to various mammalian collagens did not stain the heart sections, and antisera to streptococcal membranes used in this study did not stain collagen in the immunofluorescent test system employed by Rothbard and Watson. f . The Question of More than One Cross-Reactive Antigen. The observations of Kaplan and our own work immediately raises the question of whether there is more than one streptococcal antigen that cross-reacts with human heart tissue. The apparent distribution of the immunofluorescent staining, the protein nature of the antigen, its occurrence in Group A streptococci and not in other groups, and the unlikely possibility that two separate cross-reacting antigens would be present all suggest that we are dealing with the same antigen. On the other hand, Kaplan’s demonstration that the antigen occurs only in certain Group A streptococcal strains, its relation to Mf protein strains, and its presence in cell wall extracts suggest that these antigens are distinct ( Kaplan, 1963). The recent studies of Danilova (1966) would tend to support the latter view. Antisera to a Type 5 streptococcus exhibited sarcolemmal
MIMETIC RELATIONSHIPS
165
staining that was quite similar to the patterns described in this review, yet was absorbed only by the homologous stain. Furthermore, Lyampert et at. (1966) have observed that antisera to other streptococcal types exhibited patterns of staining that were quite different from that observed with the Type 5 streptococcus. When HCl extracts were prepared from these different strains, the authors found that they contained three or four distinct antigens when reacted with type-specific antisera in agar double-diffusion studies. Since absorption of the antisera with heart tissue, in certain instances, abolished several of the streptococcal precipitin bands, the authors felt that there were multiple cross-reactive antigens in these extracts and that these antigens were related to separate and distinct components of the cardiac myofiber. Although the possibility of two or more distinct and separate antigens must be considered, the observed discrepancies may, in reality, be based on structural variations of a single antigen from strain to strain. Thus, the position of the antigen in the streptococcal cell complex, its attachment to other structures, and the methods of extraction of the antigen could all conceivably explain what at present appears to be a multiple antigen cross-reactivity. Obviously, until we know more about the structural similarities between the streptococcal antigen and its counterpart in mammalian muscle tissue, the issue must remain unsettled. g. The Nature of Heart-Reactive Antibodies in Human Sera. The finding that a rabbit antibody to a component( s ) of the Group A streptococcus had the capacity to bind to human cardiac tissue naturally led to a search for similar antibodies in the sera of patients with recent streptococcal infections and their sequelae. Employing a combination of doublediffusion and immunofluorescent staining techniques, Kaplan and Svec (1964) reported that the majority of the sera of these patients contained precipitating antibodies which reacted with his partially purified crossreactive streptococcal antigen. In general, these lines were specifically absorbed. by both the streptococcal antigen and human heart tissue. However, certain sera contained precipitin lines that were absorbed only by the streptococcal antigen and not by heart tissue, suggesting that these antibodies were directed toward streptococcal components not related to the heart-reactive antigen. An interesting facet of these studies was the observation that paired sera differing only in their capacity to be absorbed by heart tissue gave identical precipitin lines with the streptococcal cross-reactive antigen. Kaplan has interpreted these results as indicating that the “heart-related and “nonheart-related antigens were antigenically quite similar. The problem of correlating the precipitating antibody and immuno-
166
JOHN B. ZABRISKIE
fluorescent staining activity of these sera was even more complex. Many sera formed strong precipitin lines in agar-gel studies yet produced little or no cardiac staining. Two explanations are possible to explain this discrepancy. Either the level of heart-staining antibody was insufficient to produce immunofluorescent staining of cardiac muscle, as Kaplan suggests, or these sera contain antibodies to streptococcal components other than his heart-related streptococcal antigen. Other sera, notably those obtained from patients with rheumatic heart disease or post cardiotomy sera contained both cardiac-staining and precipitating antibodies yet the immunofluorescence could not be extinguished by the streptococcal antigen preparation. Kaplan has suggested that this lack of inhibition might be attributed to the presence of heart-staining antibodies that are quite distinct from the streptococcal-induced cross-reactive antibody. Thus any form of cardiac damage, such as long-standing heart disease or cardiac surgery might invite the formation of heart-directed antibodies unrelated to the streptococcal cross-reactive antibody. In a similar fashion, precipitating antibodies found in a number of these sera could be directed toward streptococcal components other than the crossreactive antigen. Since these studies indicated the difficulties involved in attempting to correlate the results of double-diffusion studies and immunofluorescent staining reactions in these sera, our approach to heart-reactive antibodies in human sera was similar to the previously mentioned studies with the cross-reactive rabbit antibody. It was felt that a knowledge of the nature and origin of the heart-reactive antibody in human sera was crucial to a subsequent understanding of the role of the cross-reactive antigen in streptococcal infections and their sequelae. Accordingly, sera from a number of patients with recent streptococcal infections, acute rheumatic fever, and rheumatic heart disease were tested for the presence of heartstaining antibodies. Representative serum samples from these patients were layered over sections of heart tissue and stained for the presence of antibody that bound to the cardiac tissues, When the results were tabulated, it was noted that a large majority of these sera exhibited a pattern of staining (Plate I, Fig. 4, p. 158) which bore a striking resemblance to that observed with antisera to Group A streptococcal membranes (Plate I, Fig. 1).As noted previously in the staining pattern of antisera to Group A streptococcal membranes, all muscle bundles exhibited bright fluorescence while the connective tissue elements remained unstained. Plate 11, Fig. 5 demonstrates that the most intensive staining was again present in the region of the sarcolemmal lining of the myofiber but a less intense reaction was also noted in the
MIMETIC RELATIONSHIPS
167
PLATE I1 Fic. 5. Higher magnification of Fig. 4 showing that the staining is again lxightest in the region of the sarcolenima sheath. FIG. 6. Immunofluorescent staining of the muscle cells of an arteriole by the smim of a patient with acute rheumatic fever. The section was from an auricular biopsy of a patient with rheumatic heart disease. Frc;. 7 . Localization of fixed y-globulin in the wall of a small arteriole, using a fluorescein-labeled rabbit antiserum to human inimunogammaglobulin. The section was from the auricular tissue of a patient with rheumatic heart disease.
168
JOHN B. ZABRISKIE
subsarcolemmal sarcoplasm. Occasionally sera also exhibited the diffuse sarcoplasmic and intermyofibrillar patterns previously described by Kaplan et al. ( 1961a). The similarities between rabbit antiserum to streptococcal membranes and sera from patients with acute rheumatic fever was also evident in the fact that human sera stained both rheumatic and nonrheumatic heart sections with equal intensity, In addition, a number of different skeletal preparations exhibited bright staining patterns indicating that the serological reactivity of these sera was not confined exclusively to cardiac tissue. The lack of species specificity was again evidence by the fact that the human sera stained rabbit cardiac and skeletal muscle sections. Whereas rabbit sera prepared against Group A streptococcal membranes consistently bound both to cardiac muscle and to the smooth muscle layer of arteriolar walls (Kaplan, 1963; Zabriskie and Freimer, 1966), sera from patients with either streptococcal infections or rheumatic fever only occasionally demonstrated a similar pattern of staining in the smooth muscle layer of arteriolar walls. However, the pattern of staining when present was similar to that observed with rabbit antisera prepared against Group A streptococcal membranes (Plate 11, Fig. 6 ) . In fact, among 70 sera obtained in the early stages of an attack of rheumatic fever, only 7 (10%) showed serological binding to the smooth muscle layer of arterioles; yet all sera consistently reacted with cardiac myofibers. The presence or absence of rheumatic carditis or arthritis appeared to bear no relationship to the observed reactivity of these sera to the smooth muscle layer of blood vessel walls. Finally, in agreement with Kaplan’s observations (Kaplan et al., 1961b), bound y-globulin was also found in a small but significant percentage of the auricular biopsies that were studied. These deposits were present in the cardiac myofiber proper, the sarcolemmal sheaths, interstitial connective tissue, and in the muscular media of blood vessels. Deposits of this fixed y-globulin in the wall of a small arteriole is illustrated in Plate 11, Fig. 7. h. Distribution of Heart-Reactiue Antibody in the Sera of Patients with Streptococcal Infections and Rheumatic Sequelae. The presence of an antibody in human sera which bound to mammalian cardiac tissue immediately prompted an investigation into the relative frequency with which this antibody appeared in streptococcal infections and their sequelae as compared to unrelated arthritis and immunological disorders. Since undiluted sera obtained from “healthy” individuals occasionally exhibited mild degrees of nonspecific binding to cardiac tissue, all sera to be tested were first diluted 1:5 prior to testing. The intensity of the
169
MIMETIC RELATIONSHIPS
staining reaction of each serum was graded from 0 to 4f and the average intensity of staining for each disease group was then calculated. Table VII summarizes the results. It is evident that more than 80%of sera obtained from individuals with a streptococcal infection and from patients with acute rheumatic fever contain an antibody that reacts with human or rabbit heart tissue. Heart-reactive antibodies were also observed in the sera of patients with rheumatic heart disease, but, in general, the frequency with which this antibody was detected was far less than that observed during the acute disease. Serum from patients TABLE VII DEMONSTRATION OF HEART-REACTIVE ANTIBODY IN SERA m o L r INDIVIDUALS WITH STREPTOCOCCAL INFECTIONS, ACUTERHEUMATICFEVER,AND RHEUMATIC HEARTDISEASE
so. of Clinical disorder
pat ieii ts
Htreptoroccal infection Acute rheumatic fever Rheumatic heart disea.;e Post cardiotoniy Lupsu erythernatow.; Rheumatoid arthrit i.: Mill tiple niyeloma Snrmidosis
3s
so 50 12 20
1.5 10 10
Per cent with antibody 81 87 4i
100 0 0 0 0
Intensity of staining
+
4+
l+D+ 3+/4+
0 0 0 0
" These results represent the average of the individual staining reactions for each clinical disorder. The staining ic-;tction is graded from 0 ( n o staining) to 4+ (maxii m i n brightness)
.
who had undergone cardiac surgery for valvular disease or congenital defect repair produced an intense pattern of staining. However, this serological reactivity appeared to differ from that observed with acute rheumatic sera since the staining reactions of postcardiotomy sera were unaffected by absorption with streptococcal antigen but were abolished by absorption with heart tissue. In contrast, not one serum from 55 individuals with unrelated arthritic and hypergammaglobulinemia disorders exhibited serological binding to the cardiac tissue. In general, Kaplan's report (Kaplan et al., 1961a) of the incidence of heart-reactive antibodies in human sera agree quite closely with the figures reported here. The only exception was his observation that a high percentage of LIPU US erythematosus sera contained heart-reactive antibodies whereas none were detected in our series. This may be merely a reflection of the dilution techniques used in our studies.
170
JOHN B. ZABRISKIE
Although the sera from the group of patients with acute rheumatic fever generally gave staining reactions 4 times more intense than those from the group with an uncomplicated streptococcal infection, sera from several patients with rheumatic fever produced weak (1+ ) staining, whereas a few sera from patients with a streptococcal infection produced extremely bright (4+ ) staining. These observations were based on serum dilutions of 1:5 and, with further dilution of the serum, it was noted that the individual variations within each group tended to disappear. Indeed, at a dilution of l : l O , the sera of patients with a simple streptococcal infection rarely produced immunofluorescent staining of heart reactions. These results suggested that patients who developed acute rheumatic fever had higher titers of heart-reactive antibody than those with uncomplicated streptococcal infections. To investigate this concept, the sera from a carefully controlled patient population was examined. During an epidemic of scarlet fever among recruits at the Great Lakes Naval Training Center in 1944, sera were obtained from a large number of patients in whom the clinical diagnosis of scarlet fever had been supported by microbiological and serological studies. These sera were collected from each patient on admission to the hospital and at weekly intervals thereafter. The sera and the clinical records of several hundred of these patients have been preserved in our laboratory. From this series of scarlet fever patients it was possible to select two comparable groups. Group I was composed of individuals who subsequently developed typical rheumatic fever. Group I1 contained individuals who also had scarlet fever but did not develop rheumatic fever. Two serum samples were chosen from each patient’s file. The first serum had been drawn within 2 weeks of the onset of scarlet fever in both groups. The second serum had been collected from patients in Group I at the onset of rheumatic fever, and at a comparable time from patients in Group 11. Serial dilutions of each serum were prepared, and the staining reactions produced by the 1:5, 1: 10, and 1:20 dilutions were observed. For each dilution of serum, the average of twenty individual staining reactions was calculated both for Group I and for Group 11. These data are recorded in Table VIII. It can be seen that at the onset of scarlet fever, the group of patients who eventually developed rheumatic fever (Group I ) had titers of hrart-reactive antibody twice as high as the group without rheumatic fever (Group 11).Furthermore, at the onset of rheumatic fever, the titers of Group I were still rising while those of Group I1 had already fallen Off.
The average antistreptolysin 0 (ASO) response of each group is included in Table VIII, and the higher average titers in the group that
171
MIMETIC RELATIONSHIPS
developed rheumatic fever is consistent with the reported findings of Anderson et al. (1948). On the other hand, a number of patients in this study had markedly positive C-reactive protein in their sera with AS0 titers of more than 1000 and yet had no detectable heart-reactive antibodies and did not develop rheumatic fever. TABLE VIII HEART-REACTIVE ANTIBODYTITERSIN SERAOF SCARLETFEVER PATIENTS WITH AND WITHOUT SUBSEQUENT RHEUMATICFEVER Onset of scarlet fever=
Clinical disorder Scarlet fever with rheumatic fever (Group I) Scarlet fever without, rheumatic fever (Group 11)
Onset of rheumatic feverh
So. of Serum dilutions; Serum dilutions,< Average patients 1:5 1: 10 1:20 1 :5 1 : 10 1:20 AS0 titer
3+
20
3
0
2
+
4+
0
2 1
+
0
+
2+
+
700
0
0
200
Serum samples obtained within 14 days of onset of scarlet fever. Serum samples obtained at onset of rheumatic fever. ‘At each dilution of serum, the results represent the average of twenty individual staining reactions. The intensity of the staining reaction is graded from 0 (no staining) to 4+ (maximum brightness). ‘I
”
The presence of higher titers of heart-reactive antibody in the patients with rheumatic fever may be an important additional diagnostic tool in cases of suspected acute rheumatic fever. Although the number of samples to date have been admittedly small, it has been possible in each case without prior knowledge of the clinical history to predict whether the patient has had a streptococcal infection or acute rheumatic fever, since bright fluorescent staining of cardiac tissue at dilutions of serum greater than 1: 10 was not seen in uncomplicated streptococcal infections. Furthermore, the absence of any significant levels of heart-reactive antibody in patients with unrelated arthritic and immunological disorders coupled with the characteristic nuclear staining of sera from lupus erythematosus patients has been helpful in the differential diagnosis of other clinical disorders. i. Heart-Reactive Antibody Titers in the Sera of Patients with Acute Rheumatic Fever and Rheumatic Recurrences. Since heart-reactive antibodies were found in high titers in the sera of rheumatic fever patients during the acute stages of the disease, the question of the
172
JOHN B. ZABRISKIE
persistence of these antibodies was investigated next. Serial dilutions of representative samples obtained from 39 rheumatic patients during the acute and followup stages of an attack of rheumatic fever were tested for the presence of heart-reactive antibodies. These patients had been followed by the Rockefeller University rheumatic fever service for periods of time ranging from 6 months up to 10 years after the initial attack of rheumatic fever. The average intensity of staining for each group of serum samples was calculated and the results plotted against the time following the onset of the acute attack. In Fig. 8 it can be seen that the highest titers of heart-reactive antibody occur during the first 2 months and that after the third month of the disease the level of antibody gradually declines. At the end of 3 years, only a weak ( I + ) reaction remains at a 1 : s dilution. Unless a streptococcal infection or a recurrence of rheumatic fever occurred during this period, the vast majority of the patients had little or no detectable heart-reactive antibody at the end of 4 to 5 years. Examination of several sera drawn 10 years after the initial attack of rheumatic fever also were unreactive. Since heart-reactive antibody titers appeared to be detectable during the period of greatest susceptibility to subsequent rheumatic fever attacks, namely, 2-5 years following the initial episode (Markowitz and Kuttner, 1965), it was of interest to study the relationship of this antibody to recurrent attacks of rheumatic fever. Accordingly, sera obtained from patients with well-documented recurrences of rheumatic fever were tested for the presence of heart-staining antibody during the acute episode, during the inactive period, and, finally, during the subsequent attack. The observed titers were then correlated with the clinical course and laboratory data of each patient. The heart-reactive antibody patterns in these patients was then compared with the titers observed in patients who had onIy a single rheumatic episode. Figures 9-12 are illustrative of the type of response observed in each group of patients, The decline in heart-reactive antibody titers in Fig. 9 represents the usual pattern that has been observed following a single attack of rheumatic fever. The titers, originally high during the acute episode, gradually dropped off during the first and second years and by the third year were no longer demonstrable. Although the titers of heart-reactive antibody in this patient have been recorded for only 5 years, subsequent sera obtained up to 9 years after the attack gave similar results. With respect to J.C. (Fig. 10) both attacks were clinically relatively mild and the second attack was less severe than the first. This is perhaps reflected in the decline in titers of heart-reactive antibody in this patient. Although the titers were originally high during the first admission, they
173
MIhlE7IC RELATIONSHIPS cs,
Serum dilutions
m1 u I5 10
oI 2 0
2,3+
c
04[
L
2 2+ 5
al
g a,
LLL-
I+
L
8
1
2
3
4
5
6
1
2
3
4
5
Years FIG. 8. Decline in the titers of heart-ieactive antibody in a group of 39 patients with aciitr rheumatic fever. The intensity of the immunofluorescent staining of cardiac tissue by each patient wci\ determined by grading the serum dilutions from 0 to 4+. The aver'ige tntemity of staining for each group of dilutions was then plotted Months
against the time in months or years during and following the acute rheumatic attack.
dropped off rapidly when compared to heart-reactive antibody levels in Fig. 9. They did not, however, completely disappear and with the rheumatic recurrence there was a definite, although not pronounced, increase in the level of heart-reactive antibodies in this patient's serum. Again the titers fell off quite rapidly and were no longer detectable 6 months after the second attack.
Discharge
*'
Erythrocyte sedimentation rate 46
CRPl
AS0 titer Streptococcal isolation
1
3+0OOOOOOOOI*O
o
500 300 300
250
0
250
0
250
0
I50
None
800500
300
100
5
6
None
L 9
Months
bL 1
2
I
3
4
Years
FIG.9. Patient R.W., a 10-year-old white female, was admitted with migratory poly:irthralgia, abnormal electrocardiograph, and an apical systolic murmur. Normal cardiac contour was noted on X-rays. The patient made an uneventful recovery following treatment with prednisone and salicylates. Three years later an aortic diastolic murmur was first heard and has persisted. There has been no recurrence of rheumatic fever over the past 9 years.
174
JOHN B. ZABRISKIE
When Case I (R.W.) and I1 (J.C.) are compared with respect to the presence or absence of cardiac murmurs, the relationship of heartreactive antibody to permanent valvular damage is difficult to assess. The titers in both cases were essentially the same during the first admission; yet Case 11, although showing only mild evidence of carditis, has had, to date, no evidence of permanent valvular damage. Whether the persistence of heart-reactive antibody for longer periods as was the case in patient R.W., is an important factor in the production of permanent valvular damage remains at present unanswered. J C 7 "is
I
L"
z n d admission 4/20/63
I st admission 4/9/62
Erythrocyte rate
-
_ _
___lltco6+2+0 5 o
0
2-
L
L
0
Y-
25C
15::
L
-
-4
GR A Type IF!
isolation Heart-reoctive antibody titer 2 *
+
1 1 5 flIlOnI20
I
2
L_ !
L _6
9
bLL 1 2 :
c
'2
Msntt: a 7%-year-old white male, was admitted with two wellFIG. 10. Patient J.C., documented attacks of rheumatic polyarthritis approximately 1 year apart. Physical exam on both admissions revealed bilateral ankle swelling and a soft apical systolic murmur. The second attack occurred while on oral penicillin prophylaxis. A mild increase in cardiac size was noted which reverted to normal on both occasions. The patient was treated with combination of prednisone and salicylates and had an uncomplicated recovery. Three years following the second attack, the physical exam was normal.
Figure 11 is illustrative of the type of patient in which the heartreactive antibody titers did not significantly diminish between the first and second attack, and both attacks were of approximately the same severity. The titers in this case are representative of most of the patients who had their rheumatic recurrence within the first 2 years following their initial attack and appears to coincide with the time of greatest susceptibility to rheumatic recurrences. It is of interest that as the period of time following the initial attack in rheumatic patients increased, both the susceptibility to another attack and the heart-reactive antibody titers appear to decline in a similar fashion. For example, most patients who have had a single rheumatic episode rarely contain antibodies 5 years
175
MIMETIC RELATIONSHIPS
after the initial attack. This fact coupled with the observation that the incidence of rheumatic recurrences becomes less frequent 5 years or more after the initial episode (Stollerman, 1954) is suggestive of more than a coincidental relationship between the presence of heart-reactive antibody and a rheumatic attack. Perhaps the strongest evidence of a direct relationship between heartreactive antibody and the onset of rheumatic fever can be seen in the D P I1 yI5
9
I si admission
6/12/62
2nd admission 3/18/63
300
Streptococcol isolation
250
100
GR H
Months FIG.11. Patient D.P., an 11-year-old female, was admitted with two documented attacks of polyarthritis and carditis approximately 9 months apart. Physical exam revealed basal systolic murmurs on both occasions with abnormal electrocardiographs. Treatment with prednisone and salicylates was instituted on both admissions. Recovery was complicated by mild rebounds following cessation of medication. The basal systolic murmur still persists but there has been no recurrence of rheumatic fever in the past three years.
examination of the sera of M.P. (Fig. 12). In this case a period of 11 years elapsed between the two rheumatic attacks. Following the initial episode the titers declined in much the same manner as noted for patient R.W. During the ensuing 6 years the cases are remarkably similar and there was no evidence of a rheumatic recurrence in either patient during this period. The occurrence of two intervening streptococcal infections as indicated by a rise in antistreptolysin 0 titers appears to be a crucial turning point in these cases. Unlike Fig. 9 in which the heart-reactive antibody levels remain consistently negative, Fig. 12 demonstrates a rise in heart-reactive antibody levels which is sustained during the next 2 years. The final streptococcal infection complete with isolation of a Group A streptococcus from the throat was associated with the unmistakable signs of an acute rheumatic fever attack.
176
JOHN B. ZABRISKIE
If the assumption that heart-reactive antibodies play an important role in the initial events of rheumatic fever is correct, then the crucial point in M.P.’s case was the occurrence of two intervening streptococcal infections prior to her second attack. As indicated in Fig. 12, these events coincided with the elevation of heart-reactive antibodies in M.P.’s serum and they remained elevated up to the acute rheumatic attack. Thus it is U P 3yr5
1
r
I st admission 5/26/55
2nd d
Erythrocyte sedimentat ion rote 40
-
0
Mcnlhs
L
--
__A
-
Years
~-
girPi,snn
5lZIlk
n
e
=~
_ _ - ~
Months
FIG. 12. Patient M.P.’s first admission was at age 3 with chronic carditis of 3 months. Px revealed a markedly enlarged heart with loud apical systolic murmur. Staroid therapy was started on fifth hospital day and recovery was complicated by rebound phenomena following attempts to discontinue therapy. Following discharge, oral penicillin prophylaxis was irregular and subsequent A S 0 titers indicated that two intervening streptococcal infections had occurred in the ensuing 8 years. The w c o d attack occurred 11 years later with migratory arthritis and carditis. Px revealed intensification of previous murmur and swollen left knee. Therapy with prednisone and salicylates was instituted and recovery was uneventful except for rebounds during withdrawal of medication. Physical exam ( Px ) following discharge revealed the persistence of the apical systolic murmur.
tempting to speculate that repeated streptococcal episodes (perhaps with subclinical symptoms of disease) are necessary to stimulate the production of heart-reactive antibodies and only at a point when the titers are sufficiently elevated will the full-blown disease complex appear. However, to date, there is no objective evidence to sustain this hypothesis. j , Absorption of Heart-Reactive Antibody by Streptococcal Membranes in the Sera of Patients with Acute Rheumatic Fever. Although the concept of heart-reactive antibodies playing an important role in the pathogenesis of rheumatic fever was attractive, the presence of these antibodies in other disease states unrelated to rheumatic fever, (i.e., myocardial infarction, cardiotomy ) appeared at first to be paradoxical. Among the ideas entertained to explain these discrepancies was the concept that the latter sera contained heart-reactive antibodies unrelated
XdIMETIC RELATIOSSHIPS
177
FIG.13. A comparison of the absorption patterns of inimunofluorescetit staining noted in the serum obtained from a patient with acute rheumatic fever and the serum obtained from a patient following cardiac surgery for repair of a congenital
defect. The staining observed in the acute rheumatic serum was absorbed by Both streptococcal membranes and human heart tissue ( b and c ) . The staining observed in the postcommissurotomy serum was absorbed only by heart tissue ( 6 ) . All staining reactions were carried out on sections obtained from an anricular biopsy of a patient with rheumatic heart disease.
178
JOHN B. ZABRISKIE
to the streptococcal-induced cross-reactive antibody. To test this hypothesis, sera from patients with acute rheumatic fever, chronic rheumatic heart disease, and postcardiotomy states were absorbed with weighed amounts of both streptococcal membranes and isolated human sarcolemma1 sheaths. The experiments were performed by the same method used to absorb heart-reactive antibodies from rabbit antiserum prepared against streptococcal protoplast membranes, The results of one absorption experiment with serum obtained from a patient with acute rheumatic fever and a patient who had undergone cardiac surgery for correction of a congenital defect is illustrated in Fig. 13. Both patients exhibited similar patterns of sarcolemmal staining in the unabsorbed control. However, the serum from the acute rheumatic fever patient was absorbed by both the streptococcal membranes and the cardiac sarcolemma1 sheaths, whereas the postcardiotomy serum was absorbed only by the heart tissue. No diminution of heart-staining activity in this serum was observed following incubation with streptococcal membranes even when as much as 12 mg, of membranes were used as the absorbing material. Of a total of 16 patients with either long-standing heart disease or postcardiotomy states tested in this manner, all sera were absorbed by heart tissue; yet none of the sera were extinguished by absorption with streptococcal membranes. In contrast, 10 sera of acute rheumatic fever patients consistently were absorbed by both sarcolemmal sheaths and streptococcal membranes. In addition, HCl extracts of these membranes were also quite effective in abolishing the heart-staining reaction of acute rheumatic fever patients. These results suggest that sera from patients with disorders unrelated to acute rheumatic fever may contain antibodies to heart tissue unrelated to the streptococcal-induced antibody. By the same token, it may be suspected that rheumatic sera will contain antibodies to streptococcal components unrelated to the streptococcal cross-reactive antigen. Obviously, further purification of the components involved in these cross-reactions will be needed in order to gain a clearer picture of the nature of the observed precipitating and heart-staining antibodies in these sera. Furthermore, knowledge concerning the nature and specificity of the bound y-globulin in the histological lesions of rheumatic fever will be crucial to an understanding of the role these antibodies play in the pathological process. 2. Group-Specific Carbohydrate
So far the major emphasis of this section has been on the possible involvement of heart-reactive antigens in the pathogenesis of rheumatic
MIMETIC RELATIONSHIPS
179
fever. However, the possiblity that other streptococcal antigens may play a similar inciting role must also be considered. One of the more attractive hypotheses concerns the relationship of the Group A streptococcal carbohydrate to mammalian tissue antigens. A major component of the streptococcal cell wall, it accounts for almost 10% of the dry weight of the cell ( McCarty, 1966). The two principal sugars of this structure, rhamnose and glucosamine, are linked together in a branched structure and the serological specificity is largely dependent on the N-acetyl glucosamine residues ( McCarty, 1958).In addition, McCarty has demonstrated that synthetic antigens prepared by coupling p-aminopheny1-Nacetyl glucosaminide to various proteins cross-reacted strongly in precipitin reactions with Group A streptococcal antisera. These synthetic antigens were immunologically related to the group-specific carbohydrate yet differed in all other respects except for the artificially introduced Nacetyl glucosamine determinants. In view of these findings McCarty (1964) suggested that any macromolecular structure that contained N acetyl glucosamine in the proper configuration in a terminal position might cross-react with antibody to the Group A carbohydrate. For example, N-acetyl glucosamine is a prominent constituent of glycoproteins and of the mucopolysaccharides of human connective tissue and it is conceivable that in certain situations the position of the N-acetyl glucosamine terminal units is such that this type of cross-reactivity could occur. Until recently there was essentially no evidence that this type of crossreactivity did, in fact, exist. However, the preliminary report by Goldstein et a2. (1967) indicates that certain mammalian tissues contain antigens that appear to be structurally similar to those of the streptococcal Group A carbohydrate. These authors obtained a “structural glycoprotein fraction” from human and bovine heart valves by first homogenizing the valves in a calcium chloride tris buffer, followed by extractions with trichloroacetic acid at 90°C. and then a series of extractions in 8 M urea. The supernatant fraction of the urea extraction contained the glycoprotein material. Immunization of rabbits with either the whole valve homogenate or the urea-extracted material produced precipitating and hemagglutinating antibodies which could be completely absorbed by the glycoprotein and partially absorbed by Group A streptococci or the isolated group-specific carbohydrate. In addition, fluorescein-labeled antisera to the glycoprotein stained only Group A streptococci and not other streptococcal groups, and the reaction could be extinguished by either Group A carbohydrate or the glycoprotein material. Furthermore N-acetyl glucosamine was capable of absorbing approximately one-third of the antibody to the glycoprotein material
180
JOHN B. ZABRISKIE
(Caravano, 1967). The reciprocal reaction was also true. Antisera to Group A streptococci were completely absorbed following incubation with streptococcal Group A carbohydrate and absorption with the ureaextracted valvular material resulted in approximately a 50%decrease in the antibody titers. Experiments with extracts prepared from skin, cartilage, aorta, and cornea gave essentially identical results. The single exception was the absence of reactivity with material extracted from myocardial tissue. Of special interest was the authors’ finding that the sera of rheumatic patients also contained antibodies that reacted with the glycoprotein material. The authors conclude that the extraction procedures used to obtain the glycoprotein material would suggest that the cross-reactive antigen lies deep in the valvular tissue. The exact mechanism whereby these antigenic sites might be uncovered is at present unknown, but the fact that focal lesions of tissue necrosis is a feature of this disease offers a possible explanation for the uncovering of new and cross-reactive antigenic determinants. To return to the concept that the group-specific carbohydrate may play an important role in the initiation of the chain of events leading to rheumatic fever, there are several pieces of evidence which would favor its participation, First the group-specific carbohydrate is common to all Group A streptococci, an important consideration in view of the apparent lack of specificity among different types of Group A streptococci to induce rheumatic fever. Second, the carbohydrate resides in an insoluble and resistant structure which is not readily disposed of by the host’s tissues. In fact, Schmidt (1967) in an experiment still in progress has been able to detect radioactivity in the liver for as long as 450 days following the intravenous injection of Group A streptococcal cell walls in mice. Assays of density gradient fractions of the liver homogenates 210 days after injection of these walls demonstrated that the major portion of the isotope persisted as particulate material which could be solubilized by the Streptomyces albus enzyme. The soluble 14C-labeledcomponents reacted with streptococcal carbohydrate antisera by antigen binding and precipitin methods. Third, the recent studies of Karakawa et 01. (19%)and Schmidt (196513) have revealed that antibodies to the groupspecific carbohydrate were present in human sera following streptococcal infections and may be detected up to 6 months after the infection. The knowledge that antibodies to the Group A carbohydrate are produced during a streptococcal infection coupled with the previously mentioned report of a cross-reaction between the group-specific carbohydrate and a mammalian glycoprotein fraction is at least suggestive of
MIMETIC RELATIONSHIPS
181
a possible role of the carbohydrate in the disease, rheumatic fever. One might summarize this hypothetical role in the initiation of the pathological events of the disease as follows: ( I ) Antibodies to the groupspecific carbohydrate are produced following a streptococcal infection. Since the cell wall is capable of persisting for long periods of time in the host’s tissues, the stimulus for the production of group-specific carbohydrate antibodies may also continue for some time. ( 2 ) These antibodies might then cross-react with specific tissue components of the host. ( 3 ) The resulting interaction between antigen and antibody might then culminate in the development of the characteristic focal lesions of the disease. Although attractive, this hypothesis is still largely unproved. Knowledge that these cross-reactive antigens are, indeed, present or uncovered in the host during the pathological process would be an important contribution to this concept as would information concerning the specific nature and cytotoxicity of the bound y-globulin seen in the histological lesions of rheumatic fever. At the risk of being repetitious, this theory of the pathogenesis of rheumatic fever is also intriguing and merits further investigation. B. POST-STREPTOCOCCAL NEPHRITIS
Among the current theories concerning the pathogenesis of acute streptococcal glomerulonephritis is the concept that the nephritogenic streptococcus and the glomeruli of nephritis-prone individuals contain a common antigen. Thus, those individuals who share the common component with the nephritogenic streptococcus might develop antibodies during the acute streptococcal infection which had the capacity to bind to components of the glomerulus. The resulting antigen-antibody complex could then lead to the histological and clinical manifestations of the disease. This concept is based mainly on the work of Markowitz and his coworkers and stems from some initial observations in experimental nephritis in rats (Markowitz et al., 1960). In these experiments diffusion chambers, containing Type 12 nephritogenic streptococci, were implanted into the peritoneal cavity of rats and removed 72 hours later. After 4 weeks the authors noted that a great majority of the animals had developed a spotty and focal glomerular nephritis. This was associated with tubular casts and histological evidence of tubular damage. Although these lesions were never as intense or diffuse as human glomerulonephritis, the lesions were consistent with acute glomerular changes. Of interest was the author’s observations that only those Type 12 strains
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that were highly virulent for mice were capable of producing the observed glomerular lesions. Among the explanations offered for the production of these lesions was the concept that a diffusible streptococcal antigen related to a similar component of the glomerulus had incited the production of nephrotoxic antibodies. Accordingly, extracts of these streptococcal strains, prepared by trypsin digestion and Trifluortrichoroethane extraction of whole heat-killed organisms, were injected into rabbits. The rabbit antiserum against these streptococcal extracts was then injected into normal rats and produced lesions that were quite similar to that observed with the diffusion chamber experiments. Rabbit antisera to isolated rat glomeruli or extracts thereof produced essentially similar results. Embarking on an immunological and chemical analysis of the crossreactive antigens in the glomerular tissues and the nephritogenic streptococcal strains, Markowitz and Lange (1962, 1964) mechanically disrupted Type 12 nephritogenic streptococci and obtained a crude cellular fraction containing both cell wall and membrane material. Using differential centrifugation techniques, a relatively clean membrane fraction was also obtained which contained less than 1%rhamnose, one of the major sugars of the cell wall (see Table I ) . Similar techniques were employed to isolate human glomeruli and glomerular basement membrane materials. In addition, extracts of all these fractions were obtained by treating the cellular fractions with trypsin and Trifluortrichoroethane. Immunization of rabbits with either the streptococcal cellular fractions or extracts thereof led to antisera which by gel diffusion, passive cutaneous anaphylaxsis, and tanned cell hemagglutination techniques reacted with extracts of human glomeruli. Apparently not all Group A streptococci contained the cross-reactive antigen since virulent Type 11 and Type 14 streptococci, grown and extracted under the same conditions as the Type 12 streptococcus, did not yield glomerular cross-reactive material. Similar cross-reactions were also observed with streptococcal extracts and rabbit antisera to human glomerular tissue. Double-diffusion studies coupled with immunofluorescent staining suggested that the cross-reactive antigens resided in the basement membrane of the glomerulus and the streptococcal membane fraction. Chemical analysis of these two fractions revealed that they were primarily protein-carbohydrate complexes and the amino acid compositions of the two antigens were quite similar. Attempts to purify further the two extracts were only partially successful, and the major peak eluted from either Sephadex G-75 or DEAE-cellulose columns still revealed two distinct antigens when tested in agar double diffusion.
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Unfortunately, the authors did not completely eliminate the question of tissue contamination of these organisms as the growth of all the strains was carried out in Todd-Hewitt medium, a nutrient source known to contain mammalian tissue products. Admittedly, the fact that only the Type 12 streptococcal strain appeared to contain the crossreactive antigen is evidence against the possibility of tissue contamination. However, it is conceivable that streptococcal strains might differ in their ability to absorb tissue products on their surfaces. Furthermore, absorption studies with specific cellular fractions including extracellular filtrates would have been of great help in more accurately determining the localization of the antigenic components involved. The amino acid compositions of the two fractions, although similar in composition was of little value in determining the structural identity of these two crossreacting components since the starting materials of both fractions were quite complex antigenic structures. Both contradictory and supporting evidence may be found for the above-mentioned hypothesis, and it will be summarized here mainly to emphasize some of the difficulties involved in the interpretation of the studies of cross-reactive relationships. In support of Markowitz’s reports, Holm (1967) has also obtained a soluble cellular fraction which is immunologically related to components of human glomerular tissue and when injected into rabbits produced an experimental nephiritis closely resembling human acute glomerulonephritis. The inciting material was isolated from the organism by growing the nephritogenic strain in an antigenfree medium and then allowing the washed cells to “autolyze” in the cold for 2 weeks. The soluble protein-containing “autolyzate” material from these cells contained the nephritogenic material. The cellular origin of this material was evidenced by the fact that neither the broth medium nor the extracellular products released during the growth of these strains gave rise to similar glomerular lesions. Agar diffusion studies with extracts prepared from human glomeruIar material and the streptococcal nephritogenic material indicated that the two fractions contained common antigenic determinants. However, in contrast to Markowitz’s findings, non-nephritogenic strains also contained a cross-reacting antigen with human kidney tissues which was identical to that found in the nephritogenic strains. In addition, extracts from both nephritogenic and non-nephritogenic strains contained another antigen cross-reacting with kidney tissue which was specifically related only to the individual strains. Thus, both the number and the speci6city of streptococcal antigens cross-reacting with human glomerular tissue must remain unsettled for the present.
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The question of whether these cross-reactive streptococcal antigens have any causal relationship to human glomerulonephritis has not yet been established. A number of investigators (Lange et al., 1949; Liu and McCrory, 1958; Kramer et al., 1961) have demonstrated the presence of antibodies reactive against kidney tissue components, not necessarily glomeruli, in the sera of patients with acute glomerulonephritis. A strong point in favor of a streptococcal-induced basis for the initiation of the events in acute glomerulonephritis would be the demonstration that these antibodies were indeed immunologically related to glomeruli as well as to the streptococcal antigen and could be specifically absorbed by both the glomerular and streptococcal components. Also the appearance of streptococcal-induced antibodies in the serum and perhaps a demonstration of their fixation at the sites of histological damage during the acute stages of the disease and not following the establishment of chronic renal disease might provide further evidence of a direct participation of these cross-reactive antigens in the disease process. IV.
Group A Streptococci
and the Transplantation Antigens
Knowledge concerning the relationship between Group A streptococci and the transplantation antigens is only just beginning to emerge. However, the pioneering efforts in this direction recently reported by Rapaport and Chase (1964) and Chase and Rapaport (1965) are worthy of consideration and may have broad biological significance in the tissue transplantation field. Using skin homograft rejection in guinea pigs as their experimental model, the authors immunized these animals with heat-killed Group A streptococci and 14 days later challenged the animal with skin homografts from normal unrelated donors. Unlike the nonimmunized controls, a large majority of the injected animals had an accelerated rejection of the skin homograft. The time of the rejection was identical to the type of guinea pig homograft rejection described following sensitization of recipients with guinea pig tissues (Sparrow, 1953). The problem of contamination by tissue products in the growth medium was excluded by the use of a dialyzate medium as the nutrient source for growth of the organisms. In addition, the broth per se when inoculated into control animals did not provoke an accelerated homograft rejection. The reaction apparently was not related to type-specific M protein as the accelerated rejection occurred when immunization was carried out with streptococci of 6 different serological types. However, group specificity was an important factor and among the streptococcal groups tested (Groups B, C, D, G) only Group A streptococci were able to
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elicit the reaction. An impressive list of other gram-positive and gramnegative organisms also failed to evoke an accelerated response with the exception of two Staphylococcus ailreus strains and one StaphyZoCOCCUS albus strain which gave reactions quite similar to those observed with the Group A streptococci. Streptococcal cellular fractionation studies ncw in progress (Chase, 1967) indicate that the antigen responsible for the accelerated reaction resides in the cellular membrane complex. The exact mechanism whereby immunization with Group A streptococci or staphylococci induces an accelerated homograft rejection is at present unknown. Limitation of the reaction to only certain bacterial species is of interest and suggests that the reaction is not related to a general hyper-reactivity produced by bacterial immunization per se. In this connection, the preliminary view that the antigen responsible for the accelerated reaction resides in the streptococcal membrane suggests the possibility that structural relationships between membranes of mammalian cells and bacteria may play an important role in this rejection. The proliferation of mononuclear cells in the region of the graft and the cellular necrosis which was described by Rapaport and Chase suggest that cell-mediated factors are also important for the actual rejection of the graft. Whether these cells are present in response to the chemotactic effects of antibodies to cross-reactive streptococcal antigens or are in some undefined manner associated with streptococcal cell-bound antibody has yet been determined. In this regard, the application of immunofluorescent techniques, so useful for the localization and specificity of y-globulin in other disease states, might be a valuable tool in the elucidation of these problems. The knowledge that specific bacterial antigens can play a role in the rejection of homografts is intriguing and indicates that exogenous bacterial structures may be involved in the acceptance or rejection of tissue transplants. Although knowledge concerning cross-reactive bacterial and transplantation antigens is still in its infancy, the importance of the findings of Rapaport and Chase cannot be overemphasized. It provides an experimental model for further exploration of the biological effect of these antigens and will undoubtedly have important implications in the future success or failure of human transpIantation experiments.
V. Summary and Conclusions It is now clear that the concept of structural similarities between the host and parasite has important implications in biology; not only in terms of enhancement of pathogenicity of the invading organism but also with
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respect to host-parasite relationships in various disease states. Although this review has primarily emphasized the role of biological mimicry in Group A streptococci and its host, it is not difficult to imagine that antigenic similarities between host and parasite may play a role in the pathogenesis of other disease states. To cite but one example, the work of Perlmann and his collaborators (1965) in ulcerative colitis suggests that structural similarities between components of certain strains of Escherichia coli and human colonic cells may be important in the initiation of pathological events in this disease. Biological mimicry may also play an important role in biological interactions between certain viruses and their hosts. This concept is based on the observation that myxoviruses acquire their envelopes from cellular material of the host during the budding process (Compans et al., 1966), and chemical and immunological analyses of certain influenza viruses have revealed that these viruses contain lipids (Kates et al., 1962) and cellular antigens (Isacson and Koch, 1965) from the host cell in which they were grown. Although only a few suggestive reports of the role this biological mimicry between virus and its host may play in viral infections have appeared (see Damian, 1964), knowledge that viruses may mimic antigenic determinants of the host through acquisition of host cellular material during the infectious process may have important implications in future studies of virus-host interactions. Implicit in this review has been the concept that biological mimicry may have important implications in a number of disease states in man, but there are certain difficult and yet intriguing questions with respect to these cross-reactions that remain at present unanswered. The presence of bacterial-induced cross-reactive antibodies now appears to be well established, but the nature of their actual relationships to the disease state remains obscure. For example, the sera of patients with acute rheumatic fever contain high levels of circulating antibodies to mammalian muscle tissue and yet do not apparently bind to the large mass of muscle tissue in the body. Whether local inflammatory factors determine the localization and penetration of these antibodies is not known, but the fact that circulating heart-reactive antibodies may be present without obvious signs of damage raises the important question of how they might exert their cytotoxic effect. Indeed, there is no evidence at present as to whether cross-reactive antibodies do, in reality, have cytotoxic properties or merely reflect the presence of a secondary response to prior damage to tissue. Knowledge concerning the nature and the specificity of both the bound 7-globulin and the circulating cross-reactive antibody in these disease states will, therefore, be crucial to our under-
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standing of various disease processes. Obviously there are a number of important gaps in our knowledge of the relationship of biological crossreactions to the pathogenesis of disease states in man.
REFERENCES Anderson, H. C., Kunkel, H. G., and McCarty, M. (1948). J. Clin. Invest. 27, 425. Barkulis, S. S., Smith, C., Boltralik, J. J., and Heyniann, H. (1964). J. Biol. Chem. 239, 4027. Brockniann, H., Brill, J., and Frendzell, J. (1937). Klin. Wochschr. 16, 507. Caravano, R. ( 1967). Personal communication. Cavelti, P. ( 1945). Proc. SOC. Exptl. Biol. Med. 60, 739. Chase, R. M. (1967). Personal communication. Chase, R. M., and Rapaport, F. T. (1965).J. Exptl. Med. 122,721. Compans, R. W., Holmes, K. V., Dales, S., and Choppin, P. W. (1966). Virology 30, 411. Damian, R. T. (1964). Am. Naturalist 98, 129. Danilova, T. A. (1966). J. Microbiol. Epidemiol. Immunobiol. ( U S S R ) (English Transl.) 43, 94. Dumonde, D. C. ( 1966). Aduan. Immunol. 5, 245. Finland, M., and Cumen, E. C. (1938). Science 87,417. Finland, M., and Curnen, E. C. (1940).J. Immunol. 38, 457. Fox, E. M., Wittner, M. K., and Dorfman, A. (1966). J. Exptl. Med. 124, 1135. Freimer, E. H. ( 1963). J. Exptl. Med. 117, 377. Freimer, E. H., Krause, R. M., and McCarty, M. (1959). J. Erptl. Med. 110, 853. Goldstein, I., Halpern, B., and Robert, L. ( 1967). Nature 213, 46. Hammerman, D., Blau, S., Janis, R., and Sandson, J. (1965). Science 150, 353. Hirsch, J. G., and Church, A. B. (1960). J. Exptl. Med. 111, 309. Hirst, G. K. ( 1941). J. Exptl. Med. 73, 493. Holm, S. E. (1967).Acta Pathol. Microbiol. Scand. (in press). Humphrey, J. H. (1943). Biochem. J. 37, 460. Isacson, A,, and Koch, A. E. ( 1965). Virology 27, 129. Jenkin, C. R . (1963). Aduan. Immunol. 3,351. Kabat, E. A. ( 1956). “Blood Group Substances.” Academic Press, New York. Kabat, E. A., and Mayer, M. M. (1961). In “Experimental Immunochemistry” (E. A. Kabat and M. M. Mayer, eds.), 2nd Ed., p. 430. Thomas, Springfield, Illinois. Kaplan, M. H. ( 1963). J. lmmunol. 90, 595. Kaplan, M. H., and Dallenbach, F. D. (1961b). J. Exptl. Med. 113, 1. Kaplan, M. H., and Meyeserian, M. (1962). Lancet 1, 706. Kaplan, M. H., and Svec, K. H. (1964).1. Exptl. Med. 119,654. Kaplan, M. H., Meyeserian, M., and Kushner, I. (1961a). J. Exptl. Med. 113, 17. Karakawa, \!’. W., Osterland, C. K., and Krause, R. M. (1965). J. Exptl. Med. 122, 195. Kass, E. H., and Seastone, C. V. (1944).1. Exptl. Med. 79,319. Kates, M., Allison, A, C., Tyrell, D. A. J., and James, A. T. (1962). Cold Spring Harbor Symp. Quant. Bwl. 27, 273. Kramer, N. C . , Watt, M. F., Howe, J. H., and Parrish, A. E. (1961). Am. J. Med. 30, 39. Lange, K., Gold, M. A., Wiener, D., and Simon, V. (1949). J. Clin. Inuest. 28, 50.
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Laurent, T. C., Ryan, M., and Pietruszkiewica, A. (1960). Biochim. Biophys. Acta 42, 476. Liu, C. T., and McCrory, W. W. (1958). J. Imrnttnol. 81, 492. Lyampert, I. M., Vvedenskaya, 0. I., and Danilova, T. A. (1966). Zmmunol. 11, 313. McCarty, M. (1958). J. Ex&. M e d . 29, 488. IvlcCarty, M. ( 1964). Circulation 29, 488. McCarty, M. ( 1966). I n “Bacterial and Mycotic Infections of Man” (R. J. Dubos and J. G. Hirsh, eds.), 4th Ed., p. 356. Lippincott, Philadelphia, Pennsylvania. Mackaness, G. B., Blanden, R. V., and Collins, F. M. (1966). J. Exptl. M e d . 124, 573, 585, 601. Markowitz, M., and Kuttner, A. G. (1965). “Rheumatic Fever Diagnosis, hlanagement and Prevention,” pp. 124. Sanders, Philadelphia, Pennsylvania, Markowitz, A. S., and Lange, C. F. (1962). J. Lnh. Clin. M e d . 60, 1001. Markowitz, A. S., and Lange, C. F. ( 1964). J. Immunol. 92, 565. Markowitz, A. S., Armstrong, J. S., and Kushner, D. S. (1960). Nature 187, 1095. Meyer, K. (1947). Physiol. Reo. 27, 335. Meyer, K. ( 1958). Federation Proc. 17, 1075. Meyer, E.,and Palmer, J. W. ( 1943). J. Biol. Cheni. 107, 629. Muschel, L. H., and Osawa, E. (1959). Proc. SOC. Exptl. Biol. M e d . 101, 614. Ogsten, A. G., and Stainer, J. E. (1952). Biochern. J. 52, 149. Osler, A. E., Hardy, P. H., and Sharp, J. T. (1954). A m . J. Syph. 38, 554. Perlmann, P., Hamnierstron, S., Lagercrantz, R., and Gustafsson, B. E. (1965). Ann. N.Y. A c a d . Sci. 124, 377. Quinn, R. W., and Singh, K. P. (1957). Proc. SOC. Exptl. Biol. Jfed. 95, 290. Rapaport, F. T., and Chase, R. M. (1964). Science 145,407. Rothbard, S. (1948). J. Exptl. M e d . 88, 325. Rothbard, S., and Watson, R. F. (1965). J. Exptl. Med. 122, 441. Rowley, D. (1966). Experientia 22, 1. Rowley, D., and Jenkin, C. R. (1962). Nature 193, 151. Sandson, J., and Hammerman, D. (1962). J. C h i . Incest. 41, 1817. Schmidt, W. C. (1965a). J. E r p t l . M e d . 121, 171. Schmidt, W. C. (196513). J. Exptl. Med. 121,793. Schmidt, W. C. (1967). Personal communication. Seastone, C. V. ( 1939a). J. Exptl. M e d . 10,347. Seastone, C. V. (193913). J. Exptl. M e d . 70, 361. Seastone, C. V. (1943). J. Exptl. M e d . 77, 21. Sparrow, E. M. ( 1953). J. E n d o cr i n d . 9, 101. Sprent, J. F. A. (1959). I n “The Evolution of Living Organisms,” Symp. Roy. SOC. Victoria (G. W. Luper, ed.), p. 149. Melbourne Univ. Press, Australia. Springer, G. F. ( 1956). Naturwissenschuften 43, 93. Springer, G. F., Horton, R. W., and Forbes, M. (1959). J. Exptl. M e d . 110, 221. Springer, G. F., Williamson, P., and Brands, W. C. (1961). J. Exptl. Aied. 113, 1077. Stollerman, G . H. ( 1954). A m . J. M e d . 17, 757. Stoilerman, G. H., Kantor, F. S., and Cordon, B. D. (1958). J. Exptl. Med. 108, 475. Stollerman, G. H., Rytel, M., and Ortiz, J. (1963). 1. Exptl. M e d . 117, 1. Vasquez, J. J., and Dixon, F. J. (1957). Lab. Infiest. 6, 205. Zabriskie, J. B., and Freimer, E. H. (1966). J. Exptl. M e d . 124, 661.
Lymphocytes and Transplantation Immunity DARCY B. WILSON AND R. E. BILLINGHAM Departmenf o f Medical Genelics, Universify of Pennsylvania School of Medicine Phihdelphia, Pennsylvania
I. Introduction . . . . . A. Peripheral Blood Lymphocytes B. Macrophages . . .
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Part 1. In Vitio Studies of the Immune Response 11. Identification of Immunologically Competent Cells . . . . 111. The Process of Sensitization . . . . . . . . . . Site of Sensitization Process in Skin and Renal Homografts . IV. The Effector Side of the Immunological Reflex . . . . . A, Transplantation Immunity and Delayed Cutaneous Inflammatory . . . . . . . . . . . . Reactions B. Mixed Lymphocyte Reactions in the Skins of Irradiated Hamsters . . . C. Local Graft-versus-Host Reactions in the Kidney . D. Transformation of the Immunological Status of Lymphoid Cells by Ribonucleic Acid . . . . . . . . . . E. The Significance of the Cellular Infiltrate in a Homograft . . F. Manner in Which the Cytopathogenic Effect of Lymphocytes is h4ediated . . , . . . . . . . . .
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Part 2. In Vitro Studies of the Immune Response Lymphocytes as Effectors of Immunity . . . . . . A. Destructive Activity of Sensitized Lymphoid Cells . . . B. Destruction of Cells in Culture with Normal Lymphoid Cells Macrophages and Antigen . . . . . . . . The Blastogenic Response of Lymphocytes in Culture . . . A. Responses to Nonspecific Mitogens . . . . . . B. Specific Mitogens: Secondary or “Recall” Response . . C. Specific hlitogens : Primary Response and Mixed Leukocyte Reactions . . . . . . . . . Cooperative Interaction of Lymphocytes and Macrophages . . References . . . . . . . . . .
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Introduction
About a decade ago the most generally held thesis concerning the sequence of events responsible for the rejection of vascularized homografts of skin, other solid tissues, and organs was as follows. Antigenic material escapes from the healing-in graft, is carried via the draining or afferent lymphatics to the regional nodes, where it stimulates the production of immunologically activated lymphocytes. These cells-the putative 189
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“effector” cells-enter the blood circulation via the efferent lymphatics, eventually coming into contact with the vasculature of the alien graft, whereupon some of them migrate through the walls of the vessels and infiltrate the parenchyma. Here, by unknown means, they bring about the destruction of the foreign cell population. That the regional lymph nodes are the principal anatomical seats of the reaction against a local homograft was suggested initially by morphological studies and subsequently confirmed by experiments which showed that these organs acquired the power to transfer sensitivity adoptively to other normal isologous hosts ( Mitchison, 1954; Billingham et al., 1954). The fact that transplantation immunity could be transferred by lymphoid cells and not by immune serum justified its classification with hypersensitivity reactions of the delayed type. The belief that the infiltrating mononuclear cells-principally small lymphocytes and histiocytes-are the principal agents of graft destruction rested upon the classic empirical observation that the development of lesions in a homograft is invariably preceded and accompanied by cellular infiltration. The only experimental evidence in its favor was that emerging from studies on the survival of homografts enclosed within cell-penetrable and cell-impenetrable Millipore chambers and inserted intraperitoneally into specifically sensitized and normal murine hosts (Algire et al., 1957; Weaver et al., 1955; see Billingham, 1962). Outstanding problems left open by the general hypothesis outlined above were ( I ) elucidation of the manner in which a homograft stimulates the regional nodes, ( 2 ) determination of the nature of the activated cells, and ( 3 ) the mechanism by which sensitivity is put into effect. The purpose of this review is to consider the present status of knowledge concerning the role of lymphocytes in the genesis and effectuation of hypersensitivity to homografts. Purely on the grounds of convenience, it has been divided into two parts. Part 1 deals with the evidence obtained from in uiuo studies, and Part 2 with that which has recently been forthcoming from investigations on lymphocyte reactivities as expressed in the greatly simplified, and more easily defined in vitro culture systems. A. PERIPHERAL BLOODLYMPHOCYTES A brief account of those aspects of the biology of peripheral blood lymphocytes germane to the subject matter of this review is presented here in preference to disseminating the information in the various sections to which it relates. In aggregate, the lymphoid tissues of a normal mammal constitute about 1%of its body weight (Yoffey and Courtice, 1956) and the pre-
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dominant cellular component is the so-called “small” lymphocyte. In spite of the ubiquity of small lymphocytes-scattered throughout the lymphoid tissues and the blood-and the abundant literature devoted to them, it is still difficult to interpret their functional significance either in normal or in pathological processes. In addition to immunological duties, other functions that have h e n attributed to lymphocytes include serving as trephocytes and stem cells (see Trowell, 1965) and even regulators of tissue growth and size (Burch and Burwell, 1965). On the basis of their morphology and size, lymphocytes have frequently been subdivided into “large,” “medium,” and “small” varieties. However, this classification is arbitrary since a frequency distribution of these cells has never been shown to be trimodal but, rathey, continuous (Mainland et al., 1935; Schooley and Berman, 1960). Furthermore, the dimensions of these cells can be expected to vary considerably according to their method of preparation ( Mainland and Coady, 1938). An inherent pitfall of this type of classification is that it tends to create the impression that all cells which fall into a particular subdivision have the same origins and fulfill similar biological roles. The majority of the lymphocytes present within the lymphoid tissues as well as in the blood and lymph are of the “small” variety. They are less than 8 /” in diameter, mononuclear, quite uniform in size; and, within their relatively small amount of cytoplasm, there are a few prominent mitochondria and vesicles. They do not attach to glass, do not respond to chemotactic stimuli, nor do they divide or incorporate radioactive thymidine in viuo or in vitro. They exhibit a characteristicaIly polarized motility which is unlike that of other cells; and they are readily killed by small doses of ionizing irradiation, cortisone, or mitotic poisons (see Gowans and McGregor, 1965; Trowell, 1965). The large and medium lymphocyte categories represent a much more heterogeneous population of cells, some of which possess a strongly basophilic cytoplasm. These represent not more than 10%of the normal, circulating lymphocytes. Electron microscope studies of the ultrastructure of these cells reveals two types, both distinguishable from small lymphocytes on the basis of their cytoplasmic organelles and their more abundant cytoplasm. One type contains more mitochondria, numerous cytoplasmic vesicles, and a prominent Golgi complex, whereas the other contains a well-developed tubular endoplasmic reticulum. Unlike small lymphocytes, these larger cells readily incorporate radioactive thymidine and mitotic figures are common both in V ~ U Oand in vitro (see Gowans and McGregor, 1965). Our knowledge of the life history of lymphocytes stems largely from
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the elegant studies of Gowans and his associates (for review, see Gowans and McGregor, 1965). Large numbers of these cells (more than 90% of the small variety) continually enter the blood stream of mammals via major lymphatics in the neck and upper thorax and then recirculate back into the lymph. It has been shown that the small lymphocytes which enter the blood are part of a large pool of cells, numbering about 1.5 to 2.0 x lo9 in an adult rat (Gowans and Knight, 1964), repeatedly circulating from blood to lymph. After only a very short time (about 2 hours in the rat) in the blood, these cells enter the lymph nodes and Peyer’s patches ( Gowans and Knight, 1964) by passing intracellularly through the endothelial cells lining the postcapillary venules of the midand deep zones of the cortices of the nodes in rats (Marchesi and Gowans, 1964). Thence, they migrate into the medullary sinuses leaving the nodes via the efferent lymph vessels. Gowans and Knight (1964) have shown that as quickly as 15 minutes after their transfer to isologous recipients, isotopically tagged small lymphocytes are demonstrable penetrating the endothelium of the postcapillary venules and accumulating between the endothelial wall and the periendothelial sheath. That few labeled cells were present in the marginal sinuses and outer rim of the nodal cortex was interpreted as indicating that circulating lymphocytes do not normally enter the node via the afferent lymphatics. Installation of a fistula, with chronic drainage from the thoracic duct in the rat, leads to a rapid and drastic diminution in the number of recoverable small lymphocytes ( McGregor and Gowans, 1963). However, the output of larger lymphoid cells remains constant. Taken together with the findings of studies of the decay of :<.P in the deoxyribonucleic acid ( DNA ) of human, peripheral blood lymphocytes ( Otteson, 1954), this indicates that the life span of a large percentage of small lymphocytes is surprisingly long and can be reckoned in terms of months or even years. Strong support for this notion has emerged from independent studies in man by Buckton and Pike (1964a,b), Norman et al. (1965), and Nowell (1965). Small lymphocytes from the peripheral blood of patients who, many years previously, had received therapeutic X-irradiation, were stimulated to divide in vitro with phytohemagglutinin. The appearance in the cultures of numerous “unstable” chromosomal anomalies such as acentrics, dicentrics, and rings, identified cells that were undoubtedly entering their first division since irradiation. In some instances, the time interval was as long as 10 years. Among the characteristics of small lymphocytes, their ubiquity, mobility, and longevity are desirable attributes of any cell type the role
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of which is to participate in the recognition, effector, and memory aspects of immuiiological phenomena (Fitzgerald, 1964). By contrast with that of small lymphocytes, the life span of the larger lymphocyte is apparently very short-of the order of a few days. There is some evidence that, rather than participating in the recirculatory behavior exhibited by the small lymphocytes, the larger ones migrate into the wall of the gut and lodge in the stroma of the villi where many of them come to resemble immature plasma cells (Gowans and Knight, 1964). Apparently the small percentage of large lymphocytes found in thoracic duct lymph can be accounted for on the basis of production of new cells in the nodes (Caffrey et al., 1962).
B. MACROPHAGES Since ( a ) histiocytes (or macrophages) comprise part of the infiltrating mononuclear cell population in homografts, ( b ) a marked histiocytosis is demonstrable during systemic graft-versus-host rewtions, and ( c ) there is evidence that the handling of antigen by macrophages is an important component of the immunological response, at least to some antigens, it is pertinent to consider the relationship between lymphocytes and these phagocytic cells. It can be regarded as firmly established that macrophages of inflammatory exudates derive from some cellular element of the blood (Ebert and Florey, 1939; Volkman and Gowans, 1965a). However, the identity and sites of proliferation of the leukocyte precursor is still debatable. Although the potentiality of monocytes to evolve into macrophages has been demonstrated (Ebert and Florey, 1939), emigrating monocytes are not generally r e p - d e d as the major source of macrophages in inflammatory exudates which can be sampled in areas of abraded skin by the “skin window” technique. Thus, many workers favor the small lymphocyte as the precursor (Rebuck and Crowley, 1955; Rebuck et al., 1961). The most telling evidence in favor of this thesis derives from cytological examination of the dividing cells in Kupffer cell preparations from the livers of mice which had been depleted of small lymphocytes, transfused with chromosomally tagged, thoracic duct lymphocytes, and stimulated by injection of killed Coryncbacterium parzjum ( Howard et al., 1966). However, Volkman and Gowans (1965a) have recently produced evidence that, in the rat, the macrophages that migrate into foci of nonbacterial inflammation are apparently short-lived cells derived from the blood and orizinating from rapidly and continuously dividing precursors located at sites. other than the area of inflammation. Lymphocyte depletion by thoracic duct drain-
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age or X-irradiation (400 r ) failed to suppress emigration of macrophages to sites of inflammations or reduce the proportion of them which became labeled on injection of tritiated thymidine. On the basis of studies in which labeled cells were transferred from various tissues to isogenic hosts, Volkman and Gowans (1965b) concluded that bone marrow and, to a lesser extent, spleen are the major sources of macrophages present in nonbacterial inflammatory lesions. Their various findings indicate that, at the best, only a small minority of leukocytes having the morphological characteristics of small lymphocytes may be able to evolve into macrophages. Clearly, the interrelationship between macrophages and lymphocytes awaits further clarification and, as Volkman and Gowans (1965b) point out, it remains to be determined whether the macrophages in acute inflammation induced by mild trauma to the skin and those evoked during immunological responses in sensitized animals are derived from identical precursors. PART 1 .
IN VlVO STUDIES OF THE IMMUNE RESPONSE
II. Identification of Immunologically C o m p e t e n t Cells Our knowledge of the cellular events that are set in motion by exposure of an animal to a solid-tissue homograft has been greatly enriched from studies on graft-versus-host reactions ( see McBride, 1966; Billingham, 1967). The latter occur in situations in which suspensions of adult lymphoid cells are injected into immunogenetically alien hosts which, for genetic or other reasons, are incapable of reacting against them. Provided that the host confronts the lymphoid cell graft with a major ioreign transplantation antigen (such as that determined by the H-2 locus in the mouse, by the Ag-B locus in the rat, or the B locus in the chicken), a wasting disease results which is usually fatal. It is characterized by an early splenomegaly, subsequent lymphoid tissue atrophy, and certain other abnormalities. Although extensive studies have so far failed to establish the pathogenesis of this syndrome, which is usually referred to as “runt,” “homologous,” or “transplantation” disease, it has been established unequivocally that it is the result of an immunological attack on the part of the transferred lymphoid cells against the host’s alien cellular antigens. Indeed, at least so far as transplantation immunology is concerned, a useful operational definition of an “immunologically competent” cell is one that is capable of initiating a graftversus-host reaction. The findings that inoculation of whole blood or leukocyte concentrates into very young avian or mammalian hosts
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causes runt disease established that immunologically competent cells are normal ingredients of blood. In rodents, identification of the immunologically competent cell stemmed from the observation that thoracic duct cells-almost exclusively small lymphocytes-were highly effective in producing runt disease (Anderson et al., 1960; Gowans et al., 1961; Billingham et at., 1962). The final piece of evidence was the demonstration of the immunological competence of preparations of almost pure small lymphocytes obtained by fractionation of whole blood from mice ( Hildemann et al., 1962; Hildemann, 1964). In chickens, which have no lymph nodes, Terasaki’s (1959) careful fractionation studies established that lymphocytes, and not monocytes, were responsible for the immunological competence of their blood. Whether the large or the small lymphocyte is the effective component is not yet resolved. When suspensions of adult chicken leukocytes are distributed over the “dropped” chorioallantoic membrane ( CAM ) of an unrelated 12-day host embryo, a variable number of white focal lesions or “pocks” develop within a few days. These CAM lesions have been shown to represent local reactions on the part of immunologically competent cells against histocompatibility antigens present in the membrane (see Simonsen, 1962). Using whole leukocyte suspensions, Szenberg and Warner (1962) correlated the number of pocks produced with the number of ‘‘large’’ lymphocytes present. However, Simons and Fowler (1966) have established that virtually 100%pure small lymphocytes obtained from blood by a discriminating fractionation procedure are also capable of inciting pocks. This finding is consistent with Solomon’s (1964) report that relatively pure suspensions of small lymphocytes caused splenomegaly when inoculated into chick embryos. It is obviously a matter of some theoretical importance to know what proportion of the small lymphocytes in a given inoculum are capable of reacting against their host’s antigens. Unfortunately, in mammals, sensitive in d u o assay procedures are not available to resolve this question. However, it may be noted that, with sume strain combinations of rats and mice, about 1 x 1 0 small lymphocytes seems to represent a threshold dose for the induction of overt runt disease (Hildemann et al., 1962; Billingham et al., 1962). In infant (ST X DBA/2) F, mice, the minimum number of ST lymph nude cells required to produce a significant enlargement of the spleen is of the order of 1 0 cells (Simonsen, 1962). In the chick embryo it has been estimated that the minimum number of peripheral blood lymphocytes that will cause splenomegaly upon intravenous inoculation into 14-17 day embryos is of the order of 10’ cells (Terasaki, 1959). In this species, use of the sensitive CAM assay
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method has established that as few as 2 x lo4 leukocytes can produce one pock or focus (Burnet and Burnet, 1960). It may be added that Szenberg and Warner (1962), who believe that the large lymphocytes are the immunologically competent cells in this species, have found that the number of these cells inoculated per pock produced is as low as 40. With purified suspensions of small lymphocytes, Simons and Fowler’s (1966) lowest ratio was 30 cells per pock produced. Burnet’s original postulate that each pock develops from a single immunologically competent cell has recently been confirmed by Coppleson and Michie ( 1966). It is interesting to compare the above estimates of the number of potentially reactive lymphocytes with estimates derived from studies using heterologous immunity systems. Brown and his associates ( 1966) have recently presented evidence suggesting that about 1 to 2 “immunological units,” probably single, competent cells, in 10’ mouse spleen cells, are capable of responding against rat erythrocytes. Using a different assay system, Kennedy et al. (1966) have estimated that the spleens of normal mice contain approximately lo3 precursor cells of the immune system which are capable of responding to sheep erythrocytes. Ill.
The Process of Sensitization
Since there is a very close similarity between the events responsible for graft-versus-host reactivity and those involved in conventionaI hostversus-graft reactions, it was reasonable to assume that the interactions between antigen and immunologicalIy competent cells which lead to the production of sensitized cells must be closely similar in both situations. Unfortunately, graft-versus-host reactions have proved to be much more complicated than host-versus-graft reactions and the pathogenesis of the various homologous diseases still remains almost completely enigmatic ( McBride, 1966; Billingham, 1967). However, the initial phases of graftversus-host reactivity, leading to sensitization, are particularly favorable for study since they are initiated by experimentally definable reactant cell populations that can be labeled in various ways. Studies by Gowans and his associates (Gowans et al., 1961; Gowans, 1962) with isotopically tagged small lymphocytes in rats have shown that when these are injected intravenously into F, hybrid or isologous hosts, they migrate very rapidly in large numbers into the cortices of the host’s nodes and white pulp of the spleen. Although between 3 and 6 hours the appearance and distribution of labeled cells in both types of host are similar, by ’24 hours there is a clear-cut difference. In the hybrid or “foreign” hosts, a proportion of the labeled small lymphocytes transform into large pyroninophilic cells which subseque~itly divide.
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Two observations indicate that this transformation of small lymphocytes is a response to an antigenic stimulus. First, it fails to occur in the lymphoid tissues of isogenic hosts and, second, lymphocytes from parental strain donor rats made tolerant of F, hybrid tissues do not transform into large, pyroninophilic cells or initiate graft-versus-host reactions if inoculated into F, hybrid hosts (Gowans et al., 1963; Elkins, 1964). As Gowans has emphasized, this change into large pyroninophilic cells only occurs in a small proportion of the labeled small lymphocytes, large numbers remaining unchanged and apparently unaffected by the antigenic stimulation for several days. Either these cells undergo a change within 24 hours of exposure or they are refractory, indicating functional heterogeneity at the level of the donor, small lymphocyte population. Unfortunately, disappearance of the radioactive label precluded determination of the fate of the large pyroninophilic cells in the present system. However, Gowans and his co-workers (1962) have shown that, when “small” lymphocytes, obtained from the thoracic ducts of rats, were inoculated into heavily irradiated murine recipients, pyroninophilic cells distinguishable as being rat in origin appeared in the recipient mouse spleens. These large pyroninophilic cells divided to form lymphocytes of progressively decreasing sizes. As these workers pointed out, the large pyroninophilic cells that arise from small lymphocytes during graft-versus-host reactions ( see reviews by Gowans, 1965; Gowans and McGregor, 1965) closely resemble similar cells which had previously been observed to develop in the cortex of regional nodes draining primary homografts of skin or bone (Scothorne, 1957; Binet and Mathk, 1962; Bunvell, 1962). These cells are characterized by abundant cytoplasmic particles, presumed to be ribonucleoprotein, and by a lack of an endoplasmic reticulum. On the basis of these findings with graft-versus-host reactions, Gowans and his associates (1962) suggested that the primary immune response of an animal to a skin homograft may be initiated by the interaction of certain competent small lymphocytes from the constantly recirculating pool of these long-lived cells. They then become established in the regional nodes and, via the intermediary of large pyroninophilic cells, produce more small lymphocytes which are “immunologically committed or effector cells. That these pyroninophilic cells or immunoblasts play an indispensable part in the response of animals to skin homografts, or to contact allergens, is indicated by evidence of their failure to appear in the regional nodes of animals whose capacity to respond immunologically has been abrogated by treatment with 6-mercaptopurine (Andre et al.,
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1962), cyclophosphamide (Turk and Stone, 1963), or thalidomide (Turk et al., 1966). Chronic drainage of lymphocytes from a thoracic duct fistula in rats over an &day period produces a striking depletion of small lymphocytes, though it is not feasible to remove all of these cells in this way (McGregor and Gowans, 1963; Gowans and Knight, 1964). It was found that the primary immune response of such “lymphocyte-depleted rats to sheep red cells and to tetanus toxoid was severely depressed but could be restored by transfusion of small lymphocytes from isogenic donors, suggesting that the recirculating small lymphocytes must be involved in the primary response to these antigens ( McGregor and Gowans, 1963). It was subsequently shown (McGregor and Gowans, 1964) that 8 days’ depletion of thoracic duct cells in rats produced a short prolongation of survival of skin homografts from distantly related donors and totally abolished their ability to reject grafts from more closely related donors. This prolongation of graft survival was maximal when transplantation was carried out at the time of closure of the fistula but, if it was delayed for 8 days, the animals were found to have recovered their ability to reject grafts with normal vigor. Depletion of lymphocytes from recently sensitized rats did not impair their capacity to give second-set reactions, suggesting that immunological memory does not reside exclusively with “depletable” lymphocytes. In man, too, lymphocytic depletion by thoracic duct drainage has been shown to prolong the sur1965), vival of skin homografts (Tunner et d., These important observations suggested that the recirculating small lymphocytes, rather than the sessile lymphoid cell population, play an important role in the primary response of rats to skin homografts. The finding that lymphocyte depletion is more effective in abrogating minor degrees of histoincompatibility than major ones was explicable if it was assumed that, whereas a few residual lymphocytes are sufficient to procure an attack against highly incompatible grafts, they are insufficient in the case of feebly antigenic grafts. An interesting parallel, which may have some significance here, is that abolition of tolerance of skin homografts in mice by transfer of normal “isologous” lymph node cells seems to require far fewer cells when the tolerance is in respect of strong transplantation antigens than when it pertains to “weak” transplantation antigens such as the Y-antigen (see Billingham et aE., 1963, 1965). Of course, the possibility has to be borne in mind that the proportion of an animal’s small lymphocytes which are capable of responding to strong histocompatibility antigens, such as those determined by the H-2 locus
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in the mouse, may be much greater than the proportion capable of responding to the weaker transplantation antigens. There are other lines of evidence which uphold the thesis that recirculating small lymphocytes play an important role in the primary response to homografts. Intravenous in jection of small lymphocytes from normal isogenic donors into tolerant rats or mice brings about the destruction of skin homografts of long standing (Gowans et al., 1963; Billingham et al., 1963). In studies with rats, when labeled cells were transfused, it was shown that by 24 hours many of them had transformed into large pyroninophilic cells scattered through the cortex of the lymph nodes and the white pulp of the spleen (Gowans et al., 1963). During the rejection episode, the cellular response was systemic and not limited strictly to the lymph node draining the hitherto tolerated skin homograft. This may be construed as evidence that the regional node of a tolerant animal is not involved in the rejection of a hitherto tolerated skin homograft. The irradiation of circulating blood, either during its passage through an extracorpareal arteriovenous shunt or by means of intraarterially implanted ""Y,a high-energy /?-emitting isotope, produces a marked lymphopenia which can be maintained for prolonged periods. By the former procedure, the intensity of the reaction of calves against skin homografts was reduced (Chanana et al., 1966); and, by the latter procedure, significant prolongation of the functional survival time of renal homografts in dogs has been achieved (Wolf et al., 1966). SITE OF SENSITIZATION PROCESSI N SKINAND RENAL HOMOCRAFTS
There is no evidence that antigenic matter of any kind escapes from a graft and travels through the regional lymphatic vessels or is liberated from intact tissues into the body fluids of normal animals. Woodruff (1957) was unable to sensitize rats by means of skin homografts isolated from contact with host tissue by means of a layer of cell-impezmeable Millipore membrane placed over the graft bed. Furthermore, the tissue "juice" that can be mechanically expressed from skin grafts seems to be incapable of eliciting sensitivity ( Billingham, unpublished). T'issues growing in Millipore chambers inserted intraperitoneally in homologous hosts are also antigenically ineffective, though, according to Mannick et al. ( 1964 ), subcellular antigenically active material can be recovered from the culture fluid of rabbit splenic tissue explanted and maintained in vitro for 2 weeks. Observations that relatively small first-set homografts of skin and other tissues implanted into highly vascular sites which lack a lymphatic drainage, such as the brain and the subcutaneous tissues of the cheek
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pouches of Syrian hamsters, are not rejected unless the host is or has previously been sensitized by another route is valid evidence of the ;mportance of lymphatic connections for the sensitization process ( Billingham and Silvers, 1962). Furthermore, that it is the regional nodes that are the principal seats of response to skin homografts strongly sugqests that the afferent lymphatics constitute an integral part of the afferent pathway of the immunological reflex. Certainly these regional nodes are not the exclusive seats of response. Their extirpation prior to grafting does not significantly reduce the power of the primary response. There is evidence that this is not merely caused by the participation of other pre-existing centers of response but also by the opening up of new pathways leading to lymph nodes which would not otherwise have participated in the reaction (Billingham et al., 1951). Scothome (1958) has studied the lymphatic repair at the sites of transplantation of skin autografts or homografts on the ears of guinea pigs. At various intervals after grafting, the lymphatics were revealed by injection of the host skin with India ink, the lymphatics of the graft filling by retrograde flow. He concluded that lymphatic repair occurs at the same time in both autografts and homografts and that the restoration of lymphatic drainage takes place by the anastomosis of host vessels lying in the graft bed with surviving intrinsic vessels within the graft. Particularly pertinent to the present discussion is his evidence that restoration of the continuity between graft and host vessels is not demonstrable until the fifth day after transplantation, whereas specific morphological changes in the regional (auricular) nodes draining skin homografts are demonstrable as early as 3 days after grafting (Scothorne, 1957). Since it is generally agreed that initiation of revascularization of skin grafts takes place between 24 and 48 hours (Taylor and Lehrfeld, 1853; Henry et al., 1962), and the establishment of an active circulation is even further delayed, it seems most unlikely that restoration of vascular continuity is essential for sensitization to occur. The most reasonable interpretation of the above evidence is that either antigenic material “exuding” from a skin homograft, or small lymphocytes that have been “primed as a consequence of exposure to antigenic material at the graft-host interface, must reach the regional nodes via the host lymphatics in the graft bed and that sensitization of the host can take place in the absence of an established lymphatic continuity with the graft. Some support for this interpretation comes from McKhann and Berrian’s (1959) observation that in mice removal of a skin homograft from its bed after 24 hours and its replacement by a new graft from an animal of the original donor strain every day for
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4 days did not prevent the development of sensitivity in the host. An inherent weakness of their experimental design is that it did not exclude the possibility that each consecutive transient graft left a few donorstrain leukocytes behind in the bed which might have been responsible for the immunity observed. Some idea of the aggregate number of “contaminating” leukocytes required to elicit sensitivity may be gained from the observation that, with the CBA + A mouse-strain combination, as few as 2000 spleen cells injected intraperitoneally elicit a just-perceptible level of immunity against donor-strain skin grafts transplanted 8-10 days later ( Billingham et al., 1957; see Hildemann et al., 1960). Although even the finest regional lymphatic vessels in the skin can easily be revealed with the aid of Patent blue V and other dyes (Hudack and McMaster, 1933)) no one has yet examined the peripheral lymph draining a skin homograft to characterize its content of cells and/or antigen, or its rate of flow, and compare them with those deriving from a skin autograft. There is a need for experiments along the lines of Hall and Morris’ (1962, 1963) elegant studies on the popliteal nodes of sheep. In this species, the rate of flow of afferent lymph to this node varies from 1 to 4 ml./hour and its cell content is low, ranging from 100 to 1500 white cells/mm3. The dominant leukocytes are medium-sized lymphocytes which these authors equate to the small lymphocyte of the rat. These observations indicate that appreciable numbers of lymphocytes are constantly leaving the blood vascular system via the endothelium and migrating into the tissues throughout the body. In an elegant experimmtal analysis, Frey and Wenk (1957) established that the presence an intact lymphatic drainage is essential in order for a contact allergen-dinitrochlorobenzene-to sensitize guinea pigs. Barker and Billingham (1966) have shown that this condition applies to orthotopic ’ in homografts too. This ear-skin homografts, 1-2 cm?. in area, fail to sensitize their guinea-pig hosts and survive for anomalously long periods if transplanted to raised flaps of skin on the side of the host’s body, prepared in such a way that they are only united to the animal by a single vascular bundle comprising an artery and a vein. In situations where dye injection revealed that the vascular “umbilical c o r d of such flaps retained a major lymph vessel cominunicating with the host’s regional node, a homograft implanted into the flap was rejected with virtually normal promptitude. However, graft survival was greatly prolonged if the lymph vessels of the bundle were ligated. As might be anticipated, skin homografts in the artificially created “immunologically privileged sites furnished by these skin flaps are fully susceptible to a state of sensitivity ( 1) evoked i n their hosts by parenteral
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inoculation of donor cells or by exposure to donor skin in the usual way, or ( 2 ) conferred upon them adoptively as a consequence of transfer of lymphoid cells from sensitized isologous donors. In Syrian hamsters, homografts of cheek pouch “skin,” transplanted to recipient areas in the integument of the trunk, heal in and acquire a rich blood supply but usually long outlive homografts of ordinary skin of the same genetic origin. Indeed, a significant proportion of pouch skin homografts survive indefinitely ( Billingham and Silvers, 1962). Various lines of indirect evidence suggested that the longevity of these grafts turns, a t least in part, upon the absence of lymphatic vessels within their peculiar connective tissue. Recent findings that dye injected very superficially in long-established isografts of pouch skins fails to appear in either the lymphatic vessels draining the area or in the regional nodes (Barker and Billingham, 1967) lends support to this interpretation. On the basis of the evidence that immunologically competent cells are constantly present in peripheral blood, Medawar (1958) raised the attractive possibility that the inductive stage of a host’s response to a homograft might take place within the substance of the graft. He reasoned that lymphocytes may enter the graft from the blood stream, react with its antigens, and then pass by way of the regional Iymphatics to the nodes where they then generate the “effectors” of the host’s response. It was further suggested that the occurrence of peripheral senritization would help to explain many of the curiosities and anomalies of the homograft reaction, such as the fact that a very small skin homograft on an animal as large as a cow can incite a reaction just as rapidly and violently as a graft of similar size on a mouse (Medawar, 1965). If sensitization is peripheral, he suggests, then homografts of similar size will be able to sensitize the same number of percolating lymphocytes in both hosts. A serious weakness of this argument is that, granted that similar numbers of cells may be activated by grafts of similar size in both the mouse and the cow, in terms of the total recirculating lymphocyte pools, there are no grounds for belief that the cow will produce more effector cells than the mouse. Consequently, the ratio of effector-to-noneffector cells in the latter species will be much higher than in the former. Medawar’s premise would only apply if the postulated peripheral sensitization was followed by local destructive activity within the graft on the part of the potentially reactive cells remaining within it. Hellman et al. (1965) showed that treating mice with thalidomide prolongs the survival of skin homografts. Furthermore, skin homografts treated with this agent in uitro, or derived from thalidomide-treated donors, also enjoy some prolongation of survival which is associated with
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a failure of the usual concentration of immunoblasts or large pyroninophilic cells to develop in the regional nodes (Turk et al., 1966). Since there are good grounds for belief that skin grafts from the thalidomide-treated animals contain significant amounts of drug, possibly bound to skin protein, the interesting possibility has been raised by Turk and his coworkers (1966) that it may inhibit the postulated peripheral immunization process by affecting the lymphocytes during their passage through the graft. However, as they concede, it is just as likely that small amounts of the drug are released from the graft and then pass down to the draining node and exert an immunosuppressive effect directly on the differentiation of activated small lymphocytes. This work is interesting since it raises the possibility of interference with the process of peripheral sensitization. It is worth bearing in mind that the capacity of humoral isoantibodies, acquired through either active or passive immunization, to impair the development of an effective cellular immunity against homografts in the phenomenon of immunological enhancement ( see Kaliss, 1965) may also reflect, at least in part, interference with sensitization at the peripheral level (Snell et al., 1960; Brent and Medawar, 1961). It may be pertinent that in Barker and Billingham’s skin pedicle studies ( 1966; see discussion above), small accumulations of lymphocytic cells were frequently noted in both the connective tissue of the homografts and the subjacent host skin dermis in skin flaps deprived of a lymphatic drainage. However, the presence of these cells did not seem to have any adverse effect on the well-being of the homografts. Graft death was usually the nonspecific outcome of an eventual cessation of blood flow to the entire flap due to strangulation of the umbilical cord. With large organ homografts, which require vascular anastomoses at the time of transplantation, the tactics of the sensitization process appear to be different. Hume and Egdahl (1955) presented evidence that, in dogs, renal homografts could sensitize their hosts even when special steps had been taken to prevent the establishment of lymphatic or any direct solid-tissue connections between them and their hosts. This finding has recently been confirmed in man (Lavender et al., 1967). The fact that a renal graft has a large blood flow, which is restored immediately after transplantation, certainly provides an opportunity for large numbers of blood-borne lymphocytes to interact with antigen as they pass through the extensive vascular bed of the organ. In the dog, Nathan (1964) has shown, in a carefully designed experiment, that the temporary introduction of a first-set renal homograft into a host’s circulation for as short a time as 2-6 hours may be sufficient to provoke a
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DARCY B. WILSON AND R. E. BILLINGHAM
detectable level of sensitivity in respect of a second renal homograft from the original donor transplanted 7 days later. Cogent evidence that peripheral sensitization can take place in renal homografts has recently been presented by Strober and Gowans (1965) from carefully controlled experiments with rats of an inbred strain and their F1 hybrids with an unrelated strain. They have perfused hybrid kidneys in vitro with thoracic duct lymphocytes from parental-strain animals for as short a time as 1 hour and subsequently injected the cellular perfusate back into isogenic parental-strain hosts. The latter were challenged with homografts of F, hybrid skin 8 days later and shown to be sensitized. Perfusion of F, homologous kidneys in vivo for 5 to 12 hours with blood issuing from the femoral artery and returning to the femoral vein of parental-strain ‘losts” likewise resulted in the latter becoming sensitized. Both Nathan’s and Strober and Gowans’ findings indicate that, so far as renal homografts are concerned, the immunological priming process can take place very rapidly-within a matter of a few hours. On the basis of their observations, Strober and Gowans suggest that in both renal homografts, and probably in other organ homografts which are immediately united to the host by surgical anastomoses of large vessels, the afferent arc of the immunological reflex is as follows. Blood-borne small lymphocytes pass several times through the vascular bed of the homograft during which time some of them engage with antigen, becoming “primed; during the migration of the lymphocytes from the blood into the lymph nodes and spleen, those which had previously interacted with the alien graft antigens transform into large pyroninophilic cells which generate the effector cell population. This attractive hypothesis of peripheral sensitization is sustained by the results of in vitm experiments with mixed lymphocyte interactions to be described in Part 2. It is also supported by Dvorak and Waksman’s (1962) finding that after 4 days’ intimate exposure of a rabbit’s lymph node cells to small fragments of homologous skin in cell-impermeable Millipore chambers, maintained in the peritoneal cavity of an indifferent host rabbit, the lymphoid cells acquired the capacity to incite typical “transfer reactions” (see Section IV,A) on injection into the rabbit that provided the skin fragments-an indication of their newly acquired immunological activity. Strober and Gowans’ (1965) conclusions are not necessarily inconsistent with the well-established principle of the longevity of small homografts of skin and other tissues in the immunologically privileged sites afforded by the brain and the hamster’s cheek pouch. The small blood flow through the relatively trivial amount of vasculature in these
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grafts may afford too little “contact time” between lymphocytes and graft endothelial cells-presumed to be the sites of the antigenic confrontation-to be effective. Nevertheless, the findings that long acceptance of homografts of pouch skin on hamsters’ chests may lead to what appears to be a state of partial tolerance and that the long-term exposure of animals to small homografts of various tissues in privileged sites may also lead to weakening of their capacity to react against the antigens concerned, indicates that under these conditions grafts can have a considerable influence on host immunological response mechanisms ( Billingham and Silvers, 1964a) . Finally, in organ grafts such as kidneys, if interaction between lymphocyte and antigen does take place at the level of the vascular endothelial cells, the capacity of a homograft to sensitize its host by the vascular route would be expected to decline if its vascular endothelium is eventually replaced by endothelial cells of the host. Such a process might take place surreptitiously in long-term renal homografts protected from rejection by long-term regimens of immunosuppressive therapy (Medawar, 1965). IV.
The Effector Side of the Immunological Reflex
It may be recalled that the view that the destruction of vascularized solid-tissue homografts is brought about by “activated lymphoid cells, rather than by humoral antibodies, was principally sustained for a long time by the transferability of transplantation immunity to animals by means of lymph node cells, but not by means of serum. However, although it was transferable by parabiosis (Harris, 1943; Falls and Kirschbaum, 1953), failure to transfer homograft sensitivity to normal, adult, isologous hosts by means of whole blood, leukocyte concentrates, or peritoneal exudate cells constituted a serious weakness of this cellular immunity theory ( Billingham et al., 1954). In a reinvestigation of “adoptive” transfer of transplantation immunity, it was found (Billingham et al., 1963) that immunologically tolerant mice and rats could be coerced to reject their grafts by the transfer of node cells from either normal or specifically sensitized isologous animals. Cells from sensitized animals brought this about more rapidly. However, with appropriate strain combinations, when relatively small numbers of cells were transferred, only those derived from sensitized animals were effective. With the aid of this test system, it was demonstrated that whole blood, or leukocyte concentrates prepared therefrom, were as effective in transferring sensitivity as regional node cells. With this system, it was possible to determine the time of appear-
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ance and persistence of “activated cells in recipients of skin homografts. These cells were first demonstrable in the regional nodes and blood at about the same time (on the sixth day after an A-strain mouse had received a CBA-strain skin homograft), suggesting that they must leave the nodes and enter the blood stream very soon after their formation in the nodes. Furthermore, these activated cells were first demonstrable in the blood stream a day or two before the onset of an overt reaction in the immunizing skin homograft and were still demonstrable in both the nodes and the blood for as long as a year after graft rejection. The capacity of thoracic duct cells to transfer sensitivity to tolerant rats suggested that the effective cell was a small lymphocyte, since such cells can reach the thoracic duct from the regional (axillary) nodes only by recirculating through the blood. Recirculation of “activated small lymphocytes could also explain the long persistence of immunity that was transferable by blood and the spread of activity from the regional node to other lymphoid organs (see Billingham et al., 1963). However, as Gowans (1965) has pointed out, an alternative interpretation of our findings is that other nodes become colonized by large lymphocytes which enter the blood via the efferent lymphatics from the stimulated regional node. The persistence of activated cells in the blood might then involve the production of short-lived cells from continuously dividing precursors. The recent evidence of Hall et al. (1967) that in sheep the immunological response to SaZmoneZZa or human red cell antigens is propagated from a regional node to other nodes throughout the body by basophilic cells in the efferent lymph bears directly upon this interpretation. The ability of a sensitized animal to give a second-set reaction probably turns upon the ready availability of a considerable number of activated cells within its blood stream rather than upon a reawakening of sensitivity, though the latter can certainly occur (Steinmuller, 1960; Hildemann et al., 1960). Chanana and his associates (1966) have compared the fates of pairs of skin homografts from unrelated donors transplanted orthotopically to different sites on cattle, so that, whereas one graft was drained solely by the thoracic duct, the other was not. In two out of two hosts, they found that if the thoracic duct lymph was chronically irradiated extracorporeally both before and after grafting, thereby reducing the lymphocyte concentration, the graft on the area exclusively drained by the thoracic duct significantly outlived the other. They tentatively ascribe the longevity of this graft to destruction of “instructed” cells as they pass from the nodes, via the thoracic duct to the blood sfYeam, which prevents them from reaching and attacking the grafts.
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A. TRANSPLANTATION IMMUNITY AND DELAYED CUTANEOUS INFLAMMATORY REACTIONS In 1958, a new approach to the analysis of the role of lymphocytes as mediators of transplantation immunity was opened by Brent and his associates. They showed that a state of transplantation immunity in guinea pigs-classically recognized in terms of a second-set reactioncould also be revealed by the development of a local, delayed inflammatory response if challenged intradermally with suspensions of living cells, or antigenic extracts prepared therefrom, from the donor strain (Brent et al., 1958, 1962). This manifestation of homograft sensitivitysimilar to the tuberculin reaction-was designated the “direct reaction.” These authors also discovered that similar inflammatory responses OCcurred when relatively few viable lymphoid cells, leukocytes, or peritoneal exudate cells from specifically sensitized hosts of skin homografts were inoculated locally into the skins of normal animals which had donated the immunizing tissue grafts. This reaction, termed the “transfer reaction” could not be provoked with serum from specifically immunized donors and was considered to be the outcome of a local graft-versus-host reaction. Both direct and transfer reactions have latent periods of 5 to 8 hours and attain their maximal intensities at 24 to 48 hours. They are highly specific immunologically, and the intensity of the direct reaction is correlated with the strength of the homograft reaction that evoked it. Like sensitivity to orthotopic skin homografts in most species, a guinea pig’s capacity to give a direct reaction, if evoked initially by a skin homograft, persists for hundreds of days. This ability of guinea pigs to express immunity to homografts as a classic, delayed-type, cutaneous, inflammatory reaction, with a characteristic, mononuclear, cell infiltrate, provided support for the increasingIy popular thesis that transplantation immunity, contact allergies, and hypersensitivities to microorganisms are cognate immunological phenomena. Essentially similar cutaneous reactivities to transplantation isoantigens are also expressed by Syrian hamsters (Ramseier and Billingham, 1964, 1966) and rabbits (Dvorak et al., 1963; Kosunen and Dvorak, 1963). In hamsters, the transfer reaction takes longer to develop than the direct reaction (48-72 hours) and, having attained its peak, persists thus for several days. Partial or complete necrosis of the affected area of skin usually occurs, leading to scarring. As in the guinea pig, the size and severity of transfer reactions in hamsters depends upon: ( a ) the number of sensitized cells injected and ( b ) the level of sensitivity in the ~
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donor of these cells. It was also shown that the capacity to give a direct reaction could be transferred adoptively to a normal isologous host by means of intraperitoneally inoculated suspensions or fragments of lymph nodes from sensitized animals. If sensitized hamsters were challenged 30 hours after exposure to a high dosage of X-irradiation (1500 r ) , by which time their peripheral blood lymphocyte count had been reduced tenfold, the evokable direct reactions were of reduced intensity. If challenge was delayed until 48 hours postirradiation, when the granulocyte count had fallen to about 50%of its normal level, there was no significant direct reactivity. These findings indicate that reduction of the peripheral blood lymphocyte count down to about 4 x lo5 per milliliter does not abolish hypersensitivity to transplantation antigens in hamsters and hints that granulocytes may play a role in the development of the lesions. At first sight, the transfer reaction appears to be a relatively straightforward process-the most obvious interpretation being that it is tho expression of a graft-versus-host reaction in which the inoculated sensitized lymphoid cells react with host antigenic material confronting them in the indigenous cell population of the skin. In guinea pigs, cells or antigenic extracts from a donor animal, mixed with sensitized cells from a recipient R and injected into the skin of a normal guinea pip isogenic with R, incite cutaneous reactions (Brent et al., 1962). This indicates that both direct and transfer reactions depend essentially upon some sort of local engagement of antigen with sensitized ceIls. That the story is more complicated, however, became evident when it was found that hamster hosts which had received a dosage of 1500 r displayed a greatly reduced capacity to exhibit transfer reactions followin9 local inoculation with specifically sensitized cells. Since irradiation at the dosage levels employed does not harm skin (Ramseier and Strcilein, 1965; Brent and Medawar, 1966a), but causes a rapid decline in both the lymphocytes and granulocytes of the peripheral blood, it seemed likely that a cellular contribution from the host’s blood stream was essential for a transfer reaction to take place. Evidence upholding this premise was provided by Ramseier and Streilein’s ( 1965) demonstration that transfer reactions will develop in irradiated hosts if aliquots of sensitized node cells are mixed with an equivalent number of viable cells bearing the host’s transplantation antigens before injection. Lymph node cells from isogenic or genetically tolerant F, hybrid donors or isologous skin epithelial cells are effective as “additives.” It seems reasonable to suppose that these cells function simply as sources of antigen. Evidently the normal resident cell population of the skin is inadequate
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for this purpose and, in the unirradiated host, immigrant cells of hernatogenous origin enter the inoculation site of the transferred sensitized node cells and furnish the antigenic stimulus. Further evidence of the probable role of cells circulating in the host’s blood for the development of transfer reactions was forthcoming when it was found that, if node cells from MHA hamsters sensitized against CB-strain tissues were injected into the skins of MHA hamsters that had been made tolerant of CB tissues (and which were, therefore, presumed to be chimeric at the level of their leukocytes and other tissues; (see Billingham and Brent, 1959), strong transfer reactions developed. Here the only possible antigenic stimulus in the host were the descendants of the neonatally injected, tolerance-conferring, CB bone marrow cells. NO significant responses resulted from the intracutaneous injection of sensitized MHA node cells into normal MHA hosts. In another experiment adult (CB x MHA)F, hamsters received relatively large skin grafts from MHA and CB donors. After these grafts had healed-in, each animal had skin of three different genotypes available as sites for the transfer reaction: CB, MRA, and its own F, skin. When these different kinds of skin were injected with node cells from MHA donors sensitized against CB tissues, strong transfer reactions developed at all sites, indicating that indigenous cells in the skin are probably only of minor antigenic importance in these reactions. The only reasonable conclusion is that host cells arriving at the inoculation sites via the blood stream play an indispensable role in the genesis of these reactions. Analysis of the transfer reaction in rabbits, in which either the lymphoid cells of the host or the donor had been labeled with tritiated thymidine, has shown that the vast majority of cells contributing to these lesions are of host origin (Kosunen and Dvorak, 1963). In guinea pigs, hamsters, rabbits, and man, lymphocytes from normal adult donors injected intradermally into normal homologous hosts may also incite inflammatory reactions of delayed onset known as “normal lymphocyte transfer (NLT) reactions” (Brent and Medawar, 1963; Dvorak et al., 1963; Ramseier and Billingham, 1964, 1966; Gray and Russell, 1965). In the guinea pig, these reactions have been extensively studied by Brent and Medawar (1963, 1966a) and shown to be discernible as early as 15 hours after inoculation and reaching peak intensities after about 48 hours. In hamsters, NLT reactions do not attain their zenith until 72 hours after inoculation ( Ramseier and Billingham, 1966). In both guinea pigs and hamsters, appropriate tests, including those employing parental strain and F, hybrid animals, have established that NLT reactions are graft-versus-host reactions provoked by lymphocytes
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from immunologically normal donors. Indeed, all parties are agreed that the only meaningful distinctions that can be drawn between NLT and transfer reactions are chronological and quantitative. Transfer reactions, which employ sensitized cells, represent “accelerated” graft-versus-host reactions. When donor and recipient are unrelated-deriving from an outbred population or from two different inbred strains-host-versus-graft reactivity may superimpose itself upon the graft-versus-host reactivity of a NLT reaction after a few days (Gray and Russell, 1965; Gray, 1966). Brent and Medawar (1964, 1966a) have shown that exposure of host guinea pigs to 500 to 1500r whole-body irradiation prior to testing reduces or abolishes an immunological contribution on their part but does not impair the development of NLT reactions. In irradiated guinea pigs, NLT reactions evolve in three phases over a 6-day period: ( 1) an initial inflammatory episode, apparent after about 15 hours, peaks at 24 hours and remains stationary for an additional 48 hours; ( 2 ) the ‘‘flareup,” which commences between the third and fourth days and attains its maximal intensity by the sixth day, when the injection sites have been transformed into huge purple lesions; and ( 3 ) a final “fade-out” phase. Brent and Medawar (1966a) interpret the first inflammatory episode of the NLT reaction as the outward manifestation of a “recognition” event. A distinctively immunological process, which occurs when transferred lymphocytes are first engaged by host antigens and are committed to the sequence of events outwardly expressed by the subsequent flareup. The refractoriness of the first inflammatory episode to inhibitors of mitosis is cited as evidence that it does not depend upon cell division. The flareup which, by contrast, can be eliminated by certain immunosuppressive agents, such as methotrexate and cyclophosphamide, is attributed to the activity of the sensitized cell population postulated as having arisen through proliferation of donor cells activated by the first phase of the NLT reaction. An observation of importance for understanding the cytopathogenic potentiality of lymphocytes in different immunogenetic contexts is that the time sequence of the components of the NLT reaction remains fairly constant (Brent and Medawar, 1966a). The weak reactions incited when the hosts confront inoculated lymphocytes with minor histoincompatibilities, or when only small numbers of cells are injected into hosts that confront them with major alien antigens, differ from strong reactions in their level of intensity, rather than in the timing of their component events. This suggests that, irrespective of whether the sensitivity of a
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21 1
host is directed against strong or weak transplantation antigens, on a cellfor-cell basis, sensitized lymphocytes are equivalent with respect to their capacity to harm target cells or organs. This conclusion was reached independently by Simonsen (1962) on the basis of a quantitative analysis of graft-versus-host reactivity in the spleens of infant mice. The potencies of lymphoid cells from different sources to incite NLT reactions parallels their known capacities to cause the so-called homologous or transplantation diseases resulting from systemic graft-versus-host reactions. For example, blood lymphocytes are 2-5 times more active in inciting NLT reactions than lymph node cells, and thymocytes are 10-20 times less active than the latter. The third phase of the NLT reaction, i.e., the fade out, is ascribed mainly to revival of immunological reactivity in the host. Clearly, before Brent and Medawar’s ingenious interpretation of the NLT reaction can be fully accepted, studies with cytologically or isotopically labeled cells are required to obtain conclusive evidence concerning the identity of the cell population at various stages and to establish whether the postulated proliferative response does, indeed, involve only donor cells. It may be recalled that cytological analysis of several graft-versus-host reactions has revealed the substantial contribution of host cells to the conspicuous mitotic activity, despite the fact that they may be incapable of responding immunologically ( see Billingham, 1967; Kosunen and Dvorak, 1963). On the basis of their studies with the NLT reaction, Brent and Medawar (1966a) have put forward an ingenious quanta1 theory. The violent response produced by a population of normal lymphoid cells at the peak of the flareup phase and that produced from the outset by an inoculum of presensitized cells are held to be arithmetic multiples of the “recognition” process. In the second situation, more “sensitized” cells are supposed to be available to do what a relatively small number do in the first situation. In their view there are certain intrinsic differences of immunological competence on the part of lymphoid cells of a normal individual-a small proportion of these being already ( congenitally ? ) endowed with the capacity of reacting with a particular antigen. Sensitized individuals differ from normal ones simply in that they contain a much higher proportion of these immunologically activated cells. Lymphocytes are known to be radiosensitive cells (Trowell, 1965), and Dempster and his co-workers (1950) have shown that the survival times of first-set skin homografts is significantly prolonged by prior irradiation of the hosts. However, it is well established that X-irradiation after exposure to antigen does not impair, and may even enhance (Dixon and McConahey, 1963; Taliaferro et al., 1964), the immunological re-
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sponse. Furthermore, a dosage of irradiation that will prolong the survival of first-set homografts exerts no influence on the survival of second-set homografts. The manner in which irradiation affects transplantation immunity has been investigated by Brent and Medawar (1966b) using the NLT reaction in the guinea pig. They have obtained suggestive evidence that lymphoid cells capable of initiating an NLT reaction are more radioresistant than cells unable to do so, but were unable to determine, from their available data, whether or not this privileged status was the outcome of the cells’ belonging to an immunologically predetermined population. Radiation doses of up to 1000r did not affect the first inflammatory episode of the NLT reaction, nor did they affect the immediate immunological performance of cells from specifically presensitized donors in the transfer reaction. The effect of irradiation of the inoculated cells was to suppress (at lower doses) or completely abolish (at higher doses) the flareup component of the NLT reaction-i.e., it affected the postulated conversion of a normal into a sensitized cell population. This transformation was presumed to be the result of mitotic activity on the part of competent cells following their engagement with antigen. On the basis of these and other findings, the aiithors argue that, provided that lymphocytes remain viable and are already endowed with qualities of effector cells, irradiation does not affect any distinctively immunological performance on their part. The effect of irradiation can be explained by (a) its antiproliferative action and ( b ) the fact that lymphocytes, though abnormally radiosensitive, acquire a relatively high degree of radioresistance as soon as exposure to antigen has committed them to an immunological response. These studies on the NLT reaction are certainly exceedingly interesting and the quanta1 theory merits very careful consideration. It is unfortunate that Brent and Medawar provide no information concerning the influence of any of the immunosuppressive drugs tested, or of Xirradiation in the various doses employed, on the peripheral blood leukocyte count in their guinea pigs. It is crucially important to know whether or not host cells of hematogenous origin are necessary for the occurrence of these reactions and, if so, whether they are dividing in the lesions during the A areup. As already mentioned, there is an impressive body of evidence that, in all graft-versus-host reactions, proliferating host cells do make an important, possibly an essential, contribution to the various reactivities. This certainly applies in both the NLT and transfer reactions in hamsters and probably in rabbits (see Kosunen and Dvorak, 1963; Ramseier and Billingham, 1966). It also applies in the case of local graft-versus-host reactions incitable in the kidney, to be
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described below. In both of these situations, the presence of host cells of hematogenous origin seems to be mandatory for the development of lesions. Attention is also drawn to the fact that Brent and Medawar offer no suggestion as to how a postulated “recognition event” can cause an inflammatory reaction. There is no evidence that the incarceration of normal lymphoid cells with small fragments of homologous skin, epidermal cell suspensions, or other tissues in Millipore chambers inserted into the peritoneal cavities of neutral hosts causes any perceptible degree of destruction of the target tissue (Fujimoto et al., 1966). However, Dvorak and Waksman ( 1962) have demonstrated that, as a consequence of 4 days or more of exposure of normal rabbit lymphoid cells to homologous skin shavings, a population of specifically sensitized lymphoid cells develops. This was evidenced by the ability of the lymphoid cells to incite transfer reactions on removal from the chambers and inoculation into the skin of the donor of the skin shavings. It was subsequently shown that the addition of peritoneal exudate or alveolar macrophages to lymph node suspensions frequently resulted in an enhancement of the sensitization acquired, suggesting that, at least in this situation, macrophages may be necessary for the primary response to transplantation antigens (Hulley et al., 1964). More critical experiments are clearly needed to confirm this suggestion. Purified preparations of small lymphocytes from the blood or thoracic duct, which do not contain macrophages, should be placed in Millipore chambers with target tissue and with or without the addition of purified macrophages. Appropriate techniques for obtaining these are now available (see Bloom and Bennett, 1966). B. MIXED LYMPHOCYTE REACTIONSIN OF IRRADIATED HAMSTERS
THE
SKTNS
It was discovered by Ramseier and Streilein (1965) that, if heavily irradiated ( 1500 r ) and highly leucopenic MHA hamsters are inoculated intracutaneously with a mixture of lymph node cells in equal numbers from MHA hamsters sensitized to CB tissues and from normal CB hamsters, severe, delayed, cutaneous inflammatory reactions result which are indistinguishable from transfer reactions. Since neither component of the mixture incited a response when inoculated on its own, the authors interpreted the reactions incited by the mixed cell inocula as arising from the local engagement of the sensitized MHA cells with the alien isoantigens of the CB node cells. They found that similar reactions were incited in irradiated MHA hamsters’ skin by inocula comprised of mixed node cells from unsensitized MHA and CB hamsters. A11 the evidence indi-
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cated that a very rapid mutual sznsitization occurred-MHA immunologically competent cells reacting against the CB cells and vice versa, the inflammatory lesion observed being the outcome of a two-way reaction. The reactions incited by mixed suspensions of MHA and (MHA X CB)F, hybrid node cells, or MHA node cells and viable CB epidermal cells, must necessarily have been unidirectional since in both cases the cells bearing the CB antigens were incapable of reacting against the MHA antigens. The finding that mixtures of cells from MHA and ( MHA x CB)F, donors incite inflammatory lesions in irradiated MHA hosts has one very important implication: that skin presents itself as an indifferent milieu in which these reactions can take place. The development of the cutaneous lesions presumably results from the liberation of some agent( s ) or mediator by the reacting cells in the mixture. It was also found that heavily irradiated hamsters would sustain and manifest reactions between lymphoid cell mixtures from unrelated donors of alien species. For example, mixtures of cells from mice belonging to strains A and CBA, from guinea pigs of strains Nos. 2 and 13, or from two unrelated human beings will incite cutaneous reactions closely similar to those incited by the inoculation of cell mixtures derived from hamster donors (Ramseier and Streilein, 1965; Streilein et al., 1966). Again, it must be emphasized, the inoculation of lymphoid cells from a single donor of an alien species was ineffective. Even where heterologous donors are concerned, there is some evidence that these mixed lymphoid cell reactions do not require differences at major histocompatibility loci in order to manifest themselves, though their intensity is rah.ted to the degree of incompatibility existing between the donors of the cells. C. LOCALGRAFT-VERSUS-HOST REACTIONSIN
THE
KIDNEY
Although the various homologous or transplantation diseases, such as runt disease, are initiated by the inoculation of lymphoid cells into hosts that confront them with alien transplantation antigens, careful studies have failed to reveal exactly how the hosts are harmed by the graftversus-host reactivity of the sensitized donor cell population that presumably arises (see Billingham, 1967). Massive destruction of tissues in a manner analogous to the destruction of tissue or organ homografts by adult hosts has not been observed and the tissues most affected are those belonging to the Iymphohematopoietic system. Studies on certain local graft-versus-host reaction systems, such as
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the transfer reaction and the NLT reaction, have certainly furnished evidence as to the cytopathogenic activity of lymphocytes. However, the most informative graft-versus-host reaction system from this viewpoint has been that studied by Elkins (1964, 1966). This investigator has shown that if lymphoid cells from adult, parental strain rats are injected beneath the renal capsules of adult F, hybrid hosts, the transferred cells, or their progeny, invade the underlying cortical tissue and bring about the destruction of most of the tubules they surround. Eventually, over the course of a week, up to 25%of the renal mass may be destroyed. These reactions were found to be “self-limiting” and purely local, being confined to the area of inoculation and infiltration. Neither the contralateral kidney, the spleen, nor any other organs of the host showed abnormalities, though some evidence was obtained that the presence of these local graft-versus-host reactions in the kidneys caused increased mitotic activity in the host’s lymphoid system. Experiments with chromosomal markers indicated that the inoculated donor lymphocytes gave rise to a rapidly dividing population of large pyroninophilic cells in the active phase of the reactions which seemed to play an important role in the pathogenesis of the lesions, possibly as progenitors of immunological effector cells. Seven days after inoculation of the kidney with parentalstrain cells, practically all the dividing cells at the site were of donor origin. During the early developmental phase of these reactions-up to about the eighth day-they were transferable to the kidneys of similar secondary F, hosts. This important observation indicates: ( a ) at least a limited persistence of the donor cell population and ( b ) that it possesses the capacity to continue an effective graft-versus-host reaction for at least 7 days. Elkins (1967) has also shown that at or after day 9 there is a proliferation of host cells in the lesions. However, the cellular basis of these local graft-versus-host reactions is much more complicated than might have been anticipated, This was evident from Elkins’ (1966) observation that prior irradiation of potentially susceptible F, hosts inhibited development of renal graft-versushost reactions to an extent related to the degree of radiation damage inflicted upon the host’s lymphoid tissues. He concluded that the development of the lesions in the host’s kidney depended upon the continuous interaction of the specifically reactive donor cells with an immunologically nonspecific population of host mononuclear cells. This important finding parallels Ramseier and Billingham’s evidence of the importance of host cells of hematogenous origin for the development of both NLT and transfer reactions in hamsters’ skins (see Section IV,A),
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OF THE IMMUNOLOGICAL STATUS OF LYMPHOID D. TRANSFORMATION CELLSBY RIBONUCLEIC ACID
Mannick and Egdahl (1962) ingeniously employed the transfer reaction in the rabbit to demonstrate that the immunological status of normal lymph node cells could be altered by exposure in oitro to ribonucleic acid (RNA) extracted from the nodes of rabbits which have been actively immunized by skin homografts. In essence, they found that if a suspension of viable node cells from a normal rabbit was incubated at 37°C. for 15 minutes with phenol-extracted RNA from the draining nodes of the sensitized animal, the lymphoid cells acquired the capacity to incite transfer reactions on injection into the skin of the donor of the sensitizing grafts. No significant reactions developed if the RNA had been extracted from normal, unstimulated nodes and “reactive” RNA alone was ineffective. That the reactivity acquired by RNA-treated normal lymphoid cells was specific was evidenced by their failure to incite reactions when inoculated into “neutral” rabbits. Pretreatment of reactive RNA with RNase abolished its ability to transform normal node cells. This interesting principle of RNA conversion could also be employed to procure the “adoptive” immunization of a normal rabbit to skin homografts (Mannick, 1964a). Removal of rabbit A’s spleen, exposure of the dissociated cells (about 200 x loG)in vitro to RNA extracted from the nodes of rabbit R, which had been sensitized against tissues from rabbit D, followed by infusion of the cells back into A, conferred upon the latter the capacity to reject skin grafts from D in an immune manner. No sensitivity was acquired by rabbits that had been injected intravenously with reactive RNA preparations. Although the events underlying this conversion from normal to sensitized node cells have not been elucidated at the molecular level, the authors have tentatively concluded that their findings may be explained by the addition of some form of “messenger” RNA to the lymphoid cells and their subsequent synthesis of cell-bound antibody directed by the RNA molecules. Mannick ( 1964b ) has presented evidence that tritiumtagged RNA is incorporated by node cells during their exposure in uitro. Mannick (1964b) has recently reported that the capacity of immune node cells from a recipient rabbit R to incite transfer reactions on inoculation into its tissue homograft donor D can be inhibited if the cells are exposed in vitro to RNA extracted from D’s nodes or from the nodes of a normal, indifferent rabbit. Ribonucleic acid extracts of autologous nodes were ineffective. His attempts to inhibit rejection of skin
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homografts by intravenous infusion of hosts with RNA extracted from the nodes or livers of the future graft donors were unsuccessful, even when putative RNase inhibitors were added (Mannick, 1 9 6 4 ~ ) .It may be noted that this finding fails to substantiate claims of other workers that systemic administration of RNA may prolong the life of homografts (see Ashley et al., 1960; Axelrod and Lowe, 1961; Jolley et al., 1961). It is to be hoped that these important RNA studies will be repeated in species in which isogenic strains are available to facilitate more critical analyses of the specificities of the induced sensitivities and provide evidence concerning their durability. One interesting experiment yet to be done is to find out whether an animal’s own lymphoid cells can be altered so that they will give transfer reactions if inoculated into its own skin. An intriguing and provocative agent that seems to play a role somewhat analogous to that of RNA in the work reviewed above is transfer factor. In man, certain types of delayed hypersensitivity, including tuberculin hypersensitivity and homograft immunity, are transferable by means of relatively minute quantities of an agent extractable by lysis from peripheral blood leukocytes-designated “transfer factor” ( Lawrence et al., 1960, 1963). The passively acquired sensitivity appears to be long lasting, indicating that the host’s own cells must have become endowed with the capacity to mediate the specific sensitivity concerned. Transfer factor seems to be remarkably stable, being resistant to the action of DNase, RNase, trypsin, to freezing and thawing, and to lyophilization, etc. Its molecular weight is low, being of the order of 10,000. The most recent evidence suggests that it is a small polynucleotide, possibly associated with polypeptide (Lawrence et al., 1963). So far, the majority of attempts to demonstrate transfer factor in species other than man have been unsuccessful. Its existence in the guinea pig is certainly equivocal (see Chase, 1965; Bloom and Chase, 1966).
E. THESIGNIFICANCEOF THE CELLULAR INFILTRATE IN A HOMOCRAFT That the mononuclear cells of hematogenous origin which consistently infiltrate homografts of solid tissues and organs are in some way responsible €or procuring the destruction of the foreign cell population has long been part of the transplanter’s creed. However, many investigators have felt uneasy about a thesis that rested very largely upon evidence of a correlative nature. The important question remains as to whether the cellular infiltrate represents a specific immunological effector mechanism in operation, and, therefore is causally related to homograft rejection, or whether it is a consequence of injury previously inflicted
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DARCY B. WILSON AND R. E. BILLINCHAM
upon the tissue by some other agency, such as a humoral isoantibody of some kind (see Stetson, 1963; Hildemann and Pinkerton, 1966). Several lines of evidence deriving from in vivo studies have already been presented in support of the thesis that lymphocytes can cause pathogenic lesions, and this thesis has been greatly strengthened by the findings of in vitro experiments to be described in Part 2 of this review. The present section outlines the current status of knowledge concerning the mononuclear cellular infiltrate that constitutes the classic harbinger of the rejection process in vascularized tissue and organ homografts, with special emphasis upon skin grafts. It is well established on the basis of both light and electron microscopic studies (Henry et al., 1962; Waksman, 1963; Wiener et al., 1964) that the cellular infiltration of a skin homograft precedes the appearance of degenerative changes in its epithelial and other cells. Although it is difficult to identify the constituent cell types comprising the infiltrate, it seems to be generally agreed that lymphocytes of various sizes, macrophages, and a minority of plasma cells are the ingredients and that small lymphocytes predominate. Claims made from histologica1 studies on skin and tumor homografts that necrosis of the foreign cells is dependent upon contact or intimate association with infiltrating lymphocytes ( Kidd and Toolan, 1950; Waksman, 1963; Wiener et al., 1964) represent an oversimplification in our experience. Destruction of hyperplastic skin homograft epithelium very frequently does appear to be a consequence of its invasion by mononuclear cells ( the “invasive-destructive”process of some authors ) ; but the not infrequent appearance of degenerating epidermal cells, with typical vacuolated cytoplasm, in areas where infiltrating cells are sparse or absent, suggests that other factors may be involved. On the basis of a reinvestigation of the histopathology of the skin homograft reaction in rats, Waksman (1963) concluded that the principal contributory processes leading to death of the graft were ( a ) local accumulation of mononuclear cells both inside and outside the vessels and ( b ) a direct cytopathogenic action of these cells on the antigenically foreign cells, both in the vessel walls and throughout the graft. Considerable emphasis was placed upon the vascular arrest and consequent ischemia as contributing to the death of the graft. Porter and Calne (1960) and Gowans and his associates (1962) have presented convincing evidence that the small lymphocytes which invade primary skin homografts are mainly a specific group of new cells formed in response to the graft, rather than a random sample from the total lymphocyte pool. In rats given repeated systemic doses of tritiated thymidine, commencing at the time of grafting, Gowans and his associates
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(1962) found that in both 7- and 10-day biopsy specimens the dermis was heavily infiltrated by small lymphocytes, of which upward of 90% were labeled. Furthermore, at 7 days, about 7%of the small lymphocytes in the blood were labeled, whereas at 10 days the medullary sinuses of the node draining the skin homograft (axillary ) contained about 16%of labeled small lymphocytes. It is implicit in these important findings that the labeled cells do preferentially leave the blood for the graft. Further important evidence that cells produced in a draining node invade first-set skin homografts in significant numbers was presented by Prendergast ( 1964). This investigator transplanted single skin grafts from two unrelated donors to each ear of a host rabbit. He then injected tritiated thymidine locally at regularly spaced intervals into one of these grafts to IabeI the cells in its regional lymph node. Of this node, 18%of the cells were labeled whereas cells of other nodes were unlabeled. The cellular infiltrate in both homografts included a relatively large and similar proportion of labeled cells ( u p to 11%)though the proportion of labeled lymphocytes in the blood did not exceed 0.2%. It was inferred from this observation that, although labeled small lymphocytes of recent formation do show a predilection for homografts, it is not immunologically specific. Further evidence of the nonspecificity of the mononuclear cell infiltration process was forthcoming when Prendergast repeated his first experiment, using as the host for the grafts a rabbit in which delayed hypersensitivity to bovine serum albumin ( BSA ) had previously been induced. When the animal was challenged intracutaneously with the antigen after the isotope labeling of the regional node draining a skin homograft, the proportion of labeled cells observed in the inflammatory lesion which developed was similar to that observed in the skin homografts on its ears. Studies in which immunity to skin homografts has been transferred adoptively from tritiated thymidine-treated, sensitized animaIs to either normal isologous animals or to tolerant isologous animals bearing skin homografts of long standing, have consistently failed to reveal the presence of significant numbers of labeled cells in the recipient’s homografts (Najarian and Feldman, 1962; Billingham et al., 1963). Unless the label became diluted out of the transferred cells through rapid division following transfer, this suggests that the vast majority of the infiltrating cells in the skin grafts borne by these animals must be mononuclear cells of host origin. This interesting situation is in complete accord with numerous observations that, in essentially similar adoptive transfers of delayed hypersensitivity to tuberculin, to simple chemicals, and to other agents, with isotopically tagged lymphoid cells from sensitized donors, only
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DARCY B. WILSON AND R. E. BILLINGHAM
very small numbers of labeled donor cells are demonstrable in the lesions that develop at antigen test sites (Feldman and Najarian, 1963; Turk, 1962; McCluskey et al., 1963). Well-controlled experiments indicate that there is no specific attraction of labeled cells to the antigen but that they accumulate in the lesions in a purely random manner (see Chase, 1965; Bloom and Chase, 1966). Thus in the light of presently available evidence, it seems impossible to avoid the conclusion that, if cells originating in the regional nodes of the sensitized cell donors are solely responsible for initiating the lesions of delayed hypersensitivity in adoptively sensitized hosts, then the arrival of only a very small number of these cells at a test site is all that is required; and we are left with the problem of accounting for the role of the remaining cells. That the presence of this preponderance of putatively uncommitted cells may be very important for the development of lesions in adoptively sensitized animals is suggested by the observation that, when the capacity to give direct reactions to homologous cellular inocula is transferred adoptively in hamsters with a constant dosage of lymphoid cells from a sensitized donor, heavy preirradiation of the host diminishes the level of reactivity expressed (Ramseier and Billingham, 1966). Similar observations have attended adoptive transfer of tuberculin hypersensitivity in guinea pigs ( Cummings et al., 1955). Of course, it is not entirely inconceivable that in adoptive transfers of cellular immunities the uncommitted host cells that accumulate in test lesions may be “converted to a specific, immunoIogically committed status by transfer of information from the small minority of committed donor cells. Indeed, the conversion might take place elsewhere in the host’s body. Agents that might be capable of transferring the required information are RNA and transfer factor (see Section IV,D). It may be noted that, in the case of adoptive transfer of the ability to make humoral antibody to a heterologous antigen in rabbits, Harris ct al. (1963), using a sensitive and ingenious experimental design, found no evidence of conversion of host cells to antibody-synthesizing status. Finally, attention may be drawn to another feature of adoptive transfer of cellular immunities that is difficult to explain. This is the exceedingly large and physiologically abnormal number of lymphoid cells that must be transferred from a sensitized donor to confer a readily detectabIe level of sensitivity upon an isoIogous host-at least one donor equivalent of lymph node and splenic cells is usually necessary. So far as transplantation immunity is concerned, the only exception is when tolerant hosts are employed instead of normal ones. Possibly the fact that tolerant animals are chimeric with respect to the cells of their blood and hematopoietic tissues underlies their peculiar amenability to adoptive
LYMPHOCYTES AND TRANSPLANTATION IMMUNITY
221
transfer. The alien cells may provide immediate antigenic stimulation to the transferred cells, causing rapid proliferation of sensitized cells ( Billingham et al., 1963). The process of rejection of renal homografts has also been closely studied with both the light and the electron microscope. Bearing in mind that, whereas the events in a skin graft are complicated by its having to become revascularized naturally, and that this does not apply to kidney grafts, the behavior of the infiltrating cell population in these two types of homograft is very similar. In renal homografts of only a few hours’ standing, small lymphocytes are found in contact with endothelial cells lining the peritubular capillaries and venules of the cortex and outer medulla. Between 2, and 3 days later cells belonging to two broad, ultrastructurally distinct categories begin to appear in a few of these vessels. These are ( a ) cells with little or no endoplasmic reticulum, resembling large lymphocytes or histiocytes and ( 1 2 ) cells of the plasma cell type which have well-developed endoplasmic reticulums (Porter et al., 1964). Tritiated thyrnidine uptake experiments indicate that about 4% of the immigrant cells in a 4-day, canine, renal homograft are actively synthesizing DNA and will divide (Dempster and Williams, 1963; Porter et aZ., 1964). According to Kountz et al. (1963), cytoplasmic continuity may be temporarily estabIished between the infiltrating host cells and the endothelial cells lining peritubular capillaries of the graft; and this in some way leads to disruption of the peritubular capillaries and venules which, according to Porter (1965), is the most important event in the rejection of primary renal homografts in normal hosts. However, according to him, in transplants in immunosuppressed hosts, it may or may not be a major factor in the rejection process. Certainly in the case of renal homografts, and probably to a large extent in skin homografts too, it is at the endothelial boundary separating the graft from the host’s bloodthe obvious and most accessible target-that the host’s attack develops. Medawar (1965) has raised the interesting possibility that an imperceptible, progressive, and possibly rapid, replacement of the vascular cndothelium in a renal homograft by endothelial cells of host origin, during the course of a homograft reaction attenuated by irnmunosnppression, might account for the much better results obtained with renal transplantation in dogs and man than would have been anticipated from studies on skin homografts (Moseley et al., 1966). F. MANNERIN WHICHTHE CYTOPATHOGENIC EFFECTOF LYMPHOCYTES IS MEDIATED
The means by which sensitized Iymphocytes are able to fulfill their established cytopathogenic role in vivo in the process of homograft
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rejection, in the various, delayed, cutaneous, inflammatory manifestations of homograft sensitivity and in both systemic and local graft-versus-host reactions, are still largely a matter of speculation. The possibilities include the following: ( I ) the cells synthesized an antibody that is normally released only when they are in intimate contact with homologous “target” cells or antigenic material; ( 2 ) the cells do not synthesize antibody but carry on their surfaces an effective, cell-bound antibody having a special affinity for cells ( cytophilic antibody) which is synthesized elsewhere in the body (see Boyden, 1963) ; ( 3 ) the cells synthesize a special kind of antibody which is permanently incorporated on their surface and is only effective when an intimate contact with a target cell has been established; and ( 4 ) antibody is not the ultimate effector or mediator of graft destruction. Following some kind of recognition event which has an immunological basis and requires an intimate relationship between sensitized cell and graft antigen, the sensitized lymphocytes release or secrete, and/or possibly cause other host cells to release, some nonspecific pharmacologically active agent, analogous to histamine, which is the ultimate effector. Lysosomal enzymes should not be neglected as possible candidates for the role of mediators (see Section VII,B ) . Rather than discuss each of these possibilities in turn, let us consider some evidence, additional to that already presented, that may help to discriminate between them. With one possible exception (see below), the use of living sensitized lymphocytes to transfer transplantation immunity, or incite transfer reactions in animals, seems to be obligatory in the experience of nearly all investigators. Intact but “devitalized cells or homogenates are ineffective. This might be construed as suggestive evidence that synthesis of the effector agent is not initiated until sensitized lymphocytes are engaged by antigen. The fact that both transfer and direct reactions are of the delayed type may be significant here-apart from the time taken for them to mobilize, the mononuclear cells may also require time to synthesize effective amounts of a putative mediator. It is clearly important to determine whether, at the time they are circulating in the blood, sensitized cells are already “armed,” i.e., carry “preformed a full quota of the mediator, or whether this is synthesized following their engagement with antigen. There is little information bearing upon this important question. Bloom and co-workers (1964) observed that lymphoid cells from guinea pigs sensitized to picryl chloride are incapable of transferring hypersensitivity adoptively following treatment in vitm with mitomycin C at a concentration of 100 pg./ml. (an agent which arrests synthesis of RNA and later of protein). However, cells treated with low
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concentrations ( 10-15 pg./nil. ) of this antibiotic, which reduced the amount of RNA synthesis by about 30% of that of untreated cells, still transferred substantial levels of hypersensitivity if the recipients were challenged early. Since these workers consider it unlikely that the drugtrtetzd transferred cells could divide, th:. transferred, immunity must have arisen by mechanisms not involving cell division. Jankovic and Dvorak (1962) found that treatment with RNase of regional node cells from a rabbit sensitized by a skin homograft abolished their capacity to incite a transfer reaction when injected into the skin of the graft donor. However, culture of DNase-treated sensitized cells in a Millipore chamber resulted in the cells regaining their ability to incite transfer reactions. On the basis of these observations, the authors concluded that an intact protein anabolic activity on the part of the sensitized cells is essential for transfer reactivity. They suggest that, on second contact with antigen in the host’s skin, Sensitized cells synthesize increased amounts of a specific protein ( posibly antibody) responsible for the transfer reaction. That a diffusible, antibodylike agent associated with sensitized lymphoid cells is responsible for homograft immunity was implicit in some intriguing findings of Najarian and Feldman (1962, 1963a). They reported the successful transfer of sensitivity to skin homografts in mice and guinea pigs, as evidenced by second-set rejection of test skin homografts, by means of very large numbers of sensitized lymphoid cells from isologous donors sequestered in cell-impermeable Millipore diffusion chambers implanted either intraperitoneally or, more effectively, subcutaneously in the vicinity of the target skin grafts on the host. Similar findings were subsequently reported by Kretschmer and PQez-Tamayo ( 1962) from studies conducted on rabbits. Subsequently, Najarian and Feldman (1963b) reported the successful passive transfer of specific homograft sensitivity in mice by means of a soluble extract of sensitized lymphoid cells having y-globulinlike properties. Impressed by these exceedingly interesting observations, Billingham et al. ( 1963) and Wilson et al. (1966) sought to abrogate tolerance of skin homografts in mice by transfer of sensitized lymphoid cells in Millipore chambers, but they were unsuccessful. Furthermore, using Najarian and Feldman’s ( 1962) experimental design, they were unable to transfer sensitivity to normal mice, as were Fujimoto and his associates (1966). No explanation can be advanced to account for this discrepancy, and it is haped that other investigators will help to resolve it. Suggestive evidence that the mediator of transplantation immunity may be a nonspecific pharmacologically active agent has been forthcom-
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ing from the work of Spector and his associates, who have made notable contributions to our knowledge of endogenous mechanisms of injury in the inflammatory process (see Spector and Willoughby, 1964). They have been studying the “lymph node permeability factor”, or LNPF, an agent extractable by sonication from lymph nodes of normal rats, guinea pigs, and other species (Willoughby et al., 1963; Willoughby and Spector, 1964). The LNPF can also be extracted from the sites of tuberculin reactions where its concentration rises and falls parallel with the intensity of the local inflammatory reaction ( Willoughby et al., 1964). It has a powerful action in increasing vascular permeability and induces a massive immediate emigration of leukocytes. There is an early emigration of polymorphonuclear cells, observable within 45 minutes of injection, but finally, at 24 hours, mononuclear cells predominate outside the vessels. It has been suggested that LNPF is the mediator of certain delayed hypersensitivity reactions, and there is some evidence implicating this agent in the development of lesions in experimental autoimmune thyroiditis ( see Willoughby, 1966). Independently, Inderbitzin (1964) reported on the presence of a vasoactive factor in extracts of various organs which he termed “Permeability increasing factor” (PIF). He and his associates have recently presented evidence that PIF is a histamine-releasing agent and that it is identical with LNPF (Inderbitzin et al., 1966). Although the latter agent has been differentiated from other known permeability factors, it has yet to be defined chemically. The information presently available about LNPF is sufficient to alert us to its possible role as cytopathogenic agent in homograft sensitivity. PART 2.
IN VITRO STUDIES OF THE IMMUNE RESPONSE
Administration of antigenic material to an organism generally sets in motion a complex series of events culminating, ultimately, with an interaction between a specific immunological product and the antigen that elicited its formation. In the past, it has been operationally convenient to dissect the events associated with immune responses into three phases: (1) the afereiat phase, concerned primarily with recognition, i.e., those processes by which an organism is made aware that foreign materials have been introduced into its milieu inte’rieur; ( 2 ) the central phase, which includes those biochemical processes concerned with the synthesis of an immunospecific product; and, finally, ( 3 ) the eferent phase, in which the mechanisms of immunity, once called into existence, are put into effect. Many of the problems concerned with the second of these aspects have yielded to the persistent efforts of biochemists and microbiologists-it is generally agreed that the synthesis of antibody
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proteins occurs through metabolic pathways essentially common to all protein synthesis. Also, with the classic, humoral immunities, the role( s ) of various macromolecular immunoglobins is, at least, not a total enigma. There are, however, two fundamental aspects of immunity where little information is available and where considerable controversy exists concerning the mechanisms which may be operative: first, the modus opcrundi of the initial recognition events involved in immune responses in general and, secondly, the effector mechanisms underlying the cellular immunities-in particular, what are the agents of homograft sensitivities and how do they operate. Part 1 of this review dealt with manifestations of hypersensitivity to homografts occurring within the intact animal, There is no question that lymphocytes are a major factor in the immunological response of the whole organism and that these cells undergo fundamental cellular processes of proliferation and differentiation and are involved in the synthesis of specific macromolecular proteins. However, the exact nature of the cellular events that culminate in the existence of an immune state and the manner in which immunities are put into effect are not known. At times, results of studies concerned with these problems have been difficult to interpret, particularly in experiments employing the intact animal. The purpose of the present section is to consider these immunological problems in so far as they have been, or can be, studied with lymphocytes maintained in tissue culture and to review the evidence that has accrued from this approach. Its importance stems from the fact that some of the events occurring at the cellular level can be scrutinized more critically when cells are removed from the influence of the complex homeostatic mechanisms of the intact animal. Although cellular immunity is the subject of this review, to restrict it to considerations of research involving only delayed hypersensitivities would be foolish. It seems clear that the effector mechanisms of cellular and humoral immunities are distinctly different, but this may not be so for their inductive phases. Moreover, lymphoid cells are intimately involved in the development of both modalities of immunological response. Therefore, in considering the behavior of cells in simplified in vitro systems as models for in vivo mechanisms of immunity, any information pertinent to the immunological potentialities of the lymphocyte in vitro will be freely drawn upon. V.
Lymphocytes as Effectors of Immunity
Although numerous studies have emphasized the importance of plasma cells in the production of antibody (e.g., see Leduc et al., 1955; Attardi et al., 1964), other cells are known to contain and release anti-
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body in various experimental situations. Perhaps the best and most direct evidence of antibody synthesis by cells found circulating in the lymph or blood comes from the studies of Hummler et al. (1966). These investigators were able to identify individual cells producing anti-sheep hemolytic antibody b y their ability to lyze sheep erythrocytes, thereby causing plaques, in an agar semisolid medium. When removed and examined with the electron microscope, many of these antibody producers had the typical ultrastructure of small lymphocytes. This type of evidence clearly shows that small lymphocytes can play a significant role in humoral immunities. That mediation of homograft immunity, or other types of delayedtype hypersensitivity, is the province of cells, and not circulating antibody, is based principally on the failure to transfer an effective state of homograft sensitivity to isologous recipients with sera-and the unqualified success with lymphoid cells-from immunized donors ( Mitchison, 1954, 1955; Billingham et al., 1954). It should be pointed out, however, that there is no evidence which contradicts-in fact, some favors-the possibility that humoral antibodies may play an ancillary, supportive, but not an essential, nor exclusive, role in the rejection of tissue homografts. There have been reports that isoimmune sera can have a deleterious effect on normal tissues in some experimental stituations. Serum antibody can lyze suspensions of dissociated cells ( Winn, 1960a,b). In any case, knowledge of the effector mechanisms involved in the destruction of solid, vascularized tissue homografts has been difficult to obtain. That cells of the lymphoid system are involved is clear, but part of the difficulty has been caused by the failure to isolate an immunospecific product responsible for the destructive behavior of immunologically active lymphocytes. A. DESTRUCTIVE ACTIVITYOF SENSITIZED LYMPHOID CELLS 1. Qualitative Impressions
Some of these difficulties have recently been resolved by the recent demonstrations that lymphoid cells (Govaerts, 1960; Rosenau and Moon, 1961, 1966; Brondz, 1964; Hanaoka and Notake, 1962; Taylor and Culling, 1963; Vainio et al., 1964; Wilson, 1963, 1965a), as well as macrophages (Granger and Weizer, 1964; Old et al., 1963), procured from specifically immunized animals have a cytocidal effect on appropriate homologous “target” tissue cells in culture. A new era of investigations on the immunoIogical behavior of sensitized lymphoid cells was ushered in by Govaerts’ (1960) finding that thoracic duct lymphocytes
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obtained from dogs that had rejected renal homografts were capable of destroying cultures of target kidney cells obtained from the donor of the organ graft. The cytocidal capability of lymphocytes from immunized animals was quickly confirmed in a number of laboratories with slightly different test systems and, although it remains unconfirmed, isolates of nuclei from sensitized lymphocytes were also claimed to be cytocidal ( Svet-Moldavsky, 1964). When living cells from the thoracic lymphatic duct, regional lymph nodes, or spleen of a specifically immunized animal are added to monolayer cultures of target cells, obtained from a donor against whose tissues the sensitivity is directed, within a few hours many of the lymphocytes can be seen clustering around and over the target cells (Rosenau, 1963; Wilson, 1963). After approximately 10 hours of culture, DNA synthesis in the target cells begins to shut down (Wilson and Defendi, unpublished). With the aid of radioautographic procedures, it can be shown that at this time fewer target cells incorporate radioactive thymidine into DNA in cultures containing sensitized lymphocytes than in those exposed to normal lymphocytes. After 10 hours, target cells exposed to sensitized lymphocytes retract their cytoplasmic processes; some acquire a fragmented appearance and, by the twentieth hour, they round up and detach from the glass surface as dead cells (Wilson, 1963). The finding that cytocidally active cells can be obtained from the thoracic lymphatic duct of dogs that had rejected renal homografts (Govaerts, 1960) or from rats immunized with skin homografts (Wilson, 1963) is of particular importance. Here, the attacking cells are part of a constantly recirculating population of small lymphocytes and, by the strictest criteria, immunization was provoked in respect of transplantation isoantigens. By the use of target cells rendered incapable of division with X-irradiation, it has been shown that immune lymphoid cells actually kill the target cells rather than merely inhibiting their growth (Wilson, 1963). The immunological specificity of the cytocidal activities of sensitized lymphocytes has been clearly established. Destruction of the target cells ensues only under those circumstances where they bear the isoantigenic specificities in respect of which the lymphocyte donors were immunized. Lymphocytes obtained from animals immunized to heterologous antigens (Wilson and Wecker, 1966) or to transplantation antigens of a third party ( Brondz, 1964) are completely ineffective against specific homologous target cells. Moreover, “activated lymphocytes have no adverse effect on isologous target cells (Wilson, 1963; Brondz, 1964; Vainio et nl., 1964). Although the results of initial studies by various investigators are
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not in complete accord (for example, Govaerts, 1960), the cytocidal activities of sensitized lymphoid cells do not seem to require the participation of complement or serum antibodies. Rosenau and Moon (1961) and Wilson (1963) showed that target cells could be destroyed by sensitized lymphocytes in the absence of any kind of serum, and other workers have employed heat-inactivated sera to supplement the culture media. In spite of these precautions to rule out complement, its participation in this reaction cannot be entirely excluded since there remains the possibility that lymphoid cells themselves might contain or elaborate complement in uitro. This seems unlikely, however, since attempts to detect complement activity by means of a microcomplement fixation procedure in sonically disrupted normal or immune lymph node cells, or in media in which these cells had been cultured for 48 hours, were unsuccessful (Wilson, 1965a). Claims that immune sera may damage target cells or potentiate the effects of immune lymphocytes (Rosenau, 1963) can probably be ascribed to the presence of complement-dependent cytotoxic antibodies in noninactivated sera. That cytophilic isoantibody ( Boyden, 1963), capable of destroying cells, is the effective agent is extremely unlikely in view of Wilson’s reconstruction experiments ( 1965a), in which normal or immune sera were combined with normal or sensitized lymphocytes. Destructive reactivity was not acquired by otherwise normal lymphoid cells in the presence of immune sera nor could immune sera, even in high concentrations, augment the effect of sensitized lymphoid cells. In fact, under these conditions destruction was inhibited slightly, perhaps because of a coating of the target cells by the sera. Provisions for intimate cytoplasmic contact between the attacking lymphoid and target cells appear to be essential. Extracts prepared from sonically disrupted sensitized lymphocytes were no more deleterious to target cells than similar preparations from normal lymphoid cells (Wilson, 1965a). Also, when separated from the target cells by a Millipore membrane, immune lymphocytes were not cytocidal (Wilson, 1965a; Rosenau, 1963). This was taken to indicate that if a cell-bound immunospecific agent is involved in the destruction of homologous cells either it is not toxic by itself or it cannot be detached from the sensitized cells. The clustering behavior of lymphoid cells around target cells, which precedes their destruction, is generally complete within the first few hours of culture; it has been repeatedly described and termed contactual agglutination ( Koprowski and Fernandes, 1962). Although normal lymphoid cells exhibit a superficially similar clustering behavior, it is almost certainly nonspecific; normal lymphocytes can be dislodged by
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gentle agitation of the medium (Wilson, 1963, 1965a). Sensitized cells, on the other hand, leave a significant percentage of lymphocytes that agitation cannot remove after 5 hours' incubation. These few cells that cannot be detached after 8 hours' culture accomplish as much destruction in the succeeding 48 hours as all the lymphocytes if left undisturbed for a similar period in parallel cultures. It is interesting that this contactual behavior of immune lymphocytes is relatively temperatureindependent. Thus, with cultures maintained at different temperatures, similar degrees of clustering occur in cultures at 27" and at 37"C., although at the lower temperature there is no subsequent destruction of the target-cell monolayer ( Wilson, 1967a). The apparent insensitivity of contactual agglutination to changes in temperature suggests that binding of sensitized lymphocytes to target cells occurs via some preformed substance present on the surface of the lymphocytes. On the other hand, destruction of the target cells may occur only under conditions conducive to active lymphocyte metabolism. The findings outlined above show that within a population of cells obtained from a sensitized animal there exists a small minority-of the order of a few per cent-which, as immunological effectors, have the capacity to adhere to and destroy homologous target cells. As to the mechanism(s) of destruction, there is very little information. It has been suggested that a two-step process may be involved: first, lymphocytes specifically bind to target cells and, then, by means of some metabolic process, lyze the target cells (Wilson, 1965a). Whether lysosomes are involved remains to be demonstrated. Close contact between lymphoid and target cells is essential; it may be, in fact, the only step which is immunologically specific, although clustering itself is not cytotoxic. It is pertinent to mention here that certain drugs with immunosuppressive properties in v i m can effectively curtail the destructive reaction in culture. Thus, cortisone inhibits the killing reaction but not the clustering of lymphocytes (Rosenau and Moon, 1962); also Imuran has a similar effect at a concentration consistent with the viability of the attacking lymphocytes (Wilson, 196513).
2. Quantitative Relationships Quantitative studies (Wilson, 1965a) in which the number of target cells surviving were determined after various periods of incubation revealed a latent period of approximately 20 hours before demonstrable target-cell destruction occurred; within 48 hours, it was virtually complete. It goes without saying that the duration of the latent period depends on the nature of the event being scored. Thus, Taylor and Cul-
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ling ( 1963) detected morphological alterations among the target cells after a few hours, with an increasing number of cell deaths after 24 hours. Friedman (1964a) has shown that there is 5040% reduction in the numbers of target, antisheep, hemolytic, antibody-forming cells detectable with the Jerne plaque assay after 5 hours contact with specifically sensitized homologous lymphocytes. Also, Brunner and his co-workers (1966) have shown that target cell injury, as assessed by plating efficiency, begins shortly after contact with immune lymphocytes and is complete by 12 hours. Wilson (1965a) showed that increasing the ratio of attacking lymphocytes to target cells does not shorten the latent period. However, the number of target cells surviving at any one time thereafter was inversely proportional to the number of lymphoid cells employed in the cultures. Graphic plots relating the parameters of per cent survival of the target cells and the dose of attacking lymphocytes indicated that this inverse relationship is an exponential one not dissimilar to “single-hit” inactivation phenomena. These data are consistent with the interpretation that a single lymphocyte, if immunologically active, is sufficient to destroy or to have a detectably adverse effect on one target cell. From such a model, it can be computed that, of a population of lymphocytes derived from the lymph node of an immunized animal, approximately 1-2% are immunologically active. For thoracic duct lymphocytes, the percentage is lower. It should be stressed that such a figure is valid only in terms of the test system employed, i.e., the number of lymphocytes which arise as the result of a specified immunization procedure with the capacity to lyze certain homologous target cells in culture within a specified period of time. With the plating efficiency assay, Brunner et al. (1966) also demonstrated a logarithmic relationship between the number of attacking lymphocytes and their injurious effect on target cells. Examination of their data reveals’that the number of immunologically effective splenic cells from immune animals is of the order of a few per cent. It is likely that other cells, including numerous lymphocytes, having ancillary immunological activities-some of which may even contribute to the destructiveness of cytocidally active lymphocytes-are called into existence by the immunization procedures. The great increase both in the weight of the regional lymph node and absolute numbers of lymphocytes it contains following immunization is out of all proportion to the small percentage of cytocidally active lymphocytes demonstrable. Nevertheless, the figure of 1-2% or less is in line with estimates of the percentage of effector cells in other immunological test systems. Thus, the
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peak number of antisheep hemolysin-producing splenic cells detectable with the Jerne plaque assay following primary immunization with sheep erythrocytes is of the order of 0.1%(Jerne ct al., 1963). The number of hemagglutinin-producing cells following a similar immunization schedule which are detectable with Zaalberg’s cluster test is approximately 0.5% (Zaalberg et al., 1966). Studies in which the regional lymph nodes were harvested from immunized rats and mice at various times after exposure to homologous skin have shown that cytocidally active cells appear in the draining lymph node approximately 1 week after immunization. The peak 0f cytocidal activity of cell suspensions from this source was attained before the eleventh day, gradually declined, and disappeared by the twentieth day after immunization (Wilson, 1963). Small lymphocytes with cytocidal activity were nut demonstrable in the thoracic duct of rats until the ninth day after grafting (Wilson, 1965a). Whether this circulating cell population retains its cytocidal activity for longer periods of time has yet to be determined. As a means of interpreting data obtained with the normal lymphocyte transfer (NLT) reaction in guinea pigs, Brent and Medawar (1866a) have launched a “quantal” theory of the immunological behavior of lymphoid cells. According to their view, sensitization “is a property that may be predicted of a cellular population but not of a single cell, which itself either performs immunologically or does not (hence ‘quantal’).” In some respects this view is an attractive one. Although proposed to explain the activities of lymphocytes in an in vivo test system, it is consistent with the results of recent culture experiments in which the amount of destruction or injury of target cells inflicted by sensitized lymphoid cells was related to the number of attackinq cells employed (Wilson, 1965a). In these experiments, the greater cytocidal effectiveness of populations of lymphocytes from hyperimmunized animals resulted from their content of greater numbers of lymphocytes with destructive capabilities, rather than from an increased ability on the part of some cells from a hyperimmunized animal to do more damage than an equal number of effector cells from an immunized animal. According to Brent and Medawar’s proposal, the destructive action oE sensitized lymphoid cells upon target cells in vitro would not be qualitatively different from the activities of lymphoid cells obtained from a normal or nonimmune animal. The normal population simply contains fewer cells that would be cytocidally active in this type of in vitro test system. Quantitative data obtained with plaque and cluster assay system9 are consistent with this view (Zaalberg et al., 1966; Jerne et al., 1963). Thus,
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there is an easily detectable number of cells from spleens of nonimmunized animals which produce hemolytic or hemagglutinating antibody. There are good grounds for belief that, aside from its implications concerning the mechanisms of homograft immunity, the lymphocytetarget cell interaction may be equally useful as an experimental model for studies of autoimmunity. Cellular hypersensitivity almost certainly is a major factor in the pathogenesis of experimental thyroiditis and encephalomyelitis since these diseases are readily transferable to normal animals with living lymphoid cells (see Waksman, 1960). It is also possible that conditions such as rheumatic fever and ulcerative colitis may have an etiology involving the participation of cells sensitized to "self" antigens. In view of this, the finding that peripheral blood leukocytes from patients with ulcerative colitis are injurious to human fetal colon cells in culture is important (Perlmann and Broberger, 1963 ) .
B. DESTRUCTION OF CELLSIN CULTURE WITH NORMAL LYMPHOID CELLS 1. Heterologous Lymphoid Cells Stuart ( 1962) reported that lymphoid cells from normal, unsensitized murine donors had a cytopathic effect on heterologous monolayer cultures of HeLa cells; this effect was induced within 48 hours and took the form of a honeycomb pattern in the target cell monolayer. This pattern did not appear in cultures to which either no lymphoid cells or heatkilled lymphocytes were added. Lymphocytes from immunized donors produced a more rapid and severe effect than lymphocytes from normal mice. Stuart also found that the media recovered from the lymphocyte target-cell cultures and added to fresh cultures of HeLa cells was without effect. This suggested that the cytopathic effect was due to the intact lymphocyte and not to its disintegration products. Nevertheless, in view of the vast number of cells involved in these experiments, there is a very real possibility that the honeycomb appearance of the target monolayers exposed to lymphoid cells could reflect a competition both for medium and for glass surface, i.e., general limitations of an overcrowded culture. This objection can be resolved by the use of appropriate control cultures of isologous lymphoid cells and target cells. Recently, Ginsburg and Sachs (1965) reported that lymph node cells from an unimmunized population of outbred rats were capable of destroying monolayer cultures of embryonic mouse and rat cells. The
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earliest destructive effects occurred on the sixth day of culture and were preceded by the appearance of appreciable numbers of large lymphoid cells with pyroninophilic cytoplasm. When these larger lymphoid cells were transferred from the destroyed target cell monolayers onto new secondary mouse or rat monolayers, they brought about a rapid destruction of the new targets confronting them. This pronounced cytotoxicity of the transferred large lymphocytes was observed only if the secondary monolayers were derived from the same source as the primary. Because of the appearance of large transformed lymphoid cells with characteristically pyroninophilic cytoplasm, it is likely that the destructive capabilities of these lymphoid cells have an immunological basis. However, since this experimental design did not include the use of isogenic strains of animals, it was not possible for these authors to obtain conclusive evidence that the cytotoxic or cytopathic effects of nonimmunized lymphocytes had any immunological significance. 2. Homologous Lymphoid Cells
K. Hirschhorn and his co-workers (1965) have also reported on the destructive potentiality of homologous lymphocytes from normal donors. In their experiments, human peripheral blood lymphocytes had a marked cytodestructive effect by the seventh or eighth day on cultures of fibroblasts obtained from unrelated individuals. Addition of phytohemagglutinin ( PHA ) to the cultures accelerated this destructive reaction. When the lymphocytes were transferred to secondary fibroblast cultures, destruction of this second monolayer was most rapid in those instances where lymphocytes were obtained from those primary cultures to which PHA had been added. These authors felt that their results warranted the conclusions that ( 1 ) primary and secondary immunological responses were responsible for the lymphocyte-mediated destruction of the target fibroblasts-the first requiring more time than the second and ( 2 ) the course of events associated with these postulated immunological responses could be markedly accelerated in the presence of PHA. The objections concerning the lack of evidence of specificity levied against the studies described above apply here also. In 1964, Holm et al. reported that PHA-induced aggregation of normal human lymphocytes around target HeLa or human fetal kidney cells resulted in their destruction in culture. Again, whether or not isologous lymphoid cells had such an effect was not, or could not, be demonstrated. This finding triggered off a series of investigations by various investigators in Sweden (for reviews, see Hellstrom and Hell-
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strom, 1966; Moller and Moller, 1966; Hellstrom and Moller, 1965), the results of which have proven difficult to interpret and still await independent confirmation in other laboratories. Thus, E. Moller (1965) claimed that cultures of tumor target cells bearing known parental strain transplantation isoantigens could be destroyed by aggregating homologous or F, mouse lymphoid cells around them by means of heatinactivated heterologous rabbit antimouse antisera or with PHA. Since similarly aggregated isologous lymph node cells were ineffective, the clustering behavior of lymphocytes was considered to be a necessary but insufficient requirement for cytotoxicity in uitro. Prior irradiation of the added homologous or F, lymphocytes did not abrogate their cytotoxic effect ( Moller and Moller, 1965). Similarly, Hellstrom et al. (see Hellstrom and Hellstrom, 1966) claimed that homogenates or “antigenic extracts” prepared from splenic cells or tumor tissues inhibited the growth and proliferation of homologous tumor cells in uitro. This inhibition of tumor cell growth could be abrogated by cortisone in uitro as well as in uiuo ( Hellstrom et al., 1965). Isologous tumor cells cultured with antigenic preparations survived in greater numbers. Other experiments ( Hellstrom et al., 1964) indicated that death of the target cells, in addition to an inhibition of their growth potential, was involved. Furthermore, tumor cells of F, hybrid origin were not affected by homogenates or extracts of parental strain source. These authors felt that their in uitro findings provided an explanation for the phenomenon, termed syngeneic preference, and later, allogeneic inhibition, that transplanted tumor cells ( SnelI, 1958) or normal hematopoietic tissues (Cudkowicz, 1965) of parental origin do not proliferate as well in F, recipients as in hosts of the strain of origin. It was postulated that the diminished growth potential of parental tissues in foreign, yet immunologically nonresponsive, hosts, or the growth inhibition and destruction of cells in culture with foreign cells or antigenic extracts prepared therefrom, resulted from contact between cells bearing antigens of different surface patterns. These authors suggested that structural differences existing between cells bearing different transplantation antigens could result in mutual cytotoxicity based on structural rather than antigenic incompatibilities. How such cytotoxic effects can result from a “structural incompatibility” has not been formulated although, if valid, its implications are enormous. It has been suggested that allogenic inhibition might function as a homeostatic surveillance mechanism for the elimination of antigenically altered cells-including the potential progenitors of neoplasms-arising during the life of an organism. Since this process could aIso handle cells
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which had mutated to a form lacking a particular antigen, this would be a circumstance which does not engage a mechanism involving an immunological response.
3. Criticisms of Syngencic Preference It is extremely difficult to ascribe an immunological basis to allogeneic inhibition, especially in view of the claims that parental target cells can be destroyed by lymphoid cells of F, hybrid origin. However, if it is not by an immune mechanism, but one based on dserences of “structural surface patterns,” it is difficult to understand the inconsistency wherein the growth potential of F, tumor cells is unaffected by antigenic homogenates of parental origin (Hellstrom et aE., 1964), whereas F, lymphoid cells in close contact with parental fibroblasts are destructive (Moller and Moller, 1966). It may, in fact, be too early to speculate further on these findings; any attempt to evaluate their possible significance must take into account the existence of certain well-studied situations in which cells of widely different genetic origin can live harmoniously together in uiuo or in uitro. Both naturally nccmring and artificially produced mammalian or avian intraspecific chimeras, including dizygotic cattle, human and sheep twins, are perfectly healthy animals in which cells of alien origin persist indefinitely. One of the most intriguing of the artificial intraspecific chimeras is that which results from fusion of early cleavage-stage embryos in mice in which cells of different genetic origins are intimately commingled (Mintz, 1965). It is well known that skin and other tissues transplanted from parental strain donors to F, hybrid hosts become permanently incorporated in their new hosts with no evidence of lesions at the graft-host interfaces (Stimpfling, 1961). In guinea pigs, melanocytes from pigmented epidermis of parental strain donors can be caused to become intimately incorporated in white skin epidermis of F, hybrid hosts where they will proliferate and thrive indefinitely ( Billingham and Silvers, 1964b). Furthermore, with the use of infant rats rendered partially tolerant of mouse tissue, melanocytes of mouse origin can be introduced into host rat hair follicles where they may survive and function for as long as 25 days ( Silvers, 1965). Mixed cultures can he set up in ljitro in which embryonic cells from mice and chickens will live juxtaposed forming, for example, interspecific renal tubules and other histochimeras. In none of the homologous or heterologous combinations studied has any evidence of cell incompatibility been obtained (see Moscona, 1962). Finally, one might mention the most intimate of all chimeras-the interspecific hybrid cell having a
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chromosomal chimerism resulting from a viral-induced fusion of cells from different species (Harris, 1966). VI.
Macrophages and Antigen
The earliest observation that sensitized cells, derived from an animal in which a state of specific delayed hypersensitivity has been induced, behave differently than normal cells in the presence of specific antigen in culture was made by Rich and Lewis (1932) nearly 40 years ago. They showed that splenic or white blood cells, obtained from a tuberculin-sensitized guinea pig, were killed when cultured with a quantity of tuberculin antigen which was quite nontoxic to cells of normal animals. This destructive phenomenon was independent of the presence of complement and requiied several hours of contact with the antigen. These findings have been confirmed (Moen and Swift, 1936) and extended to other bacterial antigens (Heilman et al., 1960). Although there may be species differences, not all cells behave in the same manner when confronted in vitro with specific antigen. Thus, Waksman and Matoltsy ( 1958) demonstrated that peritoneal exudate cells removed from tuberculin-sensitized guinea pigs were stimulated to proliferate in cultures containing tuberculin. The interaction of peritoneal cells and antigen in tissue culture hds recently been subjected to close scrutiny by David and his colleagues (1964a,b). They employed an assay system in which peritoneal exudate cells from guinea pigs were allowed to migrate from capillary tubes onto a flat glass surface. In the presence of specific antigen, the migration of cells obtained from hypersensitive animals was markedly inhibited. This inhibition of macrophage migratory behavior was shown to be immunologically specific for the antigen used to induce the state of hypersensitivity. The admixture of small numbers (2.5%) of “sensitized” cells in a population of otherwise normal peritoneal cells in cultures with antigen resulted in an inhibition of all the celIs (David et al., 1964b). It is likely that this interesting finding has direct relevance to delayed hypersensitivity skin reactions in uivo. In particular, following the transfer of labeled cells from sensitized donors into normal recipients, the majority of cells which infiltrate and participate in the local skin reaction are unmarked and, therefore, are presumably of host origin (see Section IV,E ) . With carefully purified lymphocytes and macrophages from purified protein derivative ( PPD) -sensitized guinea pigs, Bloom and Bennett (1966) have recently shown that the presence of lymphocytes in the population of sensitized exudate cells is required for the inhibition of
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237
macrophage migration. In these studies, the migration of highly pursed suspensions of macrophages from animals was not inhibited in media containing PPD. Only by the addition of a few sensitized lymphocytes, derived from peritoneal exudates, in these cultures was inhibition demonstrable in the presence of antigen. These authors concluded that the inhibition of migration of peritoneal macrophages in this system is not a direct result of the effect of antigen but was simply an indicator of some immunological interaction between sensitized peritoneal lymphcytes and antigen. These investigators pursued this problem further by showing that cell-free supernates recovered from PPD-sensitive peritoneal lymphocytes incubated for 24 hours with that antigen inhibited migration when added to cultures of normal macrophages. Numerous control preparations including supernates: ( I) from normal lymphocytes incubated with or without PPD and (2) from PPD-sensitized lymphocytes incubated without PPD or with unrelated heterologous antigens (coccidiodin) had no inhibitory activity. Similarly, supernates from PPD-sensitized lymphocytes that had been incubated with the antigen, but to which more PPD was added after the lymphocytes were removed, were without effect. These findings support the idea that some soluble factor, termed ‘‘migratory inhibition factor” ( MIF) having an immunological specificity, is synthesized by hypersensitive lymphocytes in contact with antigen and is released into the medium where it affects the behavior of otherwise normal macrophages. The studies of Bloom and Bennett also showed that M I F is elaborated only by cells from guinea pigs with delayed-type hypersensitivity and not by cells from normal animals or animals which had been immunized so as to produce high titers of circulating antibody without delayed-type hypersensitivity. Recently, David (1966) demonstrated that lymph node cells from sensitized animals, when mixed with normal peritoneal exudate cells, cause the whole population to be inhibited by specific antigen. He also points out that preparations of sensitized lymphocytes, themselves, are not inhibited by specific antigen. With experiments similar to those of Bloom and Bennett ( 1966), David ( 1966) also demonstrated the inhibitory activity of a soluble factor in the supernates of sensitized cells incubated overnight with specific antigen ( ovalbumin or o-chlorobenzoyl chloride conjugated to bovine 7-globulin). This material proved to be nondialyzable and stable to heat treatment at 56°C. for 30 minutes. David ruled out the important possibility that sensitized lymphoid cells incubated without antigen might produce a substance which could subsequently interact with specific antigen and then have the capacity to
238
DARCY B. WILSON AND R. E. BILLINGHAM
inhibit macrophage migration. Normal peritoneal exudate cells were shown to migrate normally in supernatants of sensitized lymphocytes incubated without antigen and to which antigen was added after incubation. Both David (1966) and Bloom and Bennett (1966) have shown that incorporation of metabolic inhibitors ( puromycin or mitomycin C ) into the incubation mixtures of sensitized lymphocytes and specific antigen inhibits the production of MIF. This suggests that this inhibitory material may be a protein. VII.
The Blastogenic Response of lymphocytes in Culture
It has already been pointed out that lymphocytes are long-lived cells remaining morphologically unaltered and metabolically quiescent for extended periods of time. Generally, this also holds true when these cells are committed to culture (however, see Sabesin, 1965). That their apparently inert behavior could be altered became evident in 1960 when Nowell (1960) made the interesting observation that PHA-an extract of the red kidney bean, Phmeolus vulgaris, used to agglutinate erythrocytes and facilitate the separation of leukocyte-rich plasma from whole blood-provoked the appearance of large numbers of blastoid and dividing cells in cultures of leukocytes. Use of culture inocula consisting almost entirely of small lymphocytes indicated that it was this cell which was responding to the presence of PHA. Soon after the description of the blastogenic and mitogenic properties of PHA, several other agents with similar properties were discovered, many of which were antigens. Quite independently, Pearmain et al. (1963) and Schrek (1963) demonstrated that lymphocytes obtained from “tuberculin-sensitive’’ human donors would transform when cultured in the presence of tuberculin antigen. Moreover, the magnitude of the transformation response seemed to be directly related to the degree of delayed hypersensitivity which the donor revealed when skintested with the specific antigen. After the mitogenic properties of tuberculin and other antigens were described, it became a popular view that blastogenic transformation of cells in culture represented a secondary immunological response at the cellular level. When it was found that lymphoid cells of most species responded to PHA in culture, it became an untenable premise that the rnitogenicity of PHA had an immunological basis, i.e., that cell donors were sensitive to this agent as a ubiquitous antigen. Furthermore, responses to classic antigens as mitogenic agents were of far smaller magnitudes and totally different kinetics,
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Also, it was particularly difficult to visualize how the PHA response in culture, if it had an immunological basis, could involve the vast majority of cells that it did. Subsequently, the various mitogens were classified into one or the other of two categories, “specific” and “nonspecific,” distinguished primarily on the basis of whether or not a donor had to be immunologically presensitized in order to obtain subsequent reactivity with his cells in vitro. It was, therefore, considered that the transformations provoked by the nonspecific mitogens may not have any direct relevance to immunological mechanisms in general, whereas antigen-induced blastogenesis occurring in vitro might be directly pertinent to effector mechanisms of hypersensitivity in vivo. The finding (Bain et al., 1963, 1964) that homologous leukocytes, cell products ( Kasakura and Lowenstein, 1965), or extracts ( Hashem and Carr, 1963 ) prepared therefrom can induce transformation of lymphocytes in culture, even from nonsensitized donors, suggests that there might be yet another category of responses-perhaps related to the cellular events concerned with the induction as well as execution of hypersensitivities in vitro. Thus there are obvious and probably meaningful similarities of circumstances as well as morphology between the transformation of small lymphoid cells to large pyioninophilie cells characteristic of various immunological responses-including GVH reactions ( Section 11) and the transformation that lymphoid cells undergo when cultured in the presence of specific antigens.
A. RESPONSES TO NONSPECIFIC MITOCENS Although it is not likely, for the reasons given, that the blastoid transformation induced with nonspecific mitogens represents an immune mechanism, it will be considered in this discussion in some detail primarily because it is lymphoid cells that are so drastically affected. Thus, some of the cellular events associated with the proliferative response of lymphoid ceIIs to a specific antigen may also be triggered off by nonspecific mitogens and, therefore, made available for study.
I. Phytohemagglutinin Phytohemagglutinin was the first of these agents to be discovered and this substance, as well as the response it elicits, has been the most extensively studied of all the mitogens. Its erythroagglutinating ( Rigas and Osgood, 1955) and leukoagglutinating (Mellman et al., 1962)
240
DARCY B. WILSON AND R. E. BILLINGHAM
properties are well established; and, since it has been impossible SO far to separate the leukoagglutinating activity from its mitogenic activity, some workers [notably Hirschhorn’s group ( 1963) , Nordman et al. (1964), and Tunis ( 1964)] maintain that PHA exerts its mitogenic action through an effect on the cell surface (Rigas and Johnson, 1964; Nowell, 1960). Other nonspecific mitogens include an extract from pokeweed (PWM) (Farnes et al., 1964; Borjeson et al., 1966), staphylococcal endotoxins (Ling et al., 1965), streptolysin S (Hirschhorn et al., 19M), and antileukocyte sera ( Grasbeck et al., 1963). These mitogens characteristically induce transformation of 50 to 70%of the cultured lymphocytes by the third day. Robbins, in a recent review (1964), has provided a careful morphological description of the changes that small lymphocytes undergo in culture in the presence of PHA. On the third day, small lymphocytes cultured without PHA remain relatively unchanged, whereas with PHA, large numbers (70%)of the cells have assumed the appearance of blastoid cells. They enlarge and exhibit an agranular cytoplasm which is markedly basophilic and stains intensely with pyronin (Marshall and Roberts, 1963). The cytoplasm, which sometimes contains vacuoles, surrounds a large round nucleus with prominent nucleoli. Intermediate forms are found. Usually, the transformed cells occur in clusters (Robbins, 1964), cells on the fringe of which are actively ameboid (Cooper, 1962). Mitotic figures become increasingly prevalent during and after the third day. Electron microscopy has shown that on the third day PHA-transformed cells possess a prominent Golgi complex, ribosomal particles, and some endoplasmic reticulum ( Marshall and Roberts, 1963; Elves et al., 1964)-features typical of cells actively engaged in protein synthesis and capable of mitosis ( Bernhard and Granboulan, 1960). Studies with radioactive nucleic acid precursors have shown that RNA synthesis begins soon after the cultures are initiated and that DNA synthesis occurs during and after the second day of culture (Cooper, 1962; Byron and Lajtha, 1962). Recently, it has been demonstrated that responses of smaller magnitudes occur with cultures of purified lymphocytes rather than with culture3 containing a similar number of lymphocytes but with some contaminating granulocytes in addition (Wilson, 1966). Fluorescein-tagged PHA can be seen localized within the cytoplasm of both lymphocytes and granulocytes within 15 minutes after initiating the cultures (Razavi, 1966) and the earIiest effects on cell metabolism occur within a half hour. At this time, there is a gross degradation of lymphocyte RNA (Cooper and Rubin, 1965b), possibly a imsequence of liberating enzymes from lysosomal vacuoles prssent in
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PHA-stimulated lymphocytes (R. Hirschhorn et al., 1965). As RNA is degraded, there appears to be a simultaneous synthesis of new, probably messenger, RNA which may then regulate transition of the lymphocyte from a resting to a proliferative state (Rubin and Cooper, 1965; Cooper and Rubin, 1966). Cooper and Rubin ( 1966) have postulated that nonspecific mitogenic agents act at the biochemical level by nonspecifically derepressing lymphocyte RNA synthesis. They point out that much of the RNA synthesized in the response to PHA does not seem to be required for proliferative activities. For example, lymphocytes stimulated with RNA produce interferon ( Wheelock, 1956) ; and lymph node fragments from animals previously immunized to BSA produce specific antibodies when stimulated by PHA in vitro (Tao, 1964). 2. Pokeweed Mitogen
Extracts of the plant Phljtolncca americana (pokeweed) are also capable of inducing transformation of human lymphocytes in vitro (Farnes et al., 1964; Borjeson et al., 1966). Typical plasma cells, as well as large immature pyroninophilic lymphocytes, were observed in the peripheral blood of individuals who had accidentally been injected with pokeweed mitogen (PWM) (Barker et al., 1965). From the magnitude and kinetics of the response of lymphocytes cultured with this agent, it is clear that it can be classed as a nonspecific mitogen. Progressive changes in cellular morphology have been described by Chessin et al. (1966) and are first detectable after 16 hours. At the peak of transformation, which occurs at approximately 66 to 78 hours, approximately 50-60% of all the cells present are of the transformed type-the rest being typical small lymphocytes. Of the transformed cells, approximately 60% are indistinguishable from those found in PHA cultures and have been referred to as PWM Type I blast cells. The remainder, Type I1 intermediate cells, exhibit some of the morphological characteristics of plasma cells and have not been obFerved in PHA-transformed cultures. Histochemical procedures have been used to distinguish between these cells; Type I cells stain positively cvth Alcian blue (Chessin et al., 1966). Another distinguishing feature of cells exposed in culture to PWM is that, unlike PHA-treated cells, the sucrose density gradient sedimentation pattern for their newly synthesized RNA is typical of cells in the logarithmic phase of growth. A large proportion of the newly synthesized RNA appears to be ribosomal, like that which is present in antigenstimulated cells, rather than the nonribosomal variety found in PHAtransformed cells (Chessin et nl., 1966).
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DARCY B. WILSON AND R. E. BILLINGHAM
3. Antileukocyte Sera
Heterologous, rabbit, antihuman, leukocyte sera also induce transformation and mitosis of cultures of human leukocytes (Grasbeck et al., 1963, 1964; Elrod and Schrek, 1965). The timing as well as the magnitude of the mitogenic responses to this agent is similar in all respects to that which results from the use of PHA but not to specific antigens or homologous leukocytes. Grasbeck‘s group (1964) showed that the mitogenic effect of specific antileukocyte sera is not dependent on complement activity and, whereas absorption of the sera with erythrocytes resulted in no significant decrease in mitogenic activity, similar pretreatment with leukocyte concentrates did. These authors suggested that PHA and specific antileukocyte sera attach to some structure on the cell wall, triggering an automatic chain reaction which culminates in mitosis. A similar mechanism may underlie, or at least contribute to, the phenomenon of enhancement in tumor immunology, i.e., the increased growth and survival of homologous tumors occurring in alien hosts which have been actively or passively immunized so as to have appreciable amounts of circulating antibodies directed against transplantation antigens of the tumor ( Kaliss, 1958). One of the most provocative properties of heterologous antilymphocyte serum, which has recently been studied in some detail by Levey and Medawar (1966), is its powerful immunosuppressant effect. When administered in relatively small dosages to mice and other experimental animals, it has been shown to prolong the survival of skin homografts, sometimes indefinitely. Its capacity to reverse an already existing state of homograft sensitivity is further proof of the uniqueness of this suppressant. Although there is very little information as to its mode of action, it does not seem to depmd on a cytotoxic process since prolongation of the lives of skin homografts results in test animals without an attendant leukopenia. Levey and Medawar (1966) have postulated that antileukocyte sera may act by a process of “sterile activation,” i.e., that lymphocytes are diverted from their normal immunological pathways. Whether the mitogenic properties of antileukocyte serum contributes to its immunosuppressive activity has yet to be established. Some support for this possibility comes from Elves’ (1967) demonstration that PHA administered intraperitoneally in rats prior to their injection with sensitizing doses of chicken erythrocytes completely inhibits the appearance of hemagglutinating and hemolytic antibodies against that antigen.
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4 . Antiatlotype Sera
In a very interesting study, Sell and Gell (1965) employed isoimniune sera directed against the genetically determined antigenic differences in the 7 S globulins (1gG)-these differences termed allotypes-in the rabbit (see Dray et al., 1962). They demonstrated that blast cell transformation could be induced in rabbit peripheral blood lymphocytes cultured in the presence of specific isoimmune antiallotype antisera and that: this transformation was not dependent on the presence of complement. Furthermore, it only occurred with cells cultured in the presence of antiallotype serum specific for their donor. Heterologous antirabbit ,-globulin sera raised in goats, guinea pigs, and sheep were found to be as capable of stimulating blast cell transformation as isoimmune antiallotype sera. The effectiveness of antiallotype sera as mitogenic agents is comparable to that of antileukocyte sera and the other nonspecific mitogens, including PHA. It may be noted that as little as 15 minutes’ contact with antiallotype sera is all that is needed to stimulate rabbit leukocytes to undergo significant degrees of transformation and DNA synthesis when subsequently cultured for 48 hours in normal rabbit serum. Prior incubation of the cells with immune sera for 45 minutes initiated as much subsequent transformation in the cultures as contact with immune sera for the entire 48 hours. In this respect, the action of antiallotype sera is very similar to that of PHA (Newsome, 1963). Sell and Gell suggest that it is the small lymphocyte which responds and they contend that antiallotype antisera binds with antigenic determinants, having specificities of globulin allotypes, located on or in these cells. One interesting difference between the effect of PHA and antihuman leukocyte sera, on the one hand, and aytiallotype sera or Staphylococcus filtrate (Ling et al., 1965), on the other, is that the mitogenic capacity of the former cannot be separated from its Ieukoagglutinating property. However, since antiallotype serum is as potent a mitogen and brings about the transformation of cultured cells with the same promptitude as the leukoagglutinating mitogen, it may be concluded that leukoagglutination per se is not mandatory for blast cell transformation.
B. SPECIFICMITOGENS : SECONDARY OR
“RECALL”
RESPONSE
A superficially similar blastogenic transformation of lymphocytes from the blood of tuberculin-sensitive human donors occurs if they are cultured in the presence of PPD of tuberculin. This response has been shown to be of comparatively smaller magnitude .( 10-30%blast cells) and
244
DARCY B. WILSON AND R. E. BILLINGHAM
is somewhat more delayed in onset than the response to nonspecific mitogens (Ling and Husband, 1964). Since it can only be produced with lymphocytes from tuberculin-positive donors, it was suggested that this might be a recognition reaction in uitro of antigens previously encountered in uiuo. To this extent, the reaction may resemble that studied by Dutton and his co-workers (Dutton and Eady, 1964; Dutton and Pearce, 1962). These investigators showed that inclusion of specific antigen in cultures of splenic or lymph node cells from previously immunized rabbits resulted in the incorporation of radioactive thymidine into responding cells-an indication of increased proliferation. They found this phenomenon to be antigen specific and dose dependent. The specificity of the response suggested that it had an immunological basis, perhaps an in uitro counterpart of the proliferative activity seen in the secondary response in uiuo. Morphological and radioautographic studies of the responding cell suspensions showed that labeled thymidine was incorporated into large transformed cells which comprised 14%of the total population after 48 hours of culture. The antigens employed were albumin and globulin fractions of various animal species. When cultured in the presence of these antigens, cells from unimmunized animals did not respond. Mills (1966) showed that lymphocytes from guinea pigs sensitized to tuberculin, or other protein antigens incorporated in Freund's adjuvant and administered subcutaneously, underwent a typical blastogenic transformation when cultured in the presence of the sensitizing antigen. Furthermore, the magnitude of the response was related to the degree of hypersensitivity in the donor animals. The interesting observation was also made that cells from animals immunized intiavenously with large doses of antigen, sufficient to incite appreciable amounts of circulating antibody, did not respond in culture with the specific antigen. This finding, like that of Bloom and Bennett (1966), suggests that there are basic differences in the behavior of sensitized lymphocytes according to the type of hypersensitivity induced in the donor-delayed or immediate. Also, lymphocytes from animals immunized with hapten-protein complexes responded to the presence of immunizing conjugates in uitro but not to the same hapten complexed to a different carrier. Mills argues that these findings lend great strength to the thesis that lymphocyte transformation in culture is a manifestation of delayed-type hypersensitivity. With a different species (the rabbit) and a different route of antigen administration (intravenous), Chapman et al. (1964) demonstrated that when lymphocytes from popliteal nodes of immunized animals were cultured in the presence of the specific antigen they incorporated
LYMPHOCYTES AND TRANSPLANTATION IMMUNITY
245
thymidine into DNA. Why these findings were markedly different from Mills’s (discussed above) has yet to be resolved. There are numerous criteria according to which the transformation of lymphocytes induced by nonspecific mitogens (especially PHA) can be distinguished from the antigen-induced or specific response of lymphocytes in culture: ( I ) The specific mitogens are apparently all good antigens (Ling and Husband, 1964); they include a wide variety of fungal, bacterial, and viral products as well as certain classic protein and nonprotein allergens. ( 2 ) The magnitude of the response is considerably smaller ( 10-302 blast cells). ( 3 ) The onset seems to be delayed -up to 5 days. Finally, ( 4 ) there is some evidence to suggest that the nature of the response itself is different. Thus it has been reported that, whereas PHA induces the synthesis of messenger-like RNA (Cooper and Rubin, 1966) in antigen-stimulated cultures (but also in PWM-stimulated cultures), ribosomal RNA appears to be the type of RNA synthesized ( Chessin et al., 1966; Cooper and Rubin, 1965a). There is, however, one notable similarity in these two types of responses which, probably significant, is as yet only poorly understood. Normal, untransformed human lymphocytes contain three or four discrete lysosomal bodies occupying a perinuclear position and staining positively for acid phosphatase. Shortly after exposure to PHA or to PPD (with cells from tuberculin-sensitive donors), an increase in both the size and numbers of these lysosomal bodies occurs (Allison and Malucci, 1964; R. Hirschhorn et al., 1965). Since these changes are seen to precede blast cell transformation and mitosis, the possibility has been raised that mitogens cause a destabilization of the lysosomal membranes and the consequent release of some substance triggers cell division either directly or by inactivation of a repressor. Further evidence sustaining this concept comes from the observation that chloroquine (Hurvitz and Hirschhorn, 1965) or prednisolone ( Nowell, 1961 )-substances with a demonstrated capacity to stabilize lysosomal membranes against induced permeability changes-inhibit transformation ( Hurvitz and Hirschhorn, 1965; Elves ct a!., 1963). Diengdoh and Turk ( 1965) have recently presented evidence attributing an immunological significance to lysosomes. They showed that, associated with the massive proliferation of small lymphocytes that accompanies the development of delayed hypersensitivity to contact allergens in guinea pigs, there are significant lysosomal changes in cells of the draining node, peripheral blood, and peritoneal exudate. Whereas in normal animals about 10%of the node cells contain 3 lysosomal granules per cell, in sensitized animals 80%of the regional node cells
246
DARCY B. WILSON AND R. E. BILLINGHAM
contain twice the normal number of lysosomal granules within 2 days after exposure to antigen. Whereas 35%of the lymphocytes of the peritoneal exudates of normal animals contain an average of 3.5 granules per cell, 3-4 months after immunization with dead tubercle bacilli in waterin-oil emulsion, more than 90%of these cells contain 10 or more of these granules per cell. It seems possible, therefore, that lysosomes are involved with and may even initiate cell division under certain circumstances. Whether lysosomal changes have any specificity in an immunological sense is not certain; they might simply be a manifestation of cells about to undergo division. The contents of lysosomes are likely candidates as mediators of various lesions evokable in animals in a state of delayed hypersensitivity. However, there is no evidence that the mode of action of these substances is other than nonspecific.
C. SPECIFICMITOGENS:PRIMARY RESPONSE AND MIXED LEUKOCYTE REACTIONS Recent studies indicate that the addition of certain antigens to cultures of leukocytes derived from normal or unsensitized individuals is attended by an even later (5-8 days) and smaller (5-1Mblasts) response than with cells from sensitized donors. Johnson and Russell (1965) and Wilson (1966) demonstrated a stimulatory effect of foreign protein on human lymphocyte cultures. Whether or not this holds true for all antigens, including the specific mitogens, has yet to be determined; also, it would be useful to determine if the effective stimulatory dose of these mitogens is dependent upon the immunological status of the lymphocyte donors. Some of the most recent and interesting findings concerning antigeninduced lymphocyte proliferation in uitro have emerged from studies on the mixed leukocyte reaction. Bain and her colleagues (1963, 1964) demonstrated that when peripheral blood leukocytes from two unrelated human beings were mixed and cultured for a number of days up to 5% of the cells underwent morphological transformation to blastoid forms and incorporated radioactive thymidine. Not more than 1%of lymphocytes in control cultures of unmixed cells responded. This finding was readily confirmed by others and extended to animal cell culture systems. Dutton and his colleagues reported that mixtures of splenic or lymph node lymphocytes from outbred rabbits (Chapman and Dutton, 1965) or from different inbred strains of rats and mice (Dutton, 1966b) also manifested similar proliferative responses in short-term cultures (for review see Dutton, 1966a).
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247
Bain and her co-workers (1964) made the critical observations that mixed cultures of cells from monozygotic human twins did not undergo transformation, only slight responses occurred with cells from closely related donors, and maximal responses resulted when unrelated donors were employed. Plasma or erythrocytes from one donor did not stimulate transformation of cells from another. Frozen-thawed preparations ( Bach and Hirschhorn, 1964), extracts (Hashem and Rosen, 1964), and media in which cells from one of the donors had been cultured (Kasakura and Lowenstein 1965; Gordon and MacLean, 1965) also had an effect on the lymphocytes from another donor. The failure of cell mixtures from monozygotic twins to respond in culture suggested that the proliferation seen with cell mixtures from unrelated donors was related to their immunogenetic disparity and, therefore, that the mixed leukocyte reaction might form the basis of a useful histocompatibility matching procedure. To this end, numerous laboratories have been engaged in studies designed to compare the degree of response obtained in mixed leukocyte cultures with the rapidity with which test-skin homografts are destroyed when exchanged between the cell donors (Hirschhorn et al., 1963). It was claimed by Moynihan et al. (1965) on the basis of studies with cells from squirrel monkeys and by Bain et al. (1965) and Oppenheim et al. (1965), with human material, that an enhanced proliferative response occurred if the lymphocyte donor had previously rejected a skin homograft. The validity of this conclusion, however, is open to some criticism since ( 1 ) cultures of normal or “sensitized cells from the same donor must of necessity be done at different times and, therefore, comparisons are risky and ( 2 ) except for Oppenheim’s study, no controls for specificity were provided, i.e., the reactivity of sensitized cells was not tested against those of an indifferent homologous donor. Oppeheim reported that cells from two individuals who had rejected skin grafts from the same donor exhibited a mutual interaction of greater magnitude in culture than the pregrafting control levels. This was attributed to immunogenetic overlap. In a very interesting study, Lewis et al. (1966) demonstrated that lymphocytes from pregnant and multiparous women cultured with their husbands’ cells gave diminished degrees of blast cell transformation but responded normally to cells of unrelated males. Their data also suggested that the reactivity of cells from parous female patients having neoplasms of trophoblastic origin were as reactive against their consorts’ cells as against cells from unrelated males. This finding, if substantiated, has important implications concerning the demonstrated proliferative abili-
248
DARCY B. WILSON AND R. E. BILLINGHAM
ties of mouse trophoblastic tissues, especially in an antigenically alien and immunologically adverse environment ( Kirby, 1963; James, 1965; Billington, 1965; for review see Billingham, 1964). 1. One-way Reactivity: Importance for Histocompatibility Matching
One of the problems associated with the use of live human cells in the mixed-lymphocyte culture system is the inherent mutual or two-way stimulation and response that occurs. This has not only made results of studies on the nature of the proliferative response difficult to interpret but has also greatly complicated attempts to use the mixed-lymphocyte interaction as a histocompatibility matching procedure. Bach and Voynow (1966) sought to bypass this difficulty by prior treatment of the cells from one of the donors with mitomycin C to prevent DNA replication. Although their data do suggest that one-way responses were obtainable by this artifice, there is some question whether drug treatment of one population of cells influences the degree of proliferation of the cocultured responding population. Another important consideration stems from Nowell’s demonstration (1964) that cells in the postsynthetic G? period appear to be resistant to mitomycin C. Also, this drug can act on the genetic apparatus of mitotically inactive human leukocytes without totally inhibiting their subsequent ability to synthesize DNA. The fact that gross chromosomal aberrations appeared in PHA-stimulated cultures of drug-treated lymphocytes indicates that some new DNA synthesis must have occurred. An important question is whether treatment of lymphocytes from one donor with mitomycin C influences the magnitude of the response of the cells from the other donor in the mixed-lymphocyte interaction. This cannot be answered with the use of human cells as culture material. Indeed, pretreatment of lymphocytes from F, rat donors-which are already unresponsive in culture to parental histocompatibility antigens for genetic reasons (see below)-with mitomycin C and then culturing them with cells of parental origin drastically reduces the latter’s proliferative response ( Wilson, 1967b). At this point, therefore, estimates of the immunogenetic incompatibility of various prospective donorrecipient combinations in man, made with the use of the mixedlymphocyte interaction and prior treatment of the donor’s cells with mitomycin C, can only be based on whether or not a proliferative response occurred in culture and not on its magnitude. This restriction can be relaxed as soon as means are forthcoming for rendering a stimulatory population of cells nonresponsive without any subsequent adverse influence on proliferation of the putative responding population,
LYMPHOCYTES AND TRANSPLANTATION IMMUNITY
249
Studies have recently been conducted by Silvers et al. (1967) and Wilson ( lQ67b) to determine the extent to which mixed-lymphocyte interactions in rats reflect the degree of immunogenetic disparity between the two donors. To do this, populations of genetically defined backcross animals were obtained by mating parental strain Lewis rats to (Lewis x DA)F, hybrids. Although the progeny of this mating all receive a complete set of Lewis histocompatibility genes, they vary with respect to the number of specific DA factors received from their hybrid parent. These animals served (1) as donors of cells to be mixed with parental Lewis cells in culture and ( 2 ) as donors of test skin homografts to Lewis hosts. The culture responses were compared with the survival times of the skin homografts and also with the genotype of each of the donors. Rats of the DA and Lewis strain differ at the important A g B histocompatibility locus-the Lewis being AgB' and the DA being AgB'. These loci determine the presence of antigenic specificities which are detectable on red cells by hemagglutination with specific isoimmune sera. The results (Table Ia) of this study showed that significant incorporation of radioactive thymidine occurred in cultures when the backcross and parental strain donors were incompatible at the AgB locus; this was correlated with a very prompt (8-11 days) rejection of test skin grafts by Lewis hosts. No appreciable in vitro reactions were incited when the backcross and parental strain donors were compatible at the A g B locus, and the skin grafts survived on Lewis hosts for varying periods of time-some being destroyed as promptly as skin from A g B incompatible donors. These results indicate that, with the conditions of culture employed, the proliferative response occurring in mixed-lymphocyte cultures is highly selective in distinguishing between two classes of donor-host combinations-even when they exhibit acute homograft rejections of similar intensity. The promptitude with which Lewis animals rejected backcross skin grafts arose from the presence of A g B histocompatibility antigens in some of the grafts and, in others, probably from the presence of a multiplicity of weaker histocompatibility factors acting synergistically (see Graff et al., 1966). It was very significant that, although these two classes of acute homograft rejections-the A g B incompatible donors and some of the A g B compatible group-were indistinguishable on the basis of the length of survival time of skin grafts, prior treatment of Lewis recipients with immunosuppressive drugs (Table I b ) resulted in a prolonged survival of skin from the A g B compatible group but not from the incompatible group. These results show that mixed-lymphocyte interactions may be very useful in excluding those donor-host combinations that are the least amenable to immunosuppressive therapy.
SELECTION OF IMMUNOSUPPRESSABLE
TABLE I DONOR/HOST COMBINATIONS
a. Correlation of AgB Genotype of Backcross Animals, with Capacity to Induce Proliferation of Parental Lewis Lymphocytes in Culture, and Survival Times of Skin Homografts on Lewis Recipients"
Backcross donor
Graft survival time (days)
AgB genotype
Culture response
1
8
114
+
5* 6b 2
8 8 11
111
3 6 80
21 29 50
B Y USE OF THE MIXED
LEUCOCYTE INTERACTION
b. Effect of Cyclophosphamide in Prolonging the Survival of AgB-Compatible and -Incompatible Grafts on the Same Host"
Survival times of backcross grafts
1/4 1/1 1/1 1/1 1/1
f -
Lewis recipients
A (control) ,
genotype
days
(1/4) :
3 X8,9
(1/1) :
2 X'8, 2 X 9
-,
-
-
€3
W4):
8, 4 X 9, 2 X 10, 3 X 11, x 12 9, 2 X 10, 12, 13, 3 X 14, 15, 16,d 2 X 18
2
(drug)'
(1/1) :
'* Data compiled from experiments of Silvers et al. ( 1967).
' These animals served as donors for skin grafts reported in b. "Cyclophosphamide: This is a pooled group of animals, half of which received a single injection (7.5 mg./100 gm. body weight) 4 days after grafting; the rest were injected three times (2.0 nig./100 gm. body weight) on days 4,6, and 8 after grafting. This animal died on the sixteenth day with a healthy, intact skin graft.
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2. The Mixed-Lymphocyte Interaction. as an lmmzinological Response in Vitro One very important property of the mixed-lymphocyte interaction is that it is a response on the part of leukocytes from previously unsensitized donors. Therefore, a prima facie case exists that the antigeninduced proliferative responses in this instance represent primary immunological reactions in vitro. However, as Dutton has recently stressed (1966a), in the classic model of the immunological response, there is a newly synthesized protein antibody which specifically interacts with the stimulating antigen. These same attributes of “adaptation” and “specificity” can be demonstrated of states of delayed hypersensitivity. That the proliferative responses associated with antigens and cells in culture, in particular the mixed leukocyte interaction, are truly primary immunological responses at the cellular level also requires the demonstration of adaptation, i.e., that, as the result of contact with antigen, a product is elaborated, and of specificity, i.e., that, whatever the product is, it should specifically interact with the inducing antigen. A summary of the available information concerning the immunological nature of the mixed-lymphocyte interaction and the circumstances under which it can occur is therefore pertinent to the subject matter of this review. Dutton and his co-workers were among the first to examine the proliferative responses in mixed cultures of lymphoid cells. They employed splenic or lymph node cells from outbred rabbits (Chapman and Dutton, 1965) and inbred strains of rats and mice (Dutton, 1965, 1966b) and showed that the magnitude and kinetics of the proliferative response closely resembled those obtained with cells from hyperimmunized animals confronted with heterologous antigens in culture ( Dutton and Eady, 1964; Dutton and Pearce, 1962). With mixed cultures of lymphoid cells, up to 5% of the cells incorporated thymidine within 48 hours and neither homologous erythrocytes nor isologous tissue cells were stimulatory. Although thymus cells themselves would not respond, they did stimulate proliferation of homologous lymphoid cells (Chapman and Dutton, 1965). Studies with mixed splenic cell cultures obtained from different inbred strains of mice did not establish any marked correlation between the magnitude of the response and the strain combinations employed. However, those combinations which involved incompatibilities at the H-2 locus gave more intense reactions (Dutton, 1965). When spleen cells were mixed and cultured from various congenic pairs of donors,
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9 out of 12 of the pairs differing at the H-2 locus gave positive responses (Dutton, 1966b). Since histocompatibility factors are determined by codominant genes, cells of an F, animal should possess, in theory, all the isoantigenic determinants of the two parental strains; they should, therefore, be incapable of reacting against parental strain cells. Whether or not F, cells can respond to some other agent-and not necessarily to antigen-is important since it serves to establish the specificity and, therefore, the immunological significance and basis of the culture reaction. Dutton (1965) has carried out studies with mixed cultures of splenic cells from isogenic strains of mice and rats and their F, hybrids. He found that the magnitude of parent-F, reactions was always less than that of the homologous parent-parent responses and that the arithmetic sum of the responses obtained with F, cells cultured with the two parental strain cells separately approximated the parental-parental reaction. These findings were taken as evidence that only the parental strain cells, and not those from F, animals, were responding in mixed cultures of parental and F, hybrid cells. Since similar hybrid cells responded vigorously to unrelated-third-party strain cells, it was felt that the smaller magnitude of the parental-F, reactions could not be attributed to any defect or inability of F, cells to proliferate when appropriately stimulated. However, these findings are not conclusive; more direct evidence of the unresponsiveness of F, hybrid cells against parental-strain histocompatibility factors is essential. Some recent experiments conducted in this laboratory (Wilson, 1967b; Wilson et al., 1967) have a direct bearing on the premise that the mixed-lymphocyte interaction represents a primary immunological response at the cellular level. Despite a basic similarity in the test systems employed with that of Dutton (1965), there are a few important differences in the results obtained. Therefore, an account of our test system and findings is presented. The response of mixtures of blood leukocytes, lymph node cells, and splenic cells from donor pairs from several different inbred strains of rats was measured by the uptake of tritiated thymidine into DNA. Each mixed culture contained one million lymphoid cells from each of the unrelated donors in a total volume of 1 ml. of culture medium containing 15-20% fresh rat serum. All cultures were conducted in triplicate. The results of a typical experiment, presented in Fig. 1, show that the peak response occurs at about 6 to 7 days. Although on the second and third days there is a slightly increased uptake of thymidine in the mixed cultures (especially with splenic cells) over control levels, this is totally overshadowed by the response which occurs later. These data
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may reflect the proliferative activities of two different populations of cells -one early and one late. It is important to note that uptake af thymidine in control cultures was very low at all times in both leukocyte and lymph node cell cultures. After 4 days, however, the background levels in the splenic cell cultures were disturbingly high. In these experiments, the response patterns with the mixed peripheral blood lymphocytes tended to parallel those obtainable with mixed cultures of human cells.
L N Cells
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FIG. 1. Proliferative responses, measured in terms of tritiated thymidine incorporation, of mixed cultures of lymphoid cells obtained from the peripheral blood, lymph nodes, or spleens of Lewb and BN rats.
The reason for the discrepancy between these and Dutton’s (1965) results is not clear; it may relate to the fact that our own studies were conducted with much lower concentrations of cells of different origin than Dutton used. Certainly conclusions based upon thymidine uptake at 48 hours-perhaps reflecting the activities of the “early-responding cells”-can be expected to differ from those based on DNA synthesis from the fourth day onward. The results of studies with cells from parental-strain and F, hybrid rats also differ somewhat from those reported by Dutton (1965). As mentioned above, he found that parental-F, responses were always smaller in magnitude than parental-parental reactions, whereas, in the present culture experiments, this was not necessarily true. Thus, Fig. 2
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shows the results of parallel cultures of cells from DA and Lewis rats and from either of these parental strains with their F, hybrid. Although at certain times the Lewis-F, reactions, in terms of thymidine incorporation, are less than the DA-Lewis reactions, the DA-F, reactions were initially similar to-and subsequently became more intense than-the DA-Lewis reactions. Moreover, the pattern of parental-F, and parental-
.
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FIG. 2. Proliferative responses in mixed cultures of peripheral lyiiiphocytes from DA and Lewis rats and in mixed cultures of porental (DA or Lewis) and hylit-id (DA/Lewis F,) cells.
parental responses, although consistent for any specific strain combination, varied according to the combination used. One possible explanation for these results is that some immunospecific product elaborated by the proliferating cells curtailed further DNA synthesis in the cultures. The initial reactivity of the DA-Lewis mixtures (shown in Fig. 2 ) might be ascribed to the DA cells, since these respond with similar kinetics in parallel DA-F, cultures. After a certain time, however, the reactivity of Lewis cells intervenes, as shown by the Lewis-F, cultures, and it is pre-
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sumed that, as a result of the mutual proliferative stimulation, deleterious products-perhaps with an immunological specificity-are elaborated which inhibit continued DNA synthesis and proliferation of the “target” lymphocytes. An explanation of the difference between these and Dutton’s results with parental-F, mixed cell cultures is not immediately obvious but, again, may be related to the activities of basically different cell populations. Further studies were conducted with P-F, cultures, using donors of different sexes. This made it possible to determine the donor of the responding cells from an examination of the chromosomes of mitotic figures of cultures treated with colchicine. The results of this experiment were 1m-y clear-cut. From fifth to eighth day cultures, which exhibited from 20 to 60 mitotic figures per 1000 cells, 150 random mitotic spreads were examined and the sex determined using procedures described by Hungerford and Nowell (1963). In all these figures, the dividing cell was identified to be of the parental type. This finding constitutes direct and rather strong evidence that the P-F, responses in culture are unidirectional, i.e., that F, cells do not respond to parental antigens even in an environment of proliferating cells, and this establishes the immunological specificity of transformation and DNA synthesis occurring in a mixed-lymphocyte interaction. This being the case, it is difficult to give serious consideration to any premise that ascribes the proliferative response of the mixed-lymphocyte interaction to some nonimmunological “contact” phenomenon as, for example, syngeneic preference (Hellstrom and Moller, 1965). That some process based on mutual contact of cells with structurally different surface patterns is not involved in the mixed-lymphocyte interaction is further indicated from the results of studies (Wilson et al., 1967) with cells obtained from mutually tolerant animals (see Fig. 3 ) . Mixed cultures of leukocytes from Lewis rats rendered immunologically tolerant of BN transplantation antigens (by inoculation at birth with homologous bone marrow cells) and from BN animals tolerant of Lewis antigens showed no responses, whereas the usual amounts of thymidine incorporation occurred when cells from Lewis rats tolerant of BN antigens were mixed with cells from indifferent (Lewis x DA)F, hybrid donors. Also, cells from tolerant parental animals (Lewis tolerant of BN) did not respond in the presence of cells from (Lewis x BN)F, animals. Preliminary findings of Chapman and Dutton (1965) indicated that the reactivity of their cells in mixed spleen cell cultures could not be enhanced by prior sensitization of the donors. Our own studies (Wilson et al., 1967) bear this out (Fig. 4 ) . In fact, when sensitized cells were
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placed in mixed cultures, very slightly increased amounts of thymidine incorporation occurred during the first 2 to 3 days. The response for the next 2 days was similar to that obtained with normal cells, but then it was curtailed rather abruptly while the normal cells continued to proliferate.
DAYS
FIG. 3. Proliferative responses in mixed cultures of peripheral lymphocytes from ( a ) mutually tolerant rat donors (Lewis tolerant of BN BN tolerant of Lewis), ( b ) tolerant donors and their specific F, hybrid (Lewis tolerant of BN LewidBN F1), and ( c ) tolerant donors and an indifferent K hybrid (Lewis tolerant of BN Lewis/DA R).
+
+
+
One explanation for this finding is that the immunological activity of the sensitized lymphocytes during the first few days of culture may destroy the stimulating cells. While the magnitudes of the proliferative responses studied by various workers both in animal (Dutton, 1965; Moynihan et al., 1965; Wilson, 1967b) and in human cell culture systems (Wilson, 1966; Oppenheim et al., 1965; Bain et al., 1964) are similar, the times required for their onset differ markedly. Thus, responses are not usually detectable in mixed human blood lymphocyte cultures until after the fourth day-
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attaining their maxima on the sixth or seventh day. In Dutton’s experiments with splenic or lymph node cell mixtures of rabbits, rats, or mice, peak activity was measured before the end of the second day. Notwithstanding the observations of Chapman and Dutton (1965) of the unresponsiveness of mixed thymus cell cultures, Schwarz (1966) showed that genetically dissimilar rat thymic cells mixed in culture would proliferate
- L t X-x
L/BN F,
L(An9iBN)
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FIG. 4. Proliferative responses of peripheral lymphocytes from normal or previorisly sensitized Lewis donors cultured with Lewis/BN F, lymphocytes.
-being detectable at 3 days of culture. The transformation of leukocytes from sqnirrel monkeys was assessed after 72 hours of culture (Moynihan et al., 1965), and Rieke (1966) showed that cells obtained from the thoracic duct of inbred BN and Lewis rats exhibited some response in mixed cultures on the third day but more on the fourth. These data were based an determinations of percentage blast cell transformation, and the observations were not extended beyond 4 days. It is difficult to account for these differences, and a general explanation must take into account the following variables: ( I ) Along with
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obvious differences in the duration of culture, there are basic differences in the collation of the data. Thus some workers quantitate the response in terms of blast cell transformation, others in terms of mitotic figures, and still others give values for the incorporation of radioactive precursors into newly synthesized DNA, ( 2 ) The source, condition, and amount of serum proteins used to supplement the culture medium, i.e., whether of autologous (or isologous ) , homologous, or heterologous origin; whether serum or plasma is used; and whether it is employed fresh 01 stored can all be decisive factors (Johnson and Russell, 1965; Wilson, 1966). ( 3 ) The source, number, and means of preparation of the cells committed to culture. ( 4 ) Their degree of purity-whether other cells such as macrophages or granulocytes are present-is important. ( 5 ) The type of culture vessels (Bain and Lowenstein, 1965) and culture media used are also variables that may have some influence on the degree of response as well as the time required for its onset. All the available evidence strongly favors the belief that the mixedlymphocyte interaction has an immunological basis. That F, cells are capable of distinguishing between cells bearing parental antigens and those having nonparental histocompatibility factors, and that cells from tolerant animals accurately reflect the specific immunological debilitation of their donors constitute irrefutable evidence that some kind of immunologically specific recognition process is involved in initiating the proliferative phase of the mixed-lymphocyte interaction in culture. That the proliferative phase itself is also immunologically specific is suggested by the finding that, even in the midst of an environment of proliferating cells, F, cells themselves are not coerced into proliferation. Comparison of the behavior of lymphocytes in culture and the graftversus-host reaction in vivo reveals a marked parallel worthy of attention. Thus the absence of a proliferative response with attendant morphological transformation by cells from immunologically tolerant donors cultured in the presence of appropriate F, hybrid cells bearing specific transplantation isoantigens and the nonreactivity of F, cells themselves against parental lymphocytes, may be compared with the failure of small lymphocytes from tolerant parental donors to transform into large pyroninophilic cells in the spleens of F, hosts (see Gowans and McGregor, 1965). Furthermore, cells from tolerant parental donors, bearing skin homografts of long standing as testimony of their tolerant status, are completely incapable of producing the slightest symptoms of runt disease in F, hosts even when inoculated in dosages several times greater than those normally lethal (Gowans et al., 1963). Gowans and McGregor (1965) have argued cogently that this demon-
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strable specific nonreactivity of lymphocytes from tolerant donors in uiuo constitutes strong evidence that the cellular processes underlying the primary immunological response to transplantation antigens involves transformation of small lymphocytes into large pyroninophilic cells. Further strong support for this argument is provided by the results presented above concerning the proliferative response-or its absence --under certain telling circumstances. Although the responding population of cells in the mixed-lymphocyte interaction may be small in comparison to that which responds to nonspecific mitogens, it is surprisingly large in terms of the number of cells which are demonstrably committed immunologically in other assay systems, for example, those producing hemolytic antibody in the Jerne plaque system (Jerne et nl., 1963) or those releasing hemagglutinating antibody in the Zaalberg cluster test (Zaalberg et al., 1966). In this respect, the proliferative response observed in culture closely parallels the marked hypertrophy which occurs in the regional node draining the site of antigen adminktration. Of the cells which do respond in cultwe, it has yet to be determined how large a proportion are immunological effectors or progenitors of effector cells. In this respect, the recent experiments of Mishell and Dutton (1966) are pertinent. They included heterologous red cells in cultures of spleen cells from normal mice and observed that, after 3 to 4 days, cells were present that were capable of producing hemolytic plaques with the Jerne test system. This reaction was shown to be specific for the “immunizing” erythrocytes and quantitatively comparable to the numbers of cells recoverable from the spleens of animals immunized by the conventional intravenous route. The importance of this finding need hardly be stressed since it establishes that the proliferative response of lymphocytes in cultures, although initiated with an immunological specificity, is not without an immunological purpose, i.e., that antigen-induced proliferation of lymphocytes in culture may produce at least some cells which are immunological effectors. One important corollary of the premise that the mixed-lymphocyte interaction represents a primary immunological response at the cellular level is that, as an immune response, it is initiated and developed by cells outside the architectural framework of a regional lymph node. A natural extension of this argument is that, when confronted with a transplantation antigen, cells of the lymphoid series are capable of initiating an immune response and executing it in situ. This implies that competent lymphoid cells can be committed to the status of immunological effector
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cells without undergoing a period of residence within a draining lymph node for the purpose of proliferation or maturation. For the development of a systemic immunity demonstrable upon subsequent challenge with a specific antigen, however, involvement of lymphatic tissue may be obligatory. In this instance, sensitized cells derived from the draining lymph node may not only augment the destructive activities of cells that have already infiltrated the parenchyma of a homograft and become activated in situ hut, by “seeding” other lymphoid tissues, would be responsible for the immunological “memory” of the organism. VIII.
Cooperative Interaction of Lymphocytes and Macrophages
The idea that some cooperative interaction between macrophages and lymphocytes is a requisite step in the induction of immune responses has been sustained from many quarters. Gallily and Feldman (1967) have shown that “primed” peritoneal macrophages, i.e., cells incubated for a short time in vitro with Shigella antigen, transferred to sublethally irradiated isologous mice could trigger the formation of specific agglutinating antibody. Transfer of primed lymphoid cells, unprimed macrophages, or primed macrophages from irradiated donors or antigen alone was ineffective. It was concluded that immunologica1 suppression following sublethal doses of irradiation results, in part, from the impaired capacity of macrophages to “process” antigen. This irradiation-induced debilitation of macrophage function probably does not involve their phagocytic capabilities. Numerous investigators have shown that the capacity of macrophages to engulf colloidal gold or carbon, homologous or heterologous red cells, or bacteria is not decreased following irradiation; in fact, it may he somewhat increased (Brecher et al., 1948; Wish et al., 1952). Although the initial clearance of bacteria from the blood stream is as rapid in irradiated as unirradiated animals, in the former the bacteria are not destroyed hut are released following a short period of intracellular residence (Gordon et d.,1955). These findings strongly suggest that irradiation may severely impair important intracellular degradative processes performed by macrophages as well as have direct effects on lymphoid cells. In line with these studies, Bussards results (1966) indicate that de novo synthesis of antisheep hemolytic antibody can he procured in vitro by previously unsensitized mouse lymphoid cells only in the presence of peritoneal cells. Presumably these peritoneal cells were macrophages, although it is essential that the cell type involved be specifically identified. Also, with cdture techniques designed to facilitate the interaction between macrophages and lymphocytes, Globerson and Auerbach
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( 1966) have demonstrated the production of specific antibodies by splenic fragments in organ culture. Further support for the idea that lymphoid cells and macrophages interact during early stages of the immune response has come from Sharp and Burwell’s studies (1960) on the structure of lymph nodes. They noted the adhesion of lymphocytes to the surface of macrophages isolated from a regionally stimulated node but not from the contralateral node. From electron microscopic studies, Schoenberg et al. (1964) have described cytoplasmic bridges between lymphocytes and macrophages in immunized nodes. Time-lapse cinephotomicrography by McFarlands group ( McFarland and Heilmann, 1965; McFarland et al., 1966) of cultures of purified, human, peripheral blood lymphocytes revealed a sustained contact via the “foot appendage,” or “uropod,” of lymphocytes to other cells or to cell debris. This attachment to other cells was most striking in mixed-lymphocyte cultures. Lymphocytes gathered in the vicinity of macrophages and exhibited the characteristic behavior of “peri-” and “emperipolesis.” After 2 days of culture, clumps comprised of several lymphocytes radially attached to a single macrophage by their uropods were apparent and, by the third day, several of the attached lymphoid cells began to enlarge and assume the appearance of blastoid or transformed cells. Lymphocytes surrounding the transformed cells also began to enlarge on the fourth day. On the fifth and sixth days, some transformed cells were seen to detach from the macrophages and wander about with small lymphoid cells promptly attaching to their foot processes. These authors suggest that the uropod may be a specialized organelle for attachment to other cells for the specific purpose of transfer of subcellular material. They described threadlike extensions from the foot appendage, similar to the cytoplasmic bridges demonstrated by Schoenberg et al. ( 1964), which formed flexible connections between the lymphocyte and other cells. The foot process itself contained numerous granules; however, it can only be speculated as to what their purpose might be. Some idea of what might transpire during the cooperative interaction and contact between lymphocytes and macrophages has been put on an experimental basis by studies initiated by Fishman (1961) and Fishman and Adler (1963, 1964). They showed that an RNase-sensitive material, extractable from macrophages, which had been incubated with T2 bacteriophage, when added to rat lymph node cells, either in culture or in diffusion chambers placed intraperitoneally in X-irradiated rats, provoked the synthesis of specific antiphage antibodies. They considered this substance to be active RNA of relatively low molecular weight-as
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judged by gradient centrifugation (Fishman and Adler, 1963, 1964). It was readily incorporated into lymph node cells in culture (Fishman et al., 1963), but they did not detect any contaminating T2 antigens in their RNA preparations. Following these reports, Mannick and Egdahl (1962, 1964) showed that otherwise normal lymphoid cells could be converted to an immunologically active status with respect to transplantation isoantigens by incubation with crude RNA preparations from lymphoid cells of specifically sensitized animals. These RNA-treated cells had the capacity to cause skin lesions typical of the transfer reaction in rabbits; and, when inoculated back into donor rabbits, these animals rejected test skin homografts in the manner of a second-set reaction (see Section IV,D). With an in vitro test system, Wilson and Wecker (1966) have shown that, when lymphoid cells were treated with preparations of RNA from specifically immunized isologous rats, they developed a limited, but immunologically specific, capacity to destroy homologous target cells in culture. In this study, thri RNA could be obtained from either peritoneal exudate cells or from lymph node cells and was sensitive to RNase but not to DNase. Using essentially the same lymphocyte-target cell system, Geruhty et al. (1966) recently demonstrated that a state of sensitivity could be transferred to normal lymphoid cells with ribosome preparations from sensitized splenic cells. Treated lymphocytes were capable of destroying homologous L cells in culture. Cohen and Parks (1964) reported that splenic cells from normal mice, after incubation with antisheep erythrocyte immune RNA, produced hemolytic antibody detectable with Jerne plaque assay procedures. These findings were independently confirmed by Friedman ( 1964b), who also showed that subsequent culture of the RNA-treated cells for a few days brought about an increase in the ratio of plaque-forming cells to viable cells. Using this Jerne plaque assay procedure, Cohen (1967) has recently shown that 5-fluoro-2-deoxyuridine ( FUDR ) administered to mice with an immunizing dose of sheep erythrocytes results in significantly greater numbers of hemolysin-producing splenic cells than in mice receiving sheep erythrocytes alone. Also, a greater proportion of splenic cells from nonimmunized mice, which had received FUDR, were converted to antibody-forming cells by treatment with RNA obtained from immune cells than the same number of cells from normal mice not injected with FUDR. Interestingly enough, FUDR did not increase the number of cells spontaneously forming hemolysins. Cohen suggests that the action of
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FUDR in this system is that of an adjuvant and that administration of this agent increases the number of immunologically active lymphoid cells which can be derived from a very limited pool of primitive stem cells. From experiments in which RNA from specifically immunized animals was subjected to sucrose density gradient centrifugation, Cohen’s group (see Cohen, 1967) showed that the RNA fraction which was active in converting normal cells into an immunologically active status ( hemolysin producers in the Jerne plaque assay) had a sedimentation coefficient of 8-12 S. Also, incubation of normal lymphocytes with minimally digested (with RNase) specifically immune RNA prior to treatment with undigested immune RNA inhibited their conversion to active cells. This reaction was shown to be specific since incubation of spIeen cells with digested or nondigested RNA from animals immunized with Escherichiu coli or diphtheria toxoid failed to inhibit the conversion of cells to form sheep-celI hemolysins by RNA from mice immunized with sheep erythrocytes. Cohen (1967) presents a reasonable argument that, whether the active RNA material is an RNA-antigen complex or a messenger RNA, there are specific binding or recognition sites on the surface of recipient lymphocytes. The observations of Fishman and his group, mentioned above, were confirmed and extended by Friedman et at. (1965 ). However, these investigators did detect several phage antigens in the RNA that were isolated from the phage-treated macrophages. The presence of these antigens was revealed by means of a sensitive microcomplement fixation technique and by an anamnestic response in the production of neutralizing antibody as the result of administering the RNA preparation to mice previously primed with T2 phage. Askonas and Rhodes (1965) likewise found that macrophages can degrade hemocyanin into RNAbound antigenic fragments which are immunogenic. Furthermore, their data suggest that macrophage RNA may act as an adjuvant and thereby promote the immunogenicity of the bound antigen. Therefore, although it is possible that lymphoid cells can be instructed or induced to synthesize antibody by a specific RNA, free of antigenic determinants, it appears more likely that an antigen-RNA complex may serve as a particularly effective stimulus for lymphoid cells. Contact with macrophages, as demonstrated by the studies of McFarland and Heilman (1965) and by Schoenberg et al. (1964), might provide a means by which instructive material or complexed antigenic determinants might be transferred to lymphocytes and initiate their transformation into blastoid cells. Furthermore, contact between transformed cells and other
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lymphoid cells, with transfer of stimulatory material, might maintain a positive immunological memory for an extended period of time without the persistence of unbound antigen in the tissues. McFarland and Heilman (1965) have postulated that this intimate contact between transformed cells and lymphocytes could represent an alternative method, in addition to mitotic division, for the development of islands or clones of antibody-forming cells in lymphoid tissues after primary antigenic stimulation. Assuming that some kind of interaction between macrophages and lymphocytes is mandatory for at least some immune responses, the question remains as to the cell type in which the induction of antibody synthesis can be said to occur. Gowans (1965) points out that if lymphoid cells are passive receptors of informational molecules of RNA then the processes of immunological recognition and induction of immune responses rest with macrophages. In this light, Nossal and Ada (1964) have shown that macrophages in the nodes of immunologically tolerant rats are not deficient in their ability to ingest specific antigen. However, even with an unimpaired capacity to recognize foreign antigen, macrophages of tolerant animals may still be deficient at a later metabolic stage crucial to the degradative processing of antigenic determinants. In any case, use of the Fishman-type transfer system with macrophages from tolerant animals might provide conclusive information on this point. Rather compelling evidence that the immunological lesion of the tolerant animal resides with the lymphocyte population comes from the demonstration that cell suspensions-consisting almost exclusively of small lymphocytes and devoid of macrophages-obtained from tolerant animals are incapable of causing GVH reactions (Gowans et al., 1963) and are incapable of responding to specific transplantation isoantigens in mixed-lymphocyte cultures (Wilson et al., 1967) although they are fully responsive to the presence of third-party homologous lymphocytes in culture. On such evidence, Gowans and McGregor (1965) have argued that the existence of tolerant populations of lymphocytes is consistent with the view that the inductive phase of antibody formation occurs in lymphocytes and that the role of macrophages is to present them with an effective antigenic stimulus. ACKNOWLEDGMENTS The investigations carried out in the authors’ laboratories were supported by research grant AI-07001 from the U.S. Public Health Service. One of US ( D B W ) is a Fellow of the Helen Hay Whitney Foundation. The authors are grateful to Drs. C. F. Barker, P. C. Nowell, and W. K. Silvers for their helpful criticisms on various aspects of this review.
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H u m a n Tissue Transplantation JOHN P. MERRlll Deportment o f Medicine. Peter Bent Brighom Hospital ond Horvord Medico1 School Boston. Mossochusetts
1. Introduction I1. 111.
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Terminology . . . . . . . Biology of Human Transplantation . . Immunosuppressive Treatment . . . A . Total-Body Irradiation . . . B . Drugs . . . . . . . C Thymectomy . . . . . D Splenectomy . . . . . . E . Thoracic Duct Fistulas . . . F . Extracorporeal Irradiation . . . G . Local Irradiation . . . . H . Antilymphocyte Sera . . . . I Corticosteroid Therapy . . . J . Other Immunosuppressive Adjuncts . Complications . . . . . . Tissue Typing . . . . . . A . Skin Grafting . . . . . B . Normal Lymphocyte Transfer Test . C. Mixed Lymphocyte Cultures . . D The Irradiated Hamster Test . . E . Serotyping of Leukocytes . . . F . Red Cell Antigens . . . . C . Sumniary . . . . . . Transplantation of the Human Kidney . A . Recipient Selection . . . . B Donor Selection . . . . . C . Diagnosis of Rejection . . . D . Prognosis in Human Renal Allografting Transplantation of Other Visceral Organs A. Liver and Spleen . . . . B. Xenografts . . . . . . C . Heart . . . . . . . D. Lung . . . . . . . E . Organ Preservation . . . . Endocrine Grafts . . . . . A. Parathyroid . . . . . . B . Pituitary . . . . . . C . Thyroid . . . . . . D. Adrenal . . . . . . Corneal Grafts . . . . . . Crafts of Bone and Blood Vessels . 275
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276
JOHN P. MERRILL
XI. Transplantation of Marrow . . . . . XII. Transplantation of Human Skin . . . . XIII. Moral and Ethical Aspects of Human Transplantation References . . . . . . . . . 1.
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Introduction
Since the demonstration by Medawar (1946) that the rejection of transplanted skin results from an immune response to the graft, the study of the immunology of tissue transplantation has burgeoned mightily. The developments in transplantation immunology have been looked upon with surprise and even horror by the classic immunologist, since many of the results could not have been predicted or affirmed by the well-established thinking or methods of the earlier immunological disciplines. Nowhere has this been more obvious than in human transplantation. Of necessity in the human arena the clinician must play a role, and he is frequently a physician or surgeon with little in the way of formal immunological training. It is with some amusement that one notes that even as the classic immunologist viewed with alarm the efforts of the transplanter, now in the field of transplantation itself the “basic transplanters” look with concern at the occasionally unsophisticated efforts of the clinician. There is a real basis for this concern in many instances. On the other hand, some remarkable and unpredictable results have been obtained in the field of transplantation as a result of trial and error. In addition to this the “spinoff has been and certainly will continue to be of value to the general field of medicine. In the process of transplanting livers, kidneys, and skin in the human we have seen the development of new syndromes. The necessity bilaterally to nephrectomize the recipient before transpIantation has told us much about the role of the kidneys in erythropoiesis and in hypertension. The necessity for arteriography on the normal donor prior to nephrectomy has given us much information about normal function and structure by a technique which might not otherwise be justifiable. The development of glomerulonephritis and other “autoimmune phenomena” in the recipient has given us new insight into these syndromes as they occur spontaneously. Nor must one forget in the welter of scientific, moral, and legal criticism that there are many patients in good health today who would long ago have died of chronic renal failure.
TERMINOLOGY The confusion that attended terminology in the transplant fieId up until 1960 was nicely summarized by Gorer (1960) at that time. This
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has been subsequently clarified by Snell (1964) and others. At the present time it is generally agreed that correct usage includes the following terms. Autograft-the transplantation of tissues from one part of an individual to another part of that same individual. Isograft-the transplantation of tissues between two individuals who are of the same inbred strain. Since no histocompatibility differences exist between these individuals, no immune response to the graft occurs. A special instance of this in humans is that of identical twins. Allograft-a graft of tissue between two individuals of the same species. Histocompatibility differences may be strong or weak, depending upon the individual and the species. Xenograft-a graft between individuals of two different species. “Heterograft” is still used synonymously with xenograft, II.
Biology of Human Transplantation
The biology of tissue transplantation in man is similar although not identical to that in other animals. The general areas of tissue transplantation biology have been presented in a number of reviews (Merrill, 1961; Woodruff, 1960; Merrill, 1965) of which the most recent and comprehensive is that of Russell and Monaco (1964). Although the behavior of man to the allografting of tissues and organs is similar to that of lower mammals, it is not identical. The differences in compatibility between animals and the reasons therefor can be delineated by carefully controlled experiments involving grafting in large numbers of lower mammals. For obvious reasons this is not true for man. It is, however, possible in man to transplant skin, to inject peripheral leukocytes and platelets, and more recently to transplant kidneys. From this kind of data, a justifiable analogy to data from animal experiments and the following generalizations can be made. The rejection of an allograft in man is due to an immune response in the recipient against an antigen present on the tissues in the donor and absent in the host. Present evidence from data in man supports the view that transplantation antigens are intermediate in strength and that strong antigens may not exist (Terasaki et al., 1967). It has been clearly shown that man may be sensitized to a skin graft by prior injection of leukocytes from the skin graft donor (Friedman et al., 1961) or by a prior skin graft from the donor (Rapaport et d.,1962a). The duration of this skin graft immunity is approximately 80 days in man (Rapaport and Converse, 1958). Immunity to skin grafts induced by either prior leukocyte injection or prior skin graft is not individual specific but shows cross
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reaction in approximately 16%of random population tested in one series (Wilson et al., 1963) and 50%in another (Rapaport et al., 196213). As in the lower mammal, antigenic information in some fashion reaches lymphopoietic tissue and stimulates antibody formation. The manner in which this information reaches the lymphocyte or prelymphocyte is unclear both in the experimental animal and man. However, the participation of the lymphoid system in man is evident since patients with congenital alymphocytosis (Rosen et al., 1962) and (total) agammaglobulinemia cannot reject skin grafts (Good et al., 1957). Furthermore, as in the experimental animal, lymphocytes in large numbers are found at the site of graft rejection in man. Indeed, the first histological evidence for graft rejection elicited in serial biopsies of normal skin grafts in man have been lymphocytes around the small venules (Henry et al., 1962). Thus, the lymphocyte apparently plays a role in graft rejection in man. There is no good serological evidence in man that humoral antibodies play a role in graft rejection. However, inferential evidence that such antibodies exist and are operative in human graft rejection seems overwhelming. The rejection of a skin graft in a human sensitized by several previous skin grafts may occur in such a rapid fashion that the graft never becomes vascularized ( White graft ) . Small-vessel thrombosis occurs with rapidity, no lymphocytes are seen at the site of the rejected graft, and the polymorphonuclear leukocytes that appear seem to be a response to necrosis rather than to an immune reaction. The rapidity of the rejection and the lack of cellular infiltrate strongly suggests “humoral antibody.” Rejection characterized by acute vasculitis and minimal cellular response is also seen when kidneys are transplanted into previously immunized dogs, and this counterpart has been seen in man immunized by leukocytes or platelets in previously administered blood transfusions ( Merrill, 1965, Kissmeyer-Nielsen et al., 1966). In man the immune response to transplanted tissue may be modified by the clinical situation of the host. Thus, uremia (Dammin et al., 1957; Carpenter et al., 1966), infection, or severe burns (Rapaport et al., 1964) all may diminish the immune response to antigenic stimulation. It has been well documented in the experimental animal that a large dose of intravenous antigen produces tolerance more effectively than a small dose of antigen (such as a skin graft placed in a subcutaneous site). These observations seem true of man also since the kidney which is, indeed, a large dose of antigen administered intravenously (through the renal vein) appears to be better tolerated than a skin graft. At least one of our patients has retairred a renal allograft over 5 years following rejection of a skin graft from the same donor. In this same patient the phenomenon of tolerance, perhaps
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through the “privileged position of the kidney,” was exemplified by retention of the kidney allograft at a time when the kidney recipient’s lymphocytes injected into the kidney donor’s skin gave a violent reaction typical of injection of sensitized immunologically competent cells. Perhaps one of the most striking differences between man and animal (although some data is now evident that this appears also in animals) is the ability of the kidney recipient to support a second graft following the rejection of a first renal allograft. Although this is not a universal phenomenon, the number of patients now successfully bearing second renal allografts following rejection of the first is large enough so that this phenomenon cannot be explained on the basis of chance histocompatibility similarities alone. Ill.
Immunosuppressive Treatment
A. TOTAL-BODY IRRADIATION Attempts in man to modify the immune response against allografts and heterografts have, in general, followed the pattern suggested by success in the experimental animal. The use of corticosteroids alone or the antihistamines for this purpose has been unrewarding. Nevertheless, a combination of events, of which uremia per se was probably the principal factor, gave rise to some surprisingly long survivals in early attempts at human kidney transplantation ( Hume et aZ., 1955). The demonstration by Main and Prehn (1955) that skin could be successfully transplanted after total-body irradiation and the infusion of allogeneic bone marrow led to several attempts to apply this regimen to the human. In the experiments of Main and Prehn, whole-body irradiation was given in a dosage that destroyed bone marrow function as well as that of lymphoid tissue. To enable the animal to survive, marrow was transplanted. In the absence of effective lymphoid-initiated antibody response, the bone marrow survived, repopulating the recipient’s marrow spaces and resulting in adequate erythropoiesis and leukopoiesis. Since tolerance for nucleated cells from the donor animal had been produced in the irradiated recipient, skin grafts from the donor were also tolerated. Attempts to apply this method to three patients at the Peter Bent Brigham Hospital prior to cadaver kidney transplantation were ineffective. Pooled human marrow was used, but there was no evidence of survival of the transplanted cells although in one patient evidence of rejection of the kidney was absent. The first successful kidney allograft was accomplished in 1959 when a kidney was transplanted from a healthy nonidentical twin to his sibling. In this instance total-body irradiation was given but in a “sublethal”
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dose. It was reasoned that since donor and recipient were antigenically similar (skin grafts exchanged between the two had prolonged survival although they were eventually rejected), a less than lethal dose of irradiation might allow the kidney transplant to survive without the necessity for transplanting marrow. Although severe leukopenia followed the irradiation, the recipient’s bone marrow eventually recovered spontaneously and the renal allograft functioned normally. An episode of threatened rejection at 6 months was aborted with further irradiation and steroid therapy (Merrill et al., 1960). This appears to be the first demonstration of the production by immunosuppressive agents of partial tolerance in the absence of marrow transplantation. A renal allograft was successfully done with this same technique by Hamburger and his colleagues (1959) 6 months later. Attempts to use “sublethal” irradiation as an immunosuppressive agent met with little success thereafter. Frequently the “sublethal” dose was actually lethal, and the unpredictable antigenic disparity between the tissues of allograft donor-recipient pairs did not permit decreased dosage. Even though a number of renal allografts survived for more than 6 months with this technique (Hamburger et al., 1962; Kuss et al., 1961), whole-body irradiation has been abandoned.
B. DRUGS The basis for the immunosuppressive regimen universally used at the present time in human renal allografting was the demonstration by Schwartz and his colleagues in 1959 that the primary immune response of rabbits to the injection of bovine serum albumin could be abrogated completely by relatively small doses of Bmercaptopurine ( Schwartz and Dameshek, 1959). 6-Mercaptopurine was also shown to be capable of inducing a state closely resembling immunological tolerance to a single purified protein antigen in adult rabbits (Schwartz et al., 1959). These authors also pointed out that 6-mercaptopurine could triple the survival time of skin homografts in rabbits (Schwartz and Dameshek, 1960). Calne (1960) and Calne and Murray (1961) demonstrated the prolongation of the survival of renal allografts in dogs with 6-mercaptopurine. Pierce and Varco (1962) later suggested that 6-mercaptopurine could induce true immunological tolerance since a renal allograft in one of their dogs continued to function for 18 months, during the last 8 months of which no 6-mercaptopurine was given. Calne and his colleagues ( 1962) screened a number of immunosuppressive drugs in a program of dog kidney transplantation and demonstrated the superiority of azathiop i n e (lmuran ), the S-imidazolyl derivative of 6-mercaptopurine. Al-
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though other immunosuppressive agents appear to be more effective in other species, Imuran is clearly the most effective in the dog and human kidney transplant system and is universally utilized in human transplantation at the present time. Of the other agents screened in the dog preparation by Calne and Murray ( 1961) , azaserine, actinomycin C, and the cortisone derivatives (usually prednisone) have been the most effective. This appears also to be true of the human. 6-Mercaptopurine given to man blocks the immune response to primary antigenic stimulation by the Vi antigen of Escherichiu coli, suppresses delayed hypersensitivity to tularemia vaccine, and significantly prolongs the survival of skin grafts (Levin et al., 1964). The mechanism of action of these agents in the production of tolerance to transplanted tissue is not clearly delineated. However, “it is a fair generalization that they inhibit the synthesis of desoxyribonucleic acid (DNA) or ribonucleic acid (RNA) or b o t h ( Aisenberg, 1965). Since cell division and proliferation results as part of the immune response to antigenic stimulation, the suppression of this immune response may be mediated through the inhibition of mitosis of immunologically competent lymphoid cells via the DNA mechanism. Alternatively, this inhibition may occur through blocking the synthesis of ribonucleic acid, possibly messenger RNA. Possible mechanisms of action of the agents employed for the suppression of the immune response to renal allografts in man, dosage schedules employed, and side effects are noted in Table I. Azathioprine is the keystone of immunosuppressive therapy in man. The dosage schedule varies with the different groups using it. In theory, the action of an agent that blocks DNA and RNA synthesis as a function of antibody formation should be most effective if given after initial contact with antigen-stimulated division of cells. The experiments of Nathan suggested that 6-mercaptopurine administered 3 days after an antigenic stimulus depressed antibody formation more than a 5-day course of the drug begun the day of stimulation (Nathan, 1961). Zukowski’s studies indicated that administration of azathioprine 1-3 days after transplantation gave as much prolongation of canine renal allograft survival as it did in those animals whose therapy was begun on the day of transplantation (Zukowski, 1965). On the other hand, Starzl (1964) found that dogs with renal grafts lived longer if azathioprine was begun 7-30 days prior to the transplantation. Starzl, therefore, begins administration of azathioprine in most patients &lo days before operation. It is our custom to begin the drug 2 days prior to operation. Since azathioprine is excreted by glomerular filtration, the dose must be varied according to the functional status of the kidney. In patients who have stabilized with good
TABLE I IMMUSOSUPPRESSIVE
DRUGSUSED
IN
HUMANTRANSPLSSTATION
Action
6-MP, azathioprine
Actinomycin C
Azaserine
Inhibits synthesis of purine and pyrimidine moieties of D N S and RNA Selective inhibition of synthesis of DNA dependent RNA (messenger R N h ) Inhibition of nucleotide synthesis. Formyl glycineamide ribotide
Dose
Side effects
1-5 mg./kg./day oral
Alopecia, anemia, leucopenia, hepatitis
4-8 r g l k . l d w
Bone marrow depression, nausea, vomiting, stomatitis
IV 8-10 mg./day I V or po
Deliriiim, coma, stomatitis jaundice
1 Prednisone
(glutamine) Formylglycine amidine ribotide Antiinflammatory Lympholysis
20-800 mg./day
Hyperglycemia, peptic ulceration, (2.1. bleeding, aseptic bone necrosis.
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renal function 3 or more months after transplantation, the dose may be diminished to 1 to 2 mg./kg. It is important, however, to administer some drug even to patients with acute renal failure and anuria (commonly seen in cadaver transplants) during the anuric period since the kidney even under these circumstances receives some blood flow and rejection of the allograft is possible. Although in the experimental animal, as is noted above, it is occasionally possible to discontinue azathioprine therapy without detriment to the allograft, this has not yet been attempted in man. Even those patients with excellent function after 2 years receive small doses of azathioprine. Bone marrow depression may occur as a result of excess dosage of azathioprine, and patients must be carefully followed with white counts during the period of high-dosage therapy. Occasionally one sees jaundice which may disappear with omission of the drug or decreasing dosage. Azaserine is used as an adjunct to azathioprine now less frequently than before. The evidence is good that in the dog it is an effective adjunct. In man its efficacy is less certain: Actinomycin C is employed largely in the treatment of rejection crises. There is no question but that in both man and the dog it has reversed incipient or even advanced rejection. It is of particular value where increased prednisone dosage is not tolerated or is contraindicated. Prednisone is used by most groups either prior to or on the day of transplantation. Of all the agents employed, the effect of prednisone is the easiest to assess and in large doses it is unquestionably the most effective agent for the reversal of rejection, In general, 150 or 200 mg. of prednisone are given immediately prior to or at the time of transplantation and the dosage is tapered over a period of 2 weeks to maintenance therapy. The wellknown side effects of prednisone, particularly impairment of wound healing and predisposition to infection, make it desirable to taper the dose as rapidly as possible in the immediate postoperative period. It is used and should be used in large doses (200500 mg.) immediately upon detection of beginning rejection. When it is effective, the results are usually apparent within 24 to 48 hours and the dose may be tapered accordingly. Although most patients whose renal function is stable after 6 months or a year do not require large doses of prednisone, we have seen patients who showed increasing proteinuria when a maintenance dose of 20 mg. a day was omitted. C . THYMECTOMY It has been demonstrated that in adult mice, thymectomy combined with total-body irradiation can result in homograft tolerance (Miller, 1963). Thus, thymectomy would seem to be an effective adjunct to other
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forms of immunosuppressive therapy. Although the work quoted above was not known at the time that the first thymectomies were done in humans, the rationale for this procedure seems logical. Unfortunately, data from the human experience as well as the dog experiments yield no evidence that this procedure is helpful under the conditions in which it was studied ( Starzl, 1964; Starzl et aZ., 1963a).
D. SPLENECTOMY Splenectomy has been similarly utilized by a number of groups. The rationale is that the reduction of the total mass of lymphoid tissue might impair antibody response. Neither Starzl’s group nor our own have demonstrated that splenectomy significantly adds to the efficacy of other immunosuppressive agents (Starzl, 1964; Veith et aZ., 1965). It has been suggested that splenectomy by raising the leukocyte count allows a larger dose of azathioprine to be tolerated (Hume et d.,1964). The analysis of our patients by Veith et at. (1965) does not substantiate this. Both Hamburger et al. (1964) and Hume et al. (1964) have reported a syndrome of hypersplenism following transplantation. Possibly prior splenectomy might have obviated this complication. We have never observed this phenomenon, however, in our series of nonsplenectomized patients. E. THORACIC Durn FISTULAS The demonstration by Gowans and his associates (1962) that chronic thoracic duct fistula in rats impairs the immunological response to sheep erythrocytes and tetanus toxoid prompted the use of this technique in the larger mammals as a therapeutic adjunct in tissue grafting. Two groups of investigators (Mayer and Dumont, 1963; Samuelson et al., 1963) have shown that thoracic duct drainage in dogs prolongs the survival of skin homografts. Woodruff and Anderson (1963) combining thoracic duct fistulas and the injection of antilymphocyte serum have reported prolonged survival of skin homografts in rats. Singh and his colleagues (1965) prolonged the survival of canine renal allografts using thoracic duct drainage alone and suggested that the duration of survival of the transplant might be related to the amount and duration of lymph drainage. This technique has been applied to the human by the group at the Peter Bent Brigham Hospital (Tilney and Murray, 1967). The thoracic duct is cannulated 3-5 days prior to transplantation and drainage continued for as long as the fistula remains open postoperatively. The whole lymph is collected in plastic bags. These are spun and the cells removed. The lymph is then reinfused intravenously in order to prevent
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protein depletion that occurs with prolonged lymphatic drainage. In the human this has been used as an adjunct to other forms of immunosuppressive therapy and is, therefore, difficult to evaluate per se. However, the comparison of patients so treated with those treated with a similar regimen excepting only thoracic duct fistula shows suggestive improve. ment in their early post-transplant course in that the number of rejection crises seems to be lessened ( T h e y and Murray, 1967).
F. EXTFLKORPOREAL IRRADIATION Cronkite and his colleagues (1965) have demonstrated that significant lymphopenia can be produced by the extracorporeal irradiation of blood in animals. In calves some prolongation of skin homografts was obtained when blood was irradiated by circulation around a cesium-137 source. This prolongation was more significant when thoracic duct lymph was passed around the cesium source and returned to the animal. Our group has employed extracorporeal irradiation of blood in humans. Blood is passed around a radioactive cesium source and returned to the patient through an inlying, Teflon, silastic arteriovenous shunt of the type used for chronic intermittent hemodialysis. Treatment is begun 2-3 weeks before projected transplantation and continued until a significant drop in circulating small lymphocytes is produced and maintained. Again, because this technique is used as an adjunct to several other forms of immunosuppressive therapy, it is difficult to evaluate its effect upon the patient’s course. However, in three instances when other forms of immunosuppressive therapy were drastically reduced or stopped because of toxicity, the use of extracorporeal irradiation alone modified or reversed incipient kidney graft rejection. In these instances extracorporeal irradiation appeared to have a definite beneficial effect. Other groups, notably that of Hume in Richmond, have reported success with irradiation using a beta source placed around the extracorporeal shunt (Wolf et al., 1966). G . LOCALIRRADIATION
Hume’s group has reported their observations in the use of local irradiation to the transplant as a means of preventing and also of reversing allograft rejection (Kauffman et al., 1965). In dogs the effect of local irradiation to the graft ( 6 doses of 150 r each beginning on the day of transplantation and administered every other day for a period of 10 to 12 days) appears to prolong the Iife of the graft. On the basis of this work Hume now routinely administers local irradiation to transplant patients. The first dose is given within 24 hours after transplantation and subse-
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quent doses given on the third, fifth, and seventh post-transplant days for a total dose varying between 600 and 1200r. During periods of threatened rejection this may be repeated once or twice. Hume believes that the destruction of antibody-producing lymphocytes which have migrated to the transplant or, alternatively, the interference with antibody production through destruction of cells engaged in transport of antigen from the kidney to the lymph nodes are the possible mechanisms involved. Our experience with this technique has been limited, but we are not convinced that its value has been clearly demonstrated. Data from the most recent Transplant Registry (Murray et al., 1967), however, indicate that X-irradiated cadaver kidney transplants have a greater l-year survival than those not so treated.
H. ANTILYMPHOCYTE SERA The use of sera from animals made immune to host lymphocytes to destroy lymphoid cells and thus to suppress the immune response in the host animal has a long and interesting history. Metchnikoff first demonstrated that serum from guinea pigs immunized with rat spleen or lymph nodes destroyed rat leukocytes ( Metchnikoff, 1899). More recently Waksman and his colleagues (1961) used specific lymphocyte antisera to inhibit delayed-type hypersensitivity. Woodruff and Anderson ( 1963) showed that antilymphocyte sera in conjunction with thoracic duct fistula prolonged the survival of skin homografts in rats, and this effect was confirmed in mice by Gray et al. (1964). An excellent review of the literature and of their own work in the use of antilymphocyte serum to suppress the immune response in canine and human renal allografts has been published by the Denver transplantation team (Iwasaki et al., 1967). The reader is referred to this review for details of the preparation, route of administration, dosage, and histological findings. In a subsequent paper these authors reported twenty patients who were given daily intramuscular administration of immune horse globulin fractionated from horse antisera to human lymphocytes (Starzl et al., 1967). The lymphocyte differential count was immediately decreased in all patients, but because a total increase in white count almost invariably occurred, the result was that the absolute fall in lymphocyte count was not statistically significant. Intense pain at the site of the intramuscular injection occurred in all patients. In four of the twenty patients antiphylactic reactions occurred but the injections could be continued. Although in the dog the cortex of the lymph node contained numerous germinal centers composed of proliferating large pyroninophilic cells, the amount of lymphoid tissue appeared to be within normal limits in
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seven spleens and three human Iymph nodes examined. In the dog there was a high incidence of histological renal damage with the various antilymphoid agents utilized. The renal injury consisted of dense accumulations of horse and dog 7-globulin together with complement along with subepithelial aspects of the renal glomerular capillary basement membranes. To date the Denver group has treated twenty human allograft recipients with antilymphocyte globulin. In all of these, delayed-type skin tests disappeared. It was the impression that the mortality of the patients was lower in the antilymphocyte-globulin-treated group and that less Imuran and steroid was necessary to prevent rejection. Biopsies were made of the renal allografts in a number of patients. Ferritin and fluorescent-labeled antihorse globulin was utilized in an attempt to find horse globulin in the biopsy tissue. It is reported that none was seen. Nevertheless, the frequency with which glomerular lesions are seen in the animal treated with antiIymphocyte serum (Iwasaki et al., 1967; Guttmann et nl., 1967) suggests that this may be a problem in the human. I n addition, the concern is furthered by the reports of fatal glomerulonephritis in three human cases receiving horse antihuman cancer serum ( D e La P a w et al., 1962) and by the occurrence of glomerulonephritis in humans during an assay of bovine serum albumin as a plasma substitute in the early 1940’s. Antilymphocyte serum, however, is a potent immunosuppressive agent whose efficacy does not appear to be dependent upon lymphocyte destruction alone. Although at the present time it should be used with extreme caution in man, it seems quite probable that further exploration of this preparation may yield an effective immunosuppressive agent with relatively little hazard of serum sickness or nephrotoxic nephritis.
THERAPY I. CORTICOSTEROLD Of the agents currently used, the best known and still most effective are the corticosteroids. The efficacy of prednisone used prophylactically in the early postoperative period and to suppress subsequent rejection responses has been well documented by all observers. It does not seem necessary in this review to elaborate upon the immunosuppressive and antiinflammatory action of corticosteroids nor upon their undesirable side effects. J. OTHERIMMUNOSUPPRESSIVE ADJUNCTS Of the naturally occurring adjuncts to immunosuppressive therapy in human allograft recipients are uremia ( Dammin et a]., 1957), malnutrition, and the effects of major surgery (Graham and Peterson, 1964).
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We have utilized intravenous therapy with typhoid vaccine in aborting renal allograft rejection. Since one of the early manifestations of rejection of a kidney transplant is renal vasoconstriction, the known effect of the febrile response to typhoid vaccine in increasing renal blood flow and in causing renal vasodilation (Bradley et al., 1945) was utilized. In addition, it was postulated that possibly the immune response to the allograft might be impaired by the subsequent injection of another more disparate antigen (“antigen competition”) ( Adler, 1959). Although a rather striking improvement occurred in two cases so treated, the general results have been too inconsistent to be significant. Regional “lymphatic ablation” in the dog (Knox et al., 1964) and in the human (McIntosh et al., 1966) has had little significant result upon renal allograft survival. Prolongation of allografts by specific inhibition of complement ( Nelson, 1966; Gewurz et aE., 1966) has been effected in the animal and is of great theoretical interest but has not yet been tried in humans. Similarly, adult tolerance has been achieved in the mouse employing multiple injections of subcellular transplantation antigens, and the combination of 6-mercaptopurine and intravenous antigen has prolonged skin allografts in the rabbit ( Mannick and Southworth, 1966). This technique has not yet been employed in the human.
IV.
Complications
The complications of transplantation (renal) in the human are fascinating, unique, and somewhat terrifying. In effect, we have with our rather clumsy approach to this novel therapeutic endeavor, created a whole new host of clinical syndromes and pathophysiological phenomena. Some of the earliest of these unhappy events may occur at the operating table when, because of trauma to the kidney or because of prolonged ischemia in a cadaver kidney, the transplant may remain flabby and pale and without normal perfusion at the time of the anastomosis of donor and recipient arteries. Prolonged oliguria, due to acute tubular necrosis, may supervene and though eventually diuresis may occur, the period of oliguria requiring continued dialysis may last from days to weeks. Interestingly enough, provided adequate management is given during this stage of renal insuBciency, this complication does not always appear to impair the eventual function of the transplant. Failure of the kidney to be adequately perfused and to produce urine may also occur in instances where the recipient blood contains preformed, major blood group isoantibodies for donor antigen, although this is not invariable. Nevertheless, the statistics show clearly that crossing the major blood group barriers impairs the eventual outcome. The recipient may
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have been presensitized to human donor tissue by reason of previous exposure to plateIets or leukocytes in transfused blood (Lostumbo et al., 1966). It has been previously pointed out that sensitization to subsequent skin grafts from any donor can occur following the interdermal injection of human leukocytes, resulting in accelerated rejection of a skin graft. The uremic recipients, particularly those maintained for long periods of time on hemodialysis, need repeated transfusions and thus have repeated exposure' to formed elements in the blood capable of sensitizing the recipient to subsequent grafts. The rapid rejection of a first kidney allograft has been documented in one of our patients who presumably had been sensitized by the platelets in repeated transfusions prior to operation (Merrill, 1965). Rapid rejection of the transplant occurred on the third postoperative day. Histologically the rejected kidney showed an acute vasculitis with thrombosis of the afferent arterioles and interstitial hemorrhage. Infusion of donor platelets showed an extremely short disappearance time. Subsequently dogs immunized by platelet transfusions were shown to reject renal allografts in an accelerated fashion (Hager, 1966). Others have reported a similar phenomenon in human renal allografts in whom leukoagglutinins were shown to exist prior to transplantation ( Kissmeyer-Nielsen et al., 1966). Hypertension is a frequent complication of the post-transplant course. It may occur soon after operation and may be extremely severe and unresponsive to therapy in children in whom an adult kidney has been transplanted. Hypertension is poorly correlated with the changes in peripheral venous renin levels (Blaufox et al., 1966). It is frequently an early manifestation of rejection and if the rejection is treated promptly and effectively hypertension subsides. This is one of the few instances in which hypertension may improve with massive corticosteroid therapy. Progressive slow rises in blood pressure occur as common compIications of a later course of patients with transplanted kidneys. Almost all observers have reported vascular changes in both canine (Porter et al., 1963) and human renal allografts (Porter et al., 1964; Figueroa et al., 1964; Murray et al., 1963). Characteristically arterioles and interlobular arteries show obstruction by intimal fibroblastic thickening and marked increase in elastica. As a result, the graft itself shows widespread tubular atrophy, interstitial fibrosis, and glomerular atrophy and sclerosis ( Fig. I ) . It is generally believed that the striking vascular lesions are not secondary to the hypertension but have an immunological basis. Hypertension is secondary to the vascular lesion, but the onset of severe hypertension hastens the progress of the lesion. This combination of vascular change, hypertension, nephrosclerosis and renal failure is a
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frequent cause of death in the late (1-3 year) post-transplantation period. Modification of the host immune response by the several modalities discussed includes, of course, the modification of the host response to infectious disease, It is to be expected, then, that infection would be a common complication of human kidney transplantation. Staphylococcal infection acquired from hospital environment does occur but much more common and difficult to manage is infection arising from the’patient’s
FIG. 1. Human renal allograft from a cadaver biopsied at 8 months. The typical features are: ( 1 ) obliterative arteritis; ( 2 ) tubular and glomerular atrophy; ( 3 ) interstitial fibrosis.
own gram-negative flora. Escherichia coli, Aerobacter aerogenes, Proteus uulgaris, and Pseudomonas aeruginosa are common. For this reason the usual hospital isolation precautions preventing cross-contamination are relatively ineffective. Modification of the host immune response has also made more common in these debilitated patients infections with fungi and Pneumocystis which frequently supervened after prolonged antibiotic therapy for bacterial infection. Ureteral obstruction due to a mass of Candida albicans hyphae at the ureteropelvic junction has been reported along with its successful treatment by irrigation with amphotericin B (Shelp et al., 1966). Hill and his colleagues, reporting on
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thirty-two patients dying from 1 to 207 days after receiving an organ transplant (liver or kidney), found pulmonary infections present terminally in twenty-six of these. The responsible organisms included a large number of unusual agents not commonly producing clinical disease. Candidu, Aspergillus, Noccrrdia, Pneumocystis, and cytomegalovirus were identified in twenty-eight (Hill et al., 1964). These exotic agents are seen in other debilitating noninfectious diseases such as lymphocytic leukemia and hypogammaglobulinemia (Jacox et al., 1964), suggesting the relationship to a defect in immunological integrity. This “transplantation pneumonia” is characterized by alveolar capillary block, frequently with rapidly progressing confluent nodular densities, and arterial oxygen desaturation. It characteristically occurs during a time when steriod therapy is being decreased (lowering steriod therapy), and the possibility exists that some immunological event involving both the transplant and the lungs may be a predisposing factor. This has been a common complication in our experience. In addition, we have found that apparently healthy transplant recipients seen in the out-patient department harbor and excrete in their urine cytomegalic virus. (Contrary to expectation, however, we have not seen disseminated tuberculosis. ) The combination of chronic illness, malnutrition, uremia, anemia, and treatment with agents that block nucleoprotein synthesis again might be expected to impair wound healing. This too has been a frequent complication. The immediate use in the immediate postoperative period of high doses of corticosteriods certainly contributes. In any case, the failure of wound healing and particularly that of the ureteral anastomosis has been a common and fearful complication. In the latter instance, perhaps, the rejection process too plays a role. In addition to the complications of corticosteroid therapy listed above, the development of the Cushingoid habitus, particularly in children, diabetes, and gastrointestinal bleeding have all been seen with distressing but not prohibitive frequency. Corticosteroid therapy has been reported to cause acute hemorrhagic pancreatitis (Nelp, 1961), and we have seen two fatal instances of this in patients who received substantial immunosuppressive therapy for 9 and I1 months, respectively (Tilney et al., 1966). In addition, both patients had acute arterial changes with fibrinoid degeneration of the vessel walls in many arteries throughout the entire pancreas. This vasculitis may reflect the phenomenon previously noted in the experimental animal (Moore et al., 1959, Murray et al., 1964) in which the vessels of the host’s own organs shared in the vasculitis accompanying the rejection of an allografted organ. Aseptic necrosis of the femoral head has been associated with corticosteroid
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therapy (Boksenbaun and Mendelson, 1963). This complication has occurred in one of our patients a year and one-half following operation. Systemic fat embolism has occurred in patients with renal allografts treated with corticosteroids (Jones et al., 1965). This phenomenon is presumably secondary to hypercorticism and may result in pulmonary infiltrate, encephalopathy, and thrombophlebitis. Leukopenia and anemia are manifestations of overdosage with azathioprine. The anemia may be accompanied by the presence of many nucleated red cells in the peripheral blood and these may give a falsely elevated leukocyte count if the procedure is performed by automated techniques such as the Coulter counter. Azathioprine has been associated with jaundice of the cholestatic type which subsides when the drug dosage is reduced or discontinued. However, prolonged survival and pathogenecity of hepatitis virus has been inferred in a kidney transplant recipient manifesting smouldering hepatitis for 6 months while under continuous prednisone and azathioprine therapy (Kern et al., 1963). This factor plus multiple transfusions given prior to and during transplantation may also contribute to the incidence of jaundice. In severaI cases we have noted a “wasting” syndrome accompanied by marked esophagitis and gastritis which appears to be related to the immunosuppressive regimen, in general, but particularly to azathioprine therapy. Although 6-mercaptopurine has been reported to produce renal tubular defects manifested by glycosuria and acidosis (Butler et al., 1965), we have not seen this complication. Drug fever has also been reported following the administration of 6-mercaptopurine ( Savitsky et al., 1964). It is uncommon, however, and has not presented a problem in our series of cases following the administration of azathioprine. The development of a renal lesion in the transplanted kidney closely resembling that in the patient’s own diseased kidneys was first reported by our group in a patient with polyarteritis who received a cadaver 1955). The lesion kidney and no immunosuppressive therapy (Hume et d., of glomerulonephritis was subsequently seen in the transpIanted kidney of an identical twin recipient whose original disease had been glomerulonephritis. Of eighteen sets of identical twins whose original disease was glomerulonephritis a.nd who received a renal isograft, eleven have developed a similar histological lesion in the transplanted kidney (Glassock et al., 1967). Originally it was postulated that a continuing state of hypersensitivity existed in the recipient which resulted in the formation of “antibodies” against the isograft as a function of the continuing activity of the original nephritis. Typical membraneous and proliferative glomerulonephritis, however, have been seen in true allografts in re-
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cipients maintained constantly on immunosuppressive therapy. In some of these the original disease had not been glomerulonephritis ( O’Brien and Hume, 1966; Krieg et al., 1960; Petersen et al., 1966; Zukowski and Ende, 1965).It seems thus that one cannot implicate continuing activity of the recipient’s glomerular disease as a cause of the subsequent glomerulonephritis in transplant. Conclusive evidence of this is afforded by one of our patients who received a successful allograft from his mother following the unintentional removal of a single, normal, ectopic kidney elsewhere. This patient has developed the classic nephrotic syndrome with glomerulonephritis 2; years after transplantation. Since his original kidney was completely normal, the development of glomerulonephritis in the transplant cannot be attributed to “continuing activity.” It seems clear at the present time that in the allograft, at least, the glomerulus may share in the other vascular phenomena of chronic rejection with the production of glomerulonephritis and the classic nephrotic syndrome. A closer look at the glomerulus with more sensitive techniques also suggests that a glomerular lesion may occur with more frequency and earlier than had been thought in renal allografts in animals. Although persistent antikidney immunological activity related to the original disease must still be invoked to explain the nephritis in the identical twin transplant, this is obviously not true in the allograft. One of the interesting physiological dividends from renal allografting ias been the demonstration of autonomous secondary (renal) parathyroid hyperplasia ( McPhaul et al., 1964). Parathyroid hyperplasia secondary to prolonged stimulation by diminished ionized calcium levels in chronic renal failure has been well recognized. There have been suggestions that this prolonged stimulation might result in the development of autonomous hyperparathyroidism (Stanbury et al., 1960; Case Records Massachusetts Gen. Hosp., 1963; Cohen et ut., 1964). If this were the case, correction of the uremia and the defect in calcium metabolism by transplantation of the normally functioning kidney might be expected to “unmask” the autonomy of the parathyroid and result in the manifestations of the parathyroid hyperactivity in the absence of uremia. This has been reported by McPhaul et al. ( 1964) and by our group (Wilson et al., 1965). In both instances the removal of enlarged and histologically hyperfunctioning parathyroid tissue following transplantation has resulted in the return of serum calcium to normal. Although actinomycin (Rasmussen et al., 1964) interferes with the peripheral action of the parathyroid hormone and might thus result in continued stimulation of the gland in the post-transplant period, the amounts of actinomycin utilized in these patients does not seem to be adequate, and the relation-
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ship of the development of hyperparathyroidism to this agent is not clear cut. The development of malignant tumors in transplant recipients, presumably as a result of microscopic nests of malignant cells in the transplanted kidney have been reported a number of times (McIntosh et al., 1965; Dossetor, 1965; Wilson et al., 1967). In one of our patients, tumor tissue resembling the epidermoid carcinoma of the bronchus of which the cadaver donor died developed in the transplanted kidney 1year after transplantation. Cessation of immunosuppressive therapy in the recipient resulted in the rejection of the transplant, but also disappearance of the tumor. This fascinating observation suggests that microscopic nests of potentially malignant cells from the primary tumor may not be manifest in the individual with the normal immune response but may be allowed to multiply and propagate following transplantation in the kidney when the recipient’s immune response is modified by immunosuppressive therapy. Tumor cells transplanted as allografts are ordinarily rejected by the recipient as is the case with normal tissue (Southam and Moore, 1958), although one fatality as a result of the allografting of a melanoma without immunosuppressive therapy has been reported by Scanlon et al. (1965). In the case of the patients reported above in whom cancer tissue was transplanted with the renal allograft, it seems quite probable that immunosuppressive therapy was the factor potentiating the growth and spread of the tumor. The implications of these results for the selection of cadaver donors is obvious. Both Hume and Hamburger (Hume et al., 1964; Hamburger et al., 1964) have reported a late syndrome in patients with renal allografts characterized by splenomegaly, unexplained fever, and hypergammaglobulinemia. We have not observed this phenomenon in our patients. More recently, Bravo et al. (1967) have described a number of musculoskeletal disorders following renal allografting. These patients manifested arthralgia, diffuse musculoskeletal pain, joint swellings and effusions, and sinovitis. Avascular bone necrosis appeared in five of their patients. Antibodies to deoxyribonucleic acid and ribonucleic acid were detected in 40% and 20%of the patients, respectively. Hypergammaglobulinemia was detected in 9% of the patients but 43%had decreased y-globulin. The etiology of these disorders is unclear but findings of this sort are not uncommon in the late post-transplant period in most of the series reported. Amid this profusion of chemical and clinical complications, one must not forget the effect of psychic trauma on such patients. The constant anxiety associated with waxing and waning rejection crises, periods of
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oliguria, hypertension, and the physical deformities of hypercorticism, all take a toll. Manifestations vary depending upon the basic personality of the patient, but the effects of stress and of the abnormal environment are present at one time or another, to a greater or lesser degree in virtually all patients and members of their close family. V.
Tissue Typing
It has been clearly shown in the experimental animal that the duration of allograft survival depends upon the “strength” of the difference in histocompatibility between donor and recipient ( Streilein et al., 1966b). The rejection of an allograft is the result of the immune response of the recipient against tissue antigens present in the donor and absent in the host. If the antigen present in the donor is a “strong” antigen, the immune response to the graft is prompt and vigorous. If the antigen is weak, the response is slow and less vigorous. Indeed, whether a tissue antigen is characterized as strong or weak is, in fact, determined by the nature of the rejection of the tissue by a suitable recipient lacking the antigen in question. The genetic basis for histocompatibility has been well worked out in the experimental animal, particularly the mouse, where highly inbred strains of isogenic animals allow precise determination by tissue transplantation of the strength and of the nature of histocompatibility antigens. Fifteen histocompatibility loci are known in the moiise (Barnes and Krohn, 1957). Four of these, H-1, H-2, H-3, and H-4, have been found to exist in established autosomal linkage groups (Russell and Monaco, 1964). Of these, the antigen determined by the H-2 locus is “the strongest.” Thus grafts exchanged between mice differing at the H-2 locus are rejected more quickly and with more prominent histological evidence of the immune response than grafts exchanged between animals differing at H-1, H-3, and H-4. For obvious reasons, such a precise systematic analysis of transplantation antigens in man has not yet been possible. Nevertheless, its importance must be obvious. If the strength of the immune response to a tissue graft is proportional to the strength of the antigen present in the donor and absent in the recipient, then the immunosuppressive agents used to modify this response and secure a successful graft must also be stronger. Since most of the presently used immunosuppressive agents are toxic, it follows that as little as possible of these agents should be employed and, thus, the hazard of undesired side effects to the human recipient minimized. The ability to decrease the dose of an immunosuppressive agent would then seem to depend upon the ability to select a compatible donor-recipient pair. Surprising strides in the advancement of our knowledge in tissue typ-
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ing have been made in recent years. The ability to type human tissues depended upon the demonstration that some easily available human tissue could be studied and characterized and that the difference in the rlntigens of this tissue obtained from donor and recipient could be used to predict the differences between less readily available tissues of more clinical importance, such as kidney or skin. The demonstration that peripheral leukocytes shared many tissue antigens and that evaluation of leukocyte antigenic composition could be used to predict skin, and even kidney survival, was of the utmost importance. This relationship was pointed out by the fact that the immunity occasioned by the rejection of a skin homograft in man was manifested by delayed intradermal sensitivity following injection of donor white cells (Merrill et a,?., 1961) and that accelerated skin graft rejection in humans could be obtained by preimmunization of the recipient by peripheral leukocytes harvested from the donor (Friedman et al., 1961). An important, and somewhat unexpected, result was the finding that the reaction was not individual specific as had been previously thought. An occasional recipient rejected a donor skin graft in an accelerated fashion after having been immunized by leukocytes from an indifferent donor. These results were obtained also when the immunizing stimulus was a skin graft (Rapaport et al., 1962b). This fact suggested that the presence or absence of strong histocompatibility antigens in any two randomly selected humans might be predictable by appropriate testing methods. A number of techniques employing human peripheral lymphocytes or leukocytes and skin grafts have been devised to assay and predict histocompatibility and to find tissue antigens in man. A brief review of these facts has been published recently by Streilein et al. (1966b). A. SKIN GRAFTING Wilson et a2. (1963) approached the problem by placing a skin graft from the patient ( A ) to an ‘‘indifferent” or unrelated recipient ( B ). Following rejection of this skin graft, the host ( B ) was then putatatively sensitized to donor (A) tissue and to other human tissue ( C ) of an antigenic configuration similar to that of the donor ( A ) . The sensitivity, therefore, would result in the accelerated rejection of skin transplanted from such a donor ( C ) . A number of skin grafts were then placed from potential donors and the rejection time compared. Of this group, the donor whose skin which was rejected most rapidly was assumed to have tissue antigens most closely resembling that of the initial skin graft donor, i.e., the patient. The difficulty with this test, of course, was that it predicted similarities between the patient and prospective donor and
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what is wanted is definition of the strength of the difference between prospective donor and recipient, i.e., the strength of antigens present in the donor and lacking in the prospective recipient. For this reason, this test has now been abandoned.
TRANSFER B. NORMALLYMPHOCYTE Brent and Medawar (1963) have pointed out that this test could predict the survival of skin grafts transplanted between guinea pigs. The test depended upon the assumption that lymphocytes harvested from peripheral blood will cause an immunological reaction against the host if injected subcutaneously. The severity of this immunological reaction depends upon the strength of the antigens contained in the host tissues and absent in those of the lymphocyte donor. A delayed-type skin reaction results in which the area of erythema, and, more important, that of induration, are quantitated. This test has been applied to man by a number of groups (Carpenter et al., 1966; Bridges et al., 1964; Gray and Russell, 1963; Amos et al., 1965). In general it is agreed that the ability of the test to predict either the success of skin grafts or renal allografts is poor, and is complicated by the fact that the normal lymphocyte transfer reaction is poorIy produced by ceIls from uremic individuals, i.e., the potential renal allograft recipient.
C. MIXEDLYMPHOCYTE CULTURES Bain and Lowenstein (1964) and Bach and Hirschhorn (1964) and associates have devised a histocompatibility matching assay based upon the evaluation of lymphocytes in tissue culture media. Peripheral blood lymphocytes are harvested from donor and recipient and are cultured together. The observations of these two groups indicate that when the cells are “stimulated they are transformed into large basophilic blastlike cells which can be quantitated either by observation of the number of morphological changes or by the uptake of tritiated thymidine, both of which reactions are assumed to be the expression of antibody formation. Insofar as the cells from donor and recipient are antigenically different, “stimulation” to form antibody will be greater, and this result can be quantitated and expressed as a function of histocompatibility difference between donor and recipient. Numerous technical difficulties remain to be overcome in this test. Perhaps the most serious objection to it is that it is a “two-way test” in which under ordinary circumstances, one cannot tell whether the degree of transformation is due to stimulation of recipient cells by donor antigen or vice versa, More recently, inhibition of the immunological potential of one group of cells by the use of mito-
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mycin (Bach and Kisken, 1967) suggests that a one-way reaction, i.e., that of the recipient against the potential donor, might be evaluated. Although preliminary results with this system have shown promise, it has yet to be thoroughly evaluated.
D. THE IRRADIATED HAMSTER TEST Streilein et ul. (1966a) have found that mixtures of human lymphoid cells, injected intracutaneously into irradiated hamsters, evoke a delayedtype skin reaction. Irradiation of the hamster prevents participation of the animal’s own antibody-forming system from reacting with the human cells and thus obscuring the results. The intensities of these reactions appear to be related to the degree of immunogenetic disparity between the cell donors as revealed by the survival times of skin grafts exchanged between pairs of unrelated human volunteers. Here, too, however, the problem is that of the two-way reaction. Simonsen has shown an interesting correlation between the survival of parent-child and siblingsibling combinations which would be expected on the basis of the oneway hypothesis and the actual survival data derived from the compilation of the Transplant Registry ( Simonsen, 1965).
E. SEROTYPINC OF LEUKOCYTES Another approach, and one which currently appears to be the most profitable in the “matching” of kidney donor and recipient pairs, is that of typing by means of sera obtained from patients who have been immunized inadvertently to human leukocytes. These sera are obtained from multiparous women or patients who have received repeated transfusions containing leukocytes. Two kinds of techniques are used: (1) the ability of the antisera to agglutinate the leukocytes to be studied; and ( 2 ) the direct cytotoxic effect of the serum. The leukoagglutinating technique has been extensively studied by Van Rood and Van Leeuwen (1963). Ceppellini et ul. (1964), Dausset and Colombani (1962), and Amos (1965). The cytotoxic technique has been largely employed by Terasaki and McClelland ( 1964). Terasaki’s microdroplet technique to assay cytotoxicity employs extremely small volumes of serum and numbers of test leukocytes, thus enabling him to make use of large numbers of individual antisera and to obtain mathematically significant numbers on which to base his results. In the performance of both tests, donor and recipient cells are set up against a single antiserum and as many of these antisera as feasible are used. If agglutination or cytotoxicity is detected for both sets of cells when placed in the antisera, their antigenic configuration is assumed to be similar, as is the case if neither are affected. If one
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is affected and the other is not, a difference is assumed. Further differences may be implied by the dilution of the serum in which differences in agglutination or cytotoxicity are obtained. The assumption is also made that the antigenic specificities of the cells detected by these in uitro tests are closely related to the difference in antigenic specificities of other tissues which provoke allograft sensitivity in uiuo. The studies cited above and the cross-relationship of the sensitivity to human leukocytes and skin grafts support this assumption. By cross-absorption of the antisera, it is possible to obtain, in theory, monospecific antisera capable of recognizing a single leukocyte antigen. Several of these antigens do, indeed, appear to predict those which are characterized as strong histocompatibility antigens in man. Although some confusion in terminology still exists, extensive intercourse and interchange of sera between all of the groups have resulted in some common terminology, and it appears certain now that some of the leukocyte antigens characterized by each of the groups can be predictably determined by the others, even though the test procedure may be somewhat different.
F. RED CELLANTIGENS The human erythrocyte is generally believed not to contain human histocompatibility antigens. It should, however, be recognized that a kidney containing blood Group A antigens, as it may (Szulman, 1960), should not be expected to do well when transplanted into a recipient whose serum contains anti-A isoagglutinins. In fact, it does not, and the success rate for renal allografts where an ABO incompatibility exists between donor and recipient, has been significantly less than in compatible combinations (Murray et al., 1965). Recently Dausset and Rapaport (1966) obtained accelerated rejection of skin grafts by recipients who had previously been immunized by injections of red cells bearing antigens A and B. The results, however, are somewhat difficult to interpret since, although the red cells were “practically devoid of leukocytes and platelets,” even trace amounts of these latter cells which are known to contain histocompatibility antigens, might have been responsible for the sensitization rather than the red cells. G. SUMMARY Painstaking and extensive analyses of the correlation of these tissue typing results with the results of both skin and renal allografts have been made (Terasaki et al., 1965, 1966; Starzl et al., 1965a; Stickel et al., 1967; Colombani et al., 1963; Vredevoe et af.,1965). The results may be 6:immarized as follows: It is possible in prospective studies to demon-
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strate more rapid rejection of skin grafts where the donor and recipient pair are incompatible as determined by evaluation of leukocyte isoantigens (Colombani et al., 1963). The correlation of success or failure of renal allografts is more difEcult because of variations in immunosuppressive treatment, surgical techniques, and all the variance of medical care that pertains to as complicated a procedure as renal allografting in the human. This much, however, may be said. In retrospective studies where donor and recipient have been matched after transplantation and the results compared with duration of survival, long-term survival results are better in instances where the donor and recipient pair were well matched. Where they were poorly matched the correlation between tissue typing and long-term clinical results is less apparent. Many good long-term survivals have been obtained in individuals who were apparently poorly matched with their donor. It is also apparent that the correlation of good matching and good clinical results within the first 3 months is poorer than for good matching and long-term survival (Colombani et al., 1963). This is probably due to factors of infection, surgical technique, and clinical problems unrelated purely to histocompatibility. Nevertheless, even within the first 3-month period, survival for matched pairs is somewhat better than those for unmatched. It is apparent from analysis of most of the groups reporting to the Transplant Registry that when the donor is consanguineous with the recipient the latter sunlives longer than when the donor is unrelated. Utilizing the cytotoxic technique of Terasaki in a rather unique situation, i.e. the selection by tissue typing of appropriate ’volunteer” donors among a convict population, it has been possible in a prospective study to improve the results in nonrelated donor-recipient kidney transplants to the point where it approaches that of related individuals (Starzl at nZ., 1965a). One must always realize the inherent difficulty of such a study is that being the most recent one, it has not only the advantage of tissue typing but the advantage of more experience in improved surgical and immunosuppressive techniques. In perhaps a more significant study carried out by Stickel et d . (1967) on human renal transplantation with donor selection by leukocyte typing, seven sibling-to-sibling transplants were studied. In this combination the genetic variance should be more predictable than in the unrelated pairs. There was an excellent correlation between the severity of the rejections noted and the leukocyte incompatibility. The progress made in tissue typing in humans suggests that in the predictable future this will be a precise art. That this is so is supported not only by the preliminary factual correlations suggested above, but by
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theoretical considerations making the assumption that, in the human, histocompatibility antigens responsible for transplant survival or rejection depend upon one locus with three or possibly four alleles. Nevertheless, these techniques cannot be applied to the clinical situation as can typing of blood donors for transfusion. The donation of a kidney by even a highly compatible, volunteer, unrelated donor has social and ethical overtones which are weightier than even the consideration as to whether or not the kidney will function well. Should these in vitro tests become quick, simple, and accurate, however, it might well be possible to type potential cadavers and then to chose, not the kidney donor, but the recipient from among a pool of patients with kidney failwe being maintained by intermittent dialysis with the artificial kidney. VI.
Transplantation of the Human Kidney
A number of aspects of human kidney transplantation have been covered in previous paragraphs, In what follows an attempt will be made to synthesize these elements in a brief discussion of the clinical experience. The report by Hume and associates (1955) contains an excellent review of efforts of human renal allografting up until 1955. The publication by these authors of nine cases of “renal homotransplantation in humans” represents the first major assault on the problem. Nine cases of renal allografting in unmodified human recipients were reported. Of these, four developed measurable function and one secreted urine for 180 days. The rejection response in the human was found to be much less violent and slower to develop than in the experimental animal. Following this report, successful transplantation of the kidney was accomplished between identical twins in 1956 (Merrill et al., 1956); then between nonidentical dizygotic twins in 1960 (Merrill et al., 1960), utilizing X-irradiation as the immunosuppressive agent; and, finally, successful transplantation from the human cadaver in 1963 (Merrill et al., 1963). In this instance, azathioprine, cortisone, and actinomycin C were utilized rather than irradiation. As of 1967 the world experience includes more than 1200 renal allografts ( Murray et al., 1967). A. RECIPIENT SELECITON Table I1 lists factors to be considered in the selection of the recipient of a human renal allograft. Obviously such a procedure should be undertaken only when other methods of treatment have failed. The physician should be certain that there are no reversible elements in the patient’s renal failure and that he is too sick to be maintained with the usual methods of treatment in a comfortable useful life but that he is not sick
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enough so that a major surgical procedure apd the use of immunosuppressive therapy will be contraindicated. Such an individual should be free of other life-threatening extrarenal complications. In essence this means that successful function of a renal allograft should solve his medical problems. Obviously restoration of normal renal function will not cure emphysema, cancer, coronary artery disease, severe diabetes with vascular complications, etc. Although age may be a limiting factor it is the physiological age rather than the exact chronological age which contraindicates transplantation. Thus, the patient at 55 with excellent vasculature may well be a candidate, whereas a patient of 45 with severe TABLE I1 FACTOXS IN RECIPIENTSELECTION ~~
~
Age (physiologic rather than chronologic) Failure t o respond to good conservative management Absence of reversible features Normal lower urinary outflow tract (?) Absence of major extrarenal complications (cerebral, coronary artery disease) Absence of evidence of prior Sensitization (leukoagglutinins, cytotoxic antibodies) Absence of Active progressive glomerulonephrit,is Iliofemoral occlusive disease Infection
Severe malnutrition Paney topenia
atherosclerosis may not. Adult renal allografts have been transplanted into recipients as young as 3 years (Starzl et al., 1966~).There apparently have been no physiological or anatomical problems with the use of such a disproportionately large organ allograft. In most groups it is believed that the presence of an abnormal urinary outflow tract contraindicates renal transplantation. However, in spite of the fact that abnormalities of the bladder and urethra represent an additional hazard to the success of kidney transplantation, at least two groups working in this field have constructed an artificial bladder (i.e., ileal conduit) (Straffon, 1967) prior to transplantation and anastomosed the ureter of the allograft to this. Present infection as well as severe malnutrition contraindicate immediate renal allografting and require that these be corrected insofar as possible before surgery. The demonstration of leukoagglutinins to human leukocytes prior to transplantation is cause for alarm and, although this serological evidence has been reported to yield rapid rejection of the transplanted kidney ( Kissmeyer-Nielsen et al., 1966), the evidence is not at present conclusive that the presence of
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leukoagglutinins totally contraindicates the procedure. If, however, there is evidence for specific leukoagglutinins or cytotoxic antibodies in high titer when recipient serum and donor leukocytes are incubated, the significance is more ominous. B. DONORSELECTION
Donor sources are volunteer living donors, usually blood relatives, although the Denver group has used living convict volunteers. A healthy kidney may also become available as a result of its necessary sacrifice during the course of the surgical procedure for hydrocephalus. The third group is the cadaver donors. Living volunteer donors should have a normal physical examination and a normal pyschiatric evaluation. They should be of the same major ABO blood group, since statistics clearly indicate that crossing major blood group barriers prejudices survival of the allograft. Volunteers should have selective renal arteriography since the presence of multiple renal arteries or abnormalities thereof makes the surgical procedure and thus the ischemic time of the normal kidney prohibitively long. Cadaver donors should not have malignant neoplastic disease because of the possibiIity of transmission to the recipient. The role of tissue typing has already been discussed. At the present time, with the exception of the convict population referred to, the number of living volunteers for any given recipient are not large enough to make prospective tissue typing a practical reality. Should tissue typing prove to have a real predictive value the most practical technique in the immediate future will be to pick the recipient from among a number of uremic patients being maintained on chronic dialysis with the artificial kidney to match the available cadaver donor.
C. DIAGNOSIS OF REJECXION The immunosuppressive management of the post-transplant patient and the complications have previously been described. Several other points about the clinical problem might be made. Anuria for periods as long as 5 weeks may follow transplantation, particularly that of cadaver kidneys. Prolonged anuria even of this degree is not necessarily inconsistent with eventual good renal function and the patient must be treated with immunosuppressive therapy although in reduced dosage during this period until such time as renal biopsy gives unequivocal evidence of irreversible destruction of renal parenchyma. The early diagnosis of rejection is imperative since prompt institution of vigorous therapy may reverse this, improve renal function, and prevent irreversible damage. Clinical evidence of rejection may be manifest by fever, swelling and
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pain over the allograft, decrease in urine volume, and oliguria. Where renal function is good initially, oliguria may be accompanied by decreased urinary sodium concentration and increased urine osmolality. This may not be true in later, more chronic stages of rejection or where renal function is impaired at the onset of rejection. The appearance of proteinuria may herald either rejection or the onset of glomerulonephritis. As pointed out above the latter may be part of the rejection process. Decrease in creatinine clearance and increase in serum creatinine reflect the decreasing filtration rate, Changes in renal hernodynamics incident to rejection may be more precisely measured by the clearance of p-aminohippurate or inulin, although these techniques are cumbersome. A radiohippuran renogram, a radiomercury scintogram, and the clearance of radioactive cobalt, which reflects the filtration rate, and that of radiohippuran, which reflects renal blood flow, may be useful. The technique described by Blaufox et al. ( 1967) whereby radiohippuran clearance may be measured by counting over the head without the necessity for urine collection is useful where obtaining accurate urine samples is difficult. Because a rise in the blood urea nitrogen may reflect fever, tissue breakdown, or the administration of prednisone, it is not as precise a measurement as the serum creatinine. Changes in serum complement probably accurately reflect the rejection process but these occur late and for this reason they are not good indices of early rejection. However, stability of the serum complement level reflects stability of the immunological balance (Carpenter et al., 1967). Selective renal arteriography may be of value in ascertaining the patency of the large vessels and in giving evidence of involvement of secondary and tertiary branches of the renal artery. Increase in the activity of lactic dehydrogenase (LDH) in both serum and urine have been suggested as an index of the rejection process (Prout et al., 1964). This has not been a generally reliable procedure, however. In the first few months following rejection it is probable that the first and earliest evidence of the rejection process occurs in the renal vasculature and that this may be reflected by changes in the intrarenal distribution of blood even before there are gross changes in total renal blood flow. The measurement of intrarenal distribution of blood by the xenon washout technique has been a valuable and early sign of rejection both in the human and experimental animal (Hollenberg et d.,1967). The technique requires catheterization of the renal artery and at present is too cumbersome to be done on a daily basis. However, in at least one case a catheter has been left in the renal artery without harm for a period of a week and frequent measurements of intrarenal distribution of blood flow can be. made by this technique. Should
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it become feasible to make these measurements on a daily basis, this would certainly become the most accurate and early index of rejection. Diagnosis of “late rejection” is best made by renal biopsy and renal arteriography (see vascuIar complications ) . Psychological complications are common and depression and anxiety may reflect both the difficult and stressful postoperative course as well as the anxiety of the physician.
D. PROGNOSIS IN HUMANRENAL ALLOGRAFTING Careful and complete evaluation of data from most of the centers during kidney transplantation has been collected and analyzed by the Kidney Transplant Registry under the guidance of Dr. Joseph Murray (Murray et al., 1967). At the present time it can be said in general that where the kidney donor is a sibling or parent, the patient has a 6575% chance of l-year survival. Approximately half the patients who survive 1 year continue to do well for the 2- or 3-year period in which they have been followed. The other half undergo decline in renal function for the reasons mentioned in the section on complications. Patients who receive kidneys from unrelated donors do not do as well, their chances for l-year survival being approximately 25402. It is of considerable interest that second and even third transplants following initial failures have been successful. In general, if failure of the first transplant has been due to technical difficulties or to thrombosis, chances for a second success are greater than if failure of the initial venture was due to rejection. Nevertheless, even in the latter category a number of second transplants have done well. The influence of tissue typing in modifying these results has been discussed previously. VII.
Transplantation of Other Visceral Organs
A. LIVERAND SPLEEN Of the organs, other than the kidney, which have a potential for clinical transplantation, the liver seems the most promising. Two indications for liver transplantation in man can be considered. First, chronic liver failure, usually as a result of Lannec’s cirrhosis; second, malignancy localized to the liver. Couch (1966) has estimated the number of potential liver recipients in the U.S. in 1963 to be about 4000 and the number of potential liver donors about 6000. Unlike the kidney in human transplantation, the liver must come from a suitable cadaver. The problem of the liver recipient may also be compounded by the fact that the absence of distant metastases in liver malignancy is d8icult to prove. Similarly, patients with cirrhosis resulting from chronic alcoholism may
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be unreliable in following the program of immunosuppression necessary to prevent rejection. Early work in the dog demonstrated the feasibility of two kinds of liver transplantation: ( 1 ) orthotopic, in which the recipient’s own liver was removed and the transplant substituted for it in the anatomical position (Starzl et al., 1965b) and (2) so-called “auxiliary” liver transplantation in which the recipient’s liver was left in place, the allograft placed in the right paravertebral gutter, the donor vena cava interposed into the recipient, terminal, inferior vena cava, the hepatic artery anastomosed end-to-end with the right common iliac artery, and the end of the allograft portal vein anastamosed to the side of the recipient, superior, mesenteric vein. Various modifications of this latter technique have been utilized (Halgrimson et al., 1966; Marchioro et al., 1965). Several forms of immunosuppressive therapy have been employed including azathioprine and prednisone. One of the animals in Marchioro’s series survived for 49 days on the allograft alone ( Marchioro et al., 1965). Starzl et al. (1963b) reported the orthotopic transplantation of human liver in three patients and, in 1964, two more. Halgrimson et al. (1966) report two patients with auxiliary liver transplantations for cirrhosis. The latter two patients died of sepsis in 22 and 34 days after operation. Of the orthotopic transplants, the longest survival was 23 days. Various modes of immunosuppression were used, including azathioprine, prednisone, thymectomy ( in one instance), azaserine, and actinomycin C. Donors were prepared with total-body perfusion and, in addition, isolated perfusion of the liver with cold lactated Ringer’s solution. As of February, 1966, seven attempts at clinical orthotopic liver transplantation have been made (Starzl et at., 1966b) and nine attempts at clinical auxiliary liver transplantation. The longest survival has been 34 days. Complications have been striking and different from those encountered with renal allografts. In many instances, an initial hemorrhagic diathesis with fibrinolysis occurs. This is succeeded by a phase of hypercoagulability in the successfully transplanted recipient which, in at least one instance, has resulted in fatal pulmonary emboli. Gastrointestinal hemorrhage and pulmonary sepsis accompanying liver allografts appear to be more difficult to control. In addition, azathioprine and prednisone, both potential hepatotoxins, have been justifiable sources of concern. Moore et al. (1964) has demonstrated the occurrence of striking vascular insufficiency in the liver undergoing rejection. Areas of vasospasm and underperfusion have been striking. Of interest is the fact that the liver, unlike the kidney, should be, and probably is, capable of a graft-versus-host reaction. This is suggested by the fact that red-cell survival time in canine recipients of orthotopic livers is usually shortened
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during the first several weeks after operation. The allografted liver is noted to contain large amounts of hemosiderin (Starzl et al., 1965b). The variability of liver graft survival in the dog suggests that histocompatibility differences may play a role and that proper tissue typing of human donor and recipient may minimize the toxic immunosuppressive therapy required and thus increase survival. The use of immunosuppressive techniques which do not involve hepatotoxins may also improve results. Nevertheless, the use of antilymphocyte serum ( ALS) in liver transplants has been less effective than the use of azathioprine (Starzl et al., 1966a). More recent results, however, suggest that concentrated y-globulin purified from horse ALS may be more effective, Three of four dogs treated in this fashion have survived at least 1month. Modifications of the various immunosuppressive regimens in dogs, including the use of L-methionine and methionine-”S have increased survival in animal experimentation. In 1965, Starzl et al. (196513) reported fifteen animals still alive from 62 to 342 days postoperatively. Similar improvement has been reported from other laboratories. It is of interest that in the pig a liver allograft has survived for 4 months without immunosuppressive therapy of any sort (Calne, 1967). At the present time, however, results in human liver transplantation have not shown this degree of improvement. Transplantation of the spleen in the human has been reported in five patients (Marchioro et al., 1964). In four of these instances the purpose was to provide a continuous endogenous source of antibody against a malignant tumor. In the fifth case it was hoped that the allograft would supply the immunological deficiency in a child with congenital sex-linked hypogammaglobulinemia. The clinical experience was disappointing. In the patients with tumor, no alteration of the progression of the disease was obtained. In the child with hypogammaglobulinemia, evidence for production of y-globulin by the homografted spleen could not be obtained. Radioisotope spleen scans in one case showed little radioactivity in the allograft 3 months post-transplant. B . XENOGRAFTS
Man’s interest in the blending of limbs and organs from different species is as old as recorded history. The Sphinx shared human and lion’s body. The Chimera, part dragon, part goat, part serpent, lends its name to a product of tissue transplantation today. Ikaros lost his life when the xenografted avian appendages fixed to his limbs by his father, failed to “take” (Fox, 1928). Clinical xenografting in man probably begins with the attempt by Kissam (1844) to graft a pig’s cornea to a human eye. Within recent
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years there has been a flurry of attempts to graft organs from alien species to man. At the moment, these have largely been abandoned but the rationale behind the attempts is of historical and, probably, of future interest. Success in the suppression of the immune response in “homotransplantation” suggested that such measures might be effective in “heterotransplantation.” Since much of the pessimism associated with heterotransplants was based on skin-grafting studies in widely different species-“it seemed unwarranted to extrapolate this experience to renal heterografting among primates, including man” ( Reemtsma et al., 1964). Although kidneys might be available from volunteer human donors, certainly lungs, livers, and hearts would not be. The present unavailability of human cadaver sources undoubtedly prompted attempts to transplant these latter organs from primates. One of the first reported attempts at renal xenografting came from Lyon, France in 1900 (Carrel, 1902), and it is of interest that Perrin working also in Lyon has recently attempted a number of primate-to-human renal xenografts (Perrin et ul., 1966). Starzl (1964), in his review of renal heterotransplantation lists five clinical renal xenografts which are known to have been tried, each with a different type of animal donor. Significant renal function was not obtained in any instance and the longest survival was 9 days. Because of the assumed “insurmountable biological difficulties,” no additional attempts were made. In 1964, Reemtsma et al. (1964) reported four patients who had received kidney transplants from chimpanzees. One of these patients obtained excellent renal function for a period of almost 9 months. Starzl et al. (1964) also has reported a number of cases in which baboon kidneys have been transplanted in man. Although some function was obtained in many of these cases, all of the patients died either of rejection of the graft, hemorrhage, or infection. In none of the baboon kidney transplant cases was success obtained of the magnitude achieved by Reemstma with a chimpanzee. The onset of rejection in the former cases was more intense and more difficult to reverse than with the chimpanzee cases, as, perhaps, might be expected since the chimpanzee belongs to the same superfamily as man whereas the baboon does not. The work of Perper and Najarian (1966) shed some light on this. These authors, studying the relative antibody responses in xenografts and allografts, point out that the recipients of xenografts reject the grafts slightly faster than allografts. In sensitized recipients, xenografts were rejected wholly by humoral mechanisms but allografts were rejected both by cellular and by humoral mechanisms. The transplanted baboon kidneys showed marked infiltration with cells, interstitial edema, and scattered focal interstitial hemorrhages. In four of the transplants
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there was focal fibrinoid necrosis of the walls of the interlobular arteries and arterioles (Starzl et al., 1964). On the contrary, in one of the cases of chimpanzee transplants reported by Reemtsma, the patient died of pneumonia at 2 months at which time the transplanted kidney showed little evidence of rejection. This disparity of histological findings between baboon and chimpanzee kidneys suggests that the more closely related species is rejected with less vigor and possibly by a different mechanism. The recipients of both baboon and chimpanzee kidneys were similarly treated with azathioprine, prednisone, and in some cases actinomycin C and local X-ray. Both baboons and chimpanzees have human blood Groups A and B antigens, and although these are not present on the red cells, they probably do exist in renal tissue, as, indeed, they do in man (Szulman, 1962). In the case of the patient receiving a baboon xenograft with A and B incompatibilities, the drop in anti-A and anti-B titers suggests hemagglutinin binding by the xenograft. In addition to this, in Starzl‘s cases, the preoperative sera of six recipients contained heteroagglutinin for baboon erythrocytes, and in each case the titer fell following transplantation. Two patients received intravenous infusions of chromium-51-labeled donor erythrocytes between 4 and 6 hours following kidney transplantation. These heterologous erythrocytes were removed from circulation with a 50%clearance time of 12 minutes, an observation which suggests biological function of the preformed host heteroagglutinins. Cytotoxicity studies were not performed in the case of the baboon grafts, but Reemtsma’s recipients failed to develop cytotoxins against living chimpanzee lymphoid cells even during clinically evident rejection crises. Although the long-term survival of the chimpanzee kidney in man is unexpected, and, therefore, of great interest, it has not led to further trial in this area. Primate donors are a potential source of infection to the recipient. Viral and bacterial infections which may be subclinical in the donor may become full blown in the recipient under treatment with immunosuppressive agents. Cytomegalovirus and Pneumocystis agents, which are rarely pathogenic in the healthy adult, have been frequent and major problems in allograft recipients. Widespread tuberculosis is difficult to detect in the primate donor and may certainly be transmitted to the recipient. Although it was hoped that not only blood typing but tissue typing might be helpful in selecting primate donors in the one instance in which a potentially suitable donor was chosen by one of the currently accepted methods of tissue typing, rejection of the xenograft was rapid and complete (Reemtsma, 1967). Although efforts to find other species whose tissues may be more compatible with man (the pig,
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for example) must be continued, there is no evidence that renal xenografts transplanted to the human may be successful in the near future.
C. HEART An excellent review of heart transplantation in experimental animals and a presentation of the current problems posed by human cardiac transplantation has been recently published by Shumway and Lower ( 1966). Although the authors neglect the considerable technical contributions by the Russians in this realm, they note that “enough has been achieved in fact, to provoke expression of the concept that only the immunological barrier lies between this day and a radical new era in the treatment of cardiac diseases.” In support of this statement they point out that profiles of cardiac performance in dogs with autotransplantation of the heart surviving for more than 2 years are equal to those found in control animals. They carefully document the major problems posed by human heart transplantation and note that “in the laboratory the beating heart of the donor animal is removed and preserved while the beating heart of the host is excised and discarded.” In the case of the human, the surgeon under these circumstances must legally deprive of life both the donor and the host, leaving to the latter at the present time no precedent for survival by transplantation. This is totally unlike the problem in renal transplantation, where with reasonable safety, the healthy kidney can be removed from a healthy donor and where the recipient can be maintained by periodic dialysis with the artificial kidney long after both of his own kidneys have been removed. The authors are pessimistic about the use of cardiac xenografts. Living donors, of course, are out of the question, and the problem of the definition of death in potential donors seems almost insuperable. “The heart must be living at the time of transplantation even though the donor was dead when the organ was removed. Implied here is complete resuscitation of the heart after it has stopped beating; resuscitation which could not be affected with the heart still in the donor.” This seems a reasonable statement of the problem at present. Heart transplantation in man has been done only once (Hardy et al., 1964). The heart of a chimpanzee was inserted orthotopically in a patient with terminal myocardial failure and apparent multiple emboli from the left atrium or ventricle. Following transplantation the primate heart was paced at the rate of 100 beats per minute and was able to maintain a blood pressure ranging from 60 to 90 mm. Hg. It was, however, increasingly unable to handle a large venous return and approximately 1 hour after transplantation further support of the circula-
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tion was abandoned. No further attempts at heart transplantation in man are known at this writing. D. LUNG
Both allografts and xenografts of the lungs have been accepted by radiation mouse chimeras (Santos et al., 1960). The use of azathioprine has prolonged dog lung allografts from 7 to 8 days in the untreated animal to an average of 30 days (Hardy et al., 1963a). With improved techniques and immunosuppressive therapy, a number of dogs have now survived for 2 years with functioning allografts ( Blumenstock, 1967). Hardy et al. (1963b) transplanted a human lung in a patient who survived for 18 days following the operation to die of renal failure. Technically the procedure was successful. The studies of gas exchange showed the lung to be functioning well, and at autopsy there was little evidence of rejection. The patient had been treated with azathioprine and prednisone. A second case reported by Magovern and Yates (1964) died 7 days following transplantation of a human lung allograft. On day 2 the procedure was technically successful and the lung could be shown to have attained some function for a period of time. Severe infection in the transplant as well as the patient’s own lung obscured definite evidence of rejection. It is apparent that transplantation of lungs in the human is a technically feasible procedure with attainment of respiratory function, and that significant prolongation of survival both in the animal and man may be obtained by immunosuppressive therapy. The indications for this procedure in man and adequate donor sources are clinical problems the colution of which is less obvious than in human renal allografting.
E. ORGANPRESERVATION Were it possible to remove cadaver organs and to store them for a period of days or longer under such conditions that they could be successfully replanted, one part of the difficult problem of organ procurement would be solved. The dream of kidney or liver “banks” from which organs suitably typed could be removed and transplanted into a suitable recipient is still far from realized. At the present writing the most effective technique for preservation is hypothermic hyperbaric perfusion. The maximal period of time, however, after which an organ so treated may be replanted with preservation of life is 2 4 3 0 hours (Manax et al., 1964; Slapak et al., 1967). Further progress in this area is urgently needed if we are to improve human donor procurement.
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VIII.
Endocrine Grafts
An extensive review of the literature on endocrine transplantation up to 1960 is found in Woodruffs book ( 1 9 6 0 ~ )and in the review of Brooks (1962). In all of these tissues the same general laws relating to histocompatibility and graft rejection appear to apply although, at least in certain animal strains, ovary (Krohn, 1959) and parathyroid (Jordan et a?.,1961) may be less antigenic. Because replacement therapy for endocrine deficiencies is presently so satisfactory there is little widespread enthusiasm for attempts to transplant endocrine tissue in man. In the past, however, enthusiastic reports of successful transplantation have been made for a number of endocrine tissues. A.
PARATHYROID
Gaillard (1954) used parathyroid from fetuses and infants dying soon after birth. He cultivated the tissue for a time in a medium containing serum from prospective hosts. Thirty patients treated in this way were reported and of these seven appeared to be “cured and two showed some improvement. No biopsy specimens were examined so the significance of the apparent success is, therefore, uncertain. Murray ( 1958) describes a patient with a jwcularized fetal thyroparathyroid transplant who had dramatic relief of symptoms and a tenfold decrease in supplemental medication immediately after operation. After 2 years there had been no need for supplemental calcium, and the thyroid had begun to take up radioactive iodine in measurable degrees. Murray, however, was uncertain as to the survival of the tissues since no microscopic evidence of this had been obtained. Dunphy and Jacob (1961) also reported dramatic clinical improvement and a rise in serum calcium for a considerable period of time in a patient in whom a goat parathyroid had been transplanted. However, the authors point out that histological sections of the graft removed 7 weeks after operation showed no evidence of surviving parathyroid tissue.
B. PITUJTARY Although the literature contains many references to attempts to transplant pituitary tissue in man, there seems little reason to continue these efforts since the number of individuals with panhypopituitarism who could not be managed by replacement therapy would hardly warrent these attempts. Nevertheless, it is intriguing to note the report of a Russian author ( Bogoraz, 1938) who transplanted glands obtained from young subjects, accidentally killed, into eleven pituitary dwarfs. He re-
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ported on four patients followed for more than 18 months, one of whom had grown 15.6 cm. in height and had ceased to look like a dwarf. This dramatic result has not been duplicated, and it seems probable that allografts of either anterior or posterior pituitary tissue have not been successful. The improvement in clinical well being or in the amelioration of diabetes insipidus have either been psychogenic or due to depots of pitressin in the transplanted posterior pituitary,
C. THYROID Sporadic reports of improvement in cretins or patients with hypothyroidism following thyroid allografts or xenografts have been made. Again, success is incompletely documented. D. ADRENAL
Successful allografts of adrenal tissue in patients with Addison’s disease have been reported by a number of individuals, notably Broster and Gardiner-Hill ( 1946). Again, no histological observations were made. The cases of Woodruff (1953) are particularly pertinent in this regard. Four patients treated by adrenal transplant all showed striking clinical improvement which was maintained without further treatment for periods ranging from 9 months to 1 year. However, adrenocorticotropic hormone ( ACTH ) stimulation showed no increase in corticosteroid output and a biopsy taken 7-10 months after the operation revealed no trace whatever of the graft. The story is similar for grafts of ovaries and testicles as well as pancreatic tissue. In general it may be said that in the absence of chemical or histological evidence, clinical improvement alone cannot be an adequate criterion. Psychogenic influences play too strong a role. In addition to evidence of correction of endocrine deficiency after grafting, one requires microscopic evidence of graft survival and perhaps more important, return of the deficiency state by removal of the graft. That improvement may persist following parathyroid grafting as a result of some form of adaptation was strikingly demonstrated by Sanderson and his colleagues ( 1960). These authors showed that dogs with parathyroid tissue transplants would survive total extirpation of their own glands and would maintain relatively normal serum calcium levels. However, they reacted as did the hypoparathyroid animals to a test of calcium infusion and, more important, did not show the ordinary signs of parathyroid deficiency following removal of the transplant. Efforts to prolong grafted endocrine tissue survival have been made by implanting tissue encased in millipore filter chambers. Theoretically, the
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pores of the chamber are adequate to let in extracellular fluid but small enough to exclude antibody-forming or -containing lymphocytes of the recipient. Clinical trials with ovary, thyroid, and adrenal using this technique have been disappointing (Brooks et al., 1960). Although some temporary evidence of hormone secretion was obtained for short periods, ultimately all the grafts failed. Interestingly, the failure here was not a typical rejection by the host, a reaction which apparently was prevented by the diffusion chamber. However, the pores of the diffusion chamber became plugged with host tissue cells preventing adequate oxygenation and nutrition of the graft. Upon removal, millipore chambers frequently contained only a few apparently viable cells and much fibrous tissue. Occlusion of the pores with calcium salts and other debris also contributes to “suffocation” of the graft ( Bassett and Campbell, 1960). IX.
Corneal Grafts
A large percentage of corneal transplants survive as allografts in man. This appears to be due largely to the fact that the cornea is not vascularized, receiving oxygen and nutrition by diffusion through epithelium and endothelium, thus excluding immunologically competent cells. This “privileged position” appears to protect it from the rejection process. When, however, a successful corneal graft in an animal is transplanted to the anterior chest wall where it is vascularized, it is destroyed in much the same way as any other allograft. Presumably this fact holds also for man. Failure of vascularization thus seems to be the most probable explanation of prolonged survival of the cornea. It has also been suggested that the cells of the graft may have been systematically replaced by host cells and that in this sense it behaves more as bone and vessel grafts do and not as a true allograft (Katzin, 1950). However, although this may be true of the surface endothelium and epithelium of the graft, recent studies of transplanted corneal stroma show survival of donor, sex chromatin characteristics for at least 3 months with no invasion of cells from the host. X.
Grafts of Bone and Blood Vessels
Despite the various conflicting opinions expressed in the past, the general belief at present is that allografts of bone do not survive as functioning tissue but act as “a scaffolding” over which host material may grow. In addition to this purely mechanical function, bone grafts apparently both stimulate and control the process of regeneration. It is also almost certain that massive orthotopic autografts of cortical bone are to a large extent replaced by overgrowth from subadjacent tissue (Wood-
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ruff, 1960a). Similarly, vessel allografts, even if viable, do not survive hut are in part replaced by host tissue and in part persist as inert material. Fresh vessel autografts may survive in part as living tissue, hut they, too, appear to he replaced to a considerable extent by tissue of local origin ( Woodruff, 1960b ) , XI.
Transplantation of Marrow
The transplantation of allogeneic marrow in man is a subject of particular interest since it was the demonstration that once allogeneic marrow could he successfully transplanted following irradiation, the marrow recipient would also “tolerate” skin grafts from animals isologous with the marrow donor. These results prompted the first attempts at kidney transplantation in man. These efforts, though unsuccessful, in turn led to the use of “sublethal” radiation which affected the first successful true renal allograft in man. A wealth of knowledge about the general problems of transplantation and transplant immunology has been derived from animal experiments and human clinical experience with marrow transplantation. Interest in the field began in 1951 when Jacobson and his colleagues (1951) pointed out that the intraperitoneal injection of isologous splenic tissue decreased mortality of mice given large doses of radiation. Subsequent work by a number of investigators suggested that this was due to repopulation of marrow by primitive hemopoietic cells originating in the spleen. Later work (Lorenz et al., 1951) showed that radiation injury might he modified by bone marrow injection in mice and guinea pigs. By the use of chromosome cell markers or the specific female configuration of polymorphonuclear leukocytes in rabbits as well as weak erythrocyte antigens, it could he demonstrated without question that hematopoietic tissue destroyed by large doses of radiation might he replaced by the donor source which would then proliferate, producing erythrocytes, leukocytes, and platelets of donor origin. The link to tissue transplantation was forged by a paper of Main and Prehn (1955) who noted that once the allogeneic marrow graft had “taken,” skin grafts from the marrow donor or isologous animals would also he tolerated. This critical observation confirmed evidence from other experiments that once tolerance had been produced for one tissue, such tolerance holds for all tissues. Since, however, the donor tissue is also immunologically competent, it is quite capable of reacting against the host. The resultant reaction of graft-versus-host, variously termed “homologous” or “secondary” disease, has been extensively studied (Barnes et nl., 1958; Trentin, 1957, 1958). The resulting syndrome characterized by loss of hair, diarrhea, wasting, and dermatitis has been startlingly reproduced in
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an occasional human recipient of bone marrow. According to Woodruff, Migdalska (1958) first described the experiments of Raszek who attempted to treat lymphatic leukemia and pernicious anemia by transplantation of bone marrow in 1939. MacFarland attempted the treatment of hypoplastic anemia with bone marrow transplantation in the absence of immunosuppressive therapy and met with indifferent success (McFarland et al., 1961). The infusion of allogeneic and isogeneic marrow in man for the treatment of leukemia following large doses of radiation has received much attention. The current status of this endeavor was reviewed by Ferrebee and Thomas (1960). Marrow obtained from cadavers and fetuses, ribs removed in surgery and by multiple biopsy, or aspirations of living donors were used. The tissue was injected directly after suspension in Hanks medium and filtering or was preserved frozen in glycerol and injected at a later date. Patients with leukemia were treated with whole-body irradiation. If given slowly enough, patients could withstand 2000 r of continuous whole-body exposure. This dosage was adequate to destroy completely lymphopoietic and hematopoietic function and theoretically malignant cells in marrow and lymphoid tissue. Although some success had been reported in dogs, the therapeutic efficacy of the technique in man was extremely limited. Similar attempts at the treatment of terminal leukemic relapse by total-body irradiation and intravenous infusion of stored autologous bone marrow obtained during remission was only questionably effective ( McGovern et al., 1959). Thomas et at. (1961) reported on five patients with acute leukemia treated by total-body irradiation and bone marrow infusion. As much as 2000r were given. Two of the patients received marrow from identical twin donors. In one patient evidence of marrow repopulation by donor tissue was obtained but not in two others. In one identical twin recipient, marrow function returned but the patient died of recurrent leukemia after 72 days. In the other identical twin, partial marrow reco17ery was evident at autopsy but death from jaundice occurred in 20 days, Since fetal hematopoietic tissue was thought to be immunologically incompetent, and, therefore, incapable of graft-versus-host reaction, this tissue was infused in one recipient. This patient showed no evidence of marrow repopulation at autopsy. Infection by gram-negative bacteria and fungus were major problems. The authors were impressed by the possibility that the failure of marrow survival in one instance might have been due to prior sensitization of the recipient to homologous cells as a consequence of previous transfusion. There was some evidence also that immunologically competent lymphocytes contained in the fresh blood transfused after irradiation might lead to a graft-versus-host reaction
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and they believed that it might be possible to avoid such a reaction by exposing fresh blood to large doses of radiation prior to its administration. Such radiation in theory would destroy the capacity of the transfused lymphocytes to react against the host. The use of autologous bone marrow collected prior to radiation of patients with widely disseminated neopIastic disease and replaced following treatment as an autograft was first reported by Kurnick et al. (1958). Black and colleagues (1959) aspirated autologous marrow from cancer recipients prior to the administration of large doses of nitrogen mustard. The marrow was infused after the mustard had been administered, and the author believed that some protective effect was demonstrated in that marrow recovery seemed to be more rapid in cases so infused than in cases without autologous marrow. These results, however, could not be reproduced by Kretchmar et al. (1963). Impressive hematopoietic improvement has been demonstrated following bone marrow transfusion from a healthy identical twin into his isologous sibling with aplastic anemia. In this instance, lack of antigenic differences obviated the need for immunosuppressive therapy with all its complications and the absence of malignant cells in the recipient further assured success. Since marrow failure due to unknown causes or drugs occasionally spontaneously recovers, the cause and effect here are not entirely conclusive. In most of the instances, however, the rapid hematopoietic response is convincing (Thomas et al., 1964; Robbins and Noyes, 1961; Mills et al., 1964; Humble, 1960). Math4 has had wide experience in allogeneic marrow transplantation in man (Math6 et al., 1959). In a group of subjects studied following accidental exposure to irradiation he found some evidence of temporary survival of the transfused bone marrow, but in none of the individuals so treated was there more than temporary persistence of donor cells ( Math&, 1960). However, he concluded that the temporary repopulation might have been helpful in maintaining the patients until regeneration of their own marrow had occurred. Math6 has summarized his extensive experience with the use of wliole-body irradiation and transfusion of allogeneic hemopoietic cells in the treatment of leukemia (Math6 et d., 1960). The patients treated were infants with various forms of leukemia. Following marrow transfusion one patient had the characteristic signs of the “secondary or homologous syndrome” with fever, anorexia, diarrhea, dermatitis, and desquamation. This syndrome has also been seen by Math4 (Math6 et al., 1963) in adults in whom apparently bone marrow was repopulated by homologous marrow. In none of the published cases has there been more than temporary improvement and a review of the subject by Congdon in 1962 concludes that the “ultimate usefulness of
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bone marrow transplantation as a therapeutic technique in human clinical medicine is still not determined ( Congdon, 1962). Thus, the experience with this technique has been generally discouraging until the publication of a recent case by Math6 and his colleagues (Math6 et al., 1963). In this instance a 26-year-old man with lymphoblastic leukemia received bone marrow from multiple donors. The patient received both X-irradiation and chemical immunosuppressive therapy for marrow transplantation. He was alive and well 6 months after this experience, with evidence of survival of bone marrow obtained from one of the donors. This donor has been previously shown to be the most closely related to the recipient by the histocompatibility test of Dausset (Dausset, 1959). It was thus felt that the recipient had “selected” the most compatible donor and this was confirmed by the fact that skin grafting at a later date showed rejection of skin from all other donors but survival of both the autologous graft and that of the donor whose cells had repopulated the recipient’s marrow. It is of particular interest that the patient underwent a crisis characterized by many of the manifestations of the secondary syndrome and apparently was able to overcome this in spite of the persistence of donor marrow. The patient survived for a tctal of 1 year after transplantation and died of recurrence of his leukemia. The ability of the patient to overcome a suggestive secondary syndrome is of interest since this phenomenon has also been seen in animals. The explanation has been given that, although the host lymphocytic cells are destroyed by radiation, the donor’s more differentiated lymphocytic cells consume themselves in reaction against tissue antigens leaving less well-differentiated cells which become “tolerant” to the host. It is possible, also, that the lymphocytic cells of donor origin undergo changes similar to the transformation achievable in bacterial species ( Avery et al., 1944), allowing them to adapt themselves to host antigens. Attempts to treat lymphoma and leukemia by hematopoietic tissue grafting and the study of the graft-versus-host reaction following marrow allografting, continue to receive intense study (Oak Ridge Natl. Lab., 1964). Although the results cannot still be classified as encouraging, Math6 (Math6 et aE., 1964) has had two patients who have survived for more than 1 year at the present time. MathB’s observation of prolonged survival of grafted marrow in a recipient whose tissues could be shown to have relatively close histocompatibility to the donor, bears further study. It seems quite possible that preselection of the donor by suitable histocompatibility testing might result in a greater number of successes. Similarly, the phenomenon of survival of the more compatible marrow from multiple
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simultaneous donors, is a phenomenon that has been seen in animal work. Apparently competition by the less compatible cells for recipient antibody mechanisms allows the more compatible cell type to survive more easily than if this cell type alone had been transfused. Math6 and his colleagues have shown that the preservation of allogeneic bone marrow for 2 hours at 37°C. reduces the secondary syndrome when it is infused into recipient mice. Myeloid restoration is not affected. The authors report that it appears to reduce the incidence of the secondary syndrome in patients on the basis of a series of twelve so studied (Math4 et al., 1966). Van Bekkum summarizing the present state of the art in 1966 (Van Bekkum, 1966) points out that loss of viability with current techniques of freezing may explain the disappointing results obtained with frozen autologous bone marrow in patients. He describes the encouraging results in preventing acute secondary disease following allografts of marrow by early administration of amethopterin nnd cyclophosphamide. XII.
Transplantation of Human Skin
The ability to transplant human skin has provided a wealth of information about human transplantation immunology which could not have been obtained by transplantation of less available tissue. In therapeutic procedures, skin grafting in man is generally confined to autografts in which the immunological problem does not exist and to allografts only in the treatment of extensive burns. A persistent attempt has not been made to prolong human skin allografts with immunosuppressive therapy because of the constant problem of infection in burned patients. However, prolongation of skin allograft survival in severely burned human subjects has been noted (Dempster and Lennox, 1951; Kay, 1957), and it has been suggested (Rapaport et al., 1964) that increased production of adrenal cortical steroids in severely burned patients may explain such prolongation. Because of the large number of allografts frequently applied it has been suggested that prolongation might be a dose effect ( Lehrfeld and Taylor, 1953) comparable to “immunological paralysis.” It is probable, however, that a number of factors including antigen competition induced by the multiple grafts as well as the sharing of tissue transplantation antigens is responsible for the prolongation of skin grafts in burned patients. Rapaport et al. (1964) have confirmed the prolongation of skin allografts in open-stock male rats following thermal injury and have discussed in detail the possible explanations.
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XIII.
Moral and Ethical Aspects of Human Transplantation
A considerable amount of correspondence and editorializing has been devoted to the knotty problem of the physician’s right to remove a healthy kidney from a living normal donor when that procedure cannot be interpreted as being for the benefit of the donor (Merrill, 1964). Of similar concern are the problems of using primate organ grafts in humans where the possibility of enduring function is presently so slim. The use of cadaver kidneys seems an obvious solution but here, the problem of when the prospective donor is truly “ d e a d constitutes a dilemma. Our skill with the operation of respirators, pace makers, and various resuscitators is such that death is increasingly difficult to define and yet the prospective donor must, of course, be permanently and irrevocably dead before a kidney is removed. Most physicians and surgeons would agree that the potential cadaver donor who, in spite of support of the cardiovasculatory and respiratory systems, has a “flat electroencephalogram” and who fails to show perfusion of cerebral cortex by radio-opaque material injected into the carotid artery is effectively and irreversibly dead, and, provided renal perfusion has been maintained, it is justifiable in this instance to remove a kidney for transplantation. These problems have been dealt with at some length in a recent symposium at the Ciba Foundation ( Wolstenholme and OConnor, 1966). The various facets of the problem have been thoroughly discussed by a group comprising physicians, surgeons, lawyers, social scientists, and theologians. It is now generally agreed that it is morally defensible to remove a healthy kidney from a living donor if the donor has been completely screened from the physical and psychiatric point of view, is capable of understanding the nature of his sacrifice, and is a blood relative. Volunteer donors who are not consanguineous are not at the present time utilized although at one time a group of convicts in a Colorado penitentiary provided a pool of donors for the group working in Denver. The surgical risk to the living donor is less than 0.05%and the S-year excess mortality anticipated by the fact of having one kidney instead of two is less than 2/1000. This figure is comparable to the increased risk in commuter driving for a total distance of only 16 miles per working day (Merrill, 1!364). These facts, plus the prognosis for the recipient, I believe minimize the moral problem where well-motivated related donors are chosen by a conscientious, experienced team. ACKNOWLEDGMENT The author acknowledges with gratitude the valuable assistance of Dr. C. B. Carpenter in the preparation of the manuscript.
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326
JOHN P. MERRILL
Scanlon, E. F., Hawkins, R. A., Fox, W. W., and Smith, W. S. (1985).Cancer 18, 782. Schwartz, R., and Dameshek, W. (1959).Nature 183, 1682. Schwartz, R., and Dameshek, W. (1960).J. Clin. lnuest. 39, 952-958. Schwartz, R., Eisner, A., and Dameshek, W. (1959).J. Clin. Invest. 33, 1394. Shelp, W.D., Wen, S.-F., and Weinstein, A. B. (1966).Arch. Internal Med. 117, 401. Shumway, N. E., and Lower, R. (1966).Ann. N.Y. Acad. Sci. 120, 773. Simonsen, M. (1965).Lancet 1, 415. Singh, L. M., Vega, R. E., Makin, G. S., and Howard, J. M. (1965).J. Am. Med. Assoc. 191, 137. Slapak, M., Chir, M., Wigmore, R. A., and MacLean, L. D. (1967). Transplantation (in press). Snell, G. D. ( 1964). Transplantation 2, 655. Southam, C. M., and Moore, A. E. (1958).Ann. N.Y. Acad. Sci. 73, 635. Stanbury, S. W,, Lumb, G. A., and Nicholson, W. F. (1960).Lancet 1, 793. Starzl, T.E. ( 1964). “Experience in Renal Transplantation.” Saunders, Philadelphia, Pennsylvania. Starzl, T. E., Marchioro, T. L., Talmadge, D. W., and Waddell, W. R. (1963a). Proc. SOC. Erptl. Biol. Med. 113, 929. Starzl, T.E.,Marchioro, T. L., Von Kaulla, K. N., Hermann, G., Brittain, R. S., and Waddell, W. R. (1963b).Surg. Gynecol. Obstet. 117, 657. Starzl, T. E., Marchioro, T. L., Peters, G. N., Kirkpatrick, C. H., Wilson, W. E. C., Porter, K. A,, Rifkind, D., Ogden, D. A,, Hitchcock, C. R., and Waddell, W. R. ( 1964). Transplantation 2, 752. Starzl, T. E.,Marchioro, T. L., Terasaki, P. I., Porter, K. A., Faris, T. D., Herrman, T. J.% Vredevoe, D. L., Hutt, M. P., Ogden, D. A., and Waddell, W. R. ( 1965a). Ann. Surg. 162, 749. Starzl, T. E., Marchioro, T. L., Porter, K. A., Taylor, P. D., Faris, T. D., Herrmann, T. J., Hlad, C. J., and Waddell, W. R. (1965b).Surgery 58, 131. Starzl, T. E., Marchioro, T. L., Faris, T. D., McCardle, R. J., and Iwaski, Y. (1966a).Am. J . Surg. 112,391. Starzl, T.E.,Marchioro, T. L., and Faris, T. D. (1966b).Ann. Internal Med. 64, 473. Starzl, T.E.,Marchioro, T. L., Porter, K. A., Faris, T. D., and Carey, T. A. (1966~). Pedint. Clin. N . Am. 13, 381. Starzl, T.E.,Marchioro, T. L., Iwasaki, Y., Cerilli, G. J., and Porter, K. A. (1967). Transplantation (in press). Stickel, D. L., Amos, D. B., Zmijewski, C. M., Glenn, J. F., and Robinson, R. R. ( 1967) . TrUflSplUfltQtiOn ( in press ) . Straffon, R. ( 1967).Personal communication. Streilein, J. W., Hildreth, E. A., Ramseier, H., and Komblurn, J. (1966a).Ann. Internal Med. 65, 511. Streilein, J. W., Billingham, R. E., and Silvers, W. K. (19661,). J. Am. Med. Assoc. 195, 351. Szulman, A. E. (1960).3. Exptl. Med. 111,785. Szulman, A. E. (1962).J . Erptl. Med. 115,977. Terasaki, P. I., and McClelland, J. D. (1964).Nature 204, 998.
HUMAN TISSUE TRANSPLANTATION
327
Terasaki, P. I., Mickey, M. R., Vredevoe, D. L., and Goyette, D. R. (1965). VOX Sangvinis 11, 350. Terasaki, P. I., Vredevoe, D. L., Porter, K. A., Mickey, M. R., Marchioro, T. L., Faris, T. D., Herrmann, T. J., and Starzl, T. E. ( 1966). Transplantation 4, 688. Terasaki, P. I., Vredevoe, D. L., and Mickey, M. R. (1967). To be published. Thomas, E. D., Herman, E. C., Jr., Greenough, W. B., Hager, E. B., Cannon, J. H., Sahler, 0. D., and Ferrebee, J. W. (1961). Arch. Internal Med. 107, 829. Thomas, E. D., Phillips, J. H., and Finch, C. A. (1964). J. Am. Med. Assoc. 188, 1041. Tilney, N. L., and Murray, J. ( 1967). Transplantation (in press). Tilney, N. L., Collins, J. J., and Wilson, R. E. (1966). New Engl. J. Med. 274, 1051. Trentin, J. J. (1957). Proc. SOC. Erptl. Biol. Med. 96, 139. Trentin, J. J. (1958).Ann. N.Y. Acad. Sci. 73, 799. Van Bekkum, D. W. (1966). Oncologia 20, Suppl., 60. Van Rood, J. J., and Van Leeuwen, A. (1963). J. Clin. Invest. 42, 1382. Veith, F. J., Luck, R. J., and Murray, J. E. (1965). Surg. Gynecol. Ohstet. 212, 299. Vredevoe, D. L., Terasaki, P. I., Mickey, M. R., Glassock, R., Merrill, J. P., and Murray, J. E. ( 1965). “Histocompatibility Testing Series,” Haemotol., 25. Munksgaard, Copenhagen. Waksman, B. H., Arbouys, S., and Arnason, B. G. ( 1961). J. Exp. Mcd. 114, 997. Wilson, R. E., Henry, L., and Merrill, J. P. (1963). J. Clin. Invest. 42, 1497. Wilson, R. E., Bernstein, D. S., Murray, J. E., and Moore, F. D. (1965). Am. J. Surg. 110, 384. Wilson, R. E., Hager, E. B., Merrill, J. P., Corson, J. M., and Murray, J. E. (1967). Proc. 1st Intern. Congr. Transplant. SOC. Paris. Muntsgaard, in press. Wolf, J. S., O’Folghludha, F. T., Kauffman, 11. M., and Hurne, D. M. (1966). Surg. Gynecol. Obstet. 122, 1262. Wolstenholme, G . E. W., and OConnor, M., eds. (1966). “Ethics in Medical Progress with Special Reference to Transplantation.” Little, Brown, Boston, Massachusetts. Woodruff, M. F. A. (1953). Transplant. Bull. I, 8. Woodruff, M. F. A. (1960a). “The Transplantation of Tissues and Organs,” p. 367. Springfield, Illinois. Woodruff, M. F. A. (1960b). “The Transplantation of Tissues and Organs,” p. 434. Springfield, Illinois. Woodruff, M. F. A. ( 1 9 6 0 ~ ) “The . Transplantation of Tissues and Organs,” p. 472. Woodruff, M. F. A., and Anderson, N. F. (1963). Nature 200, 702. Zukowski, C. F. (1965).J. Am. Med. Assoc. 191, 145. Zukowski, C. F., and Ende, N. (1965). Transplantation 3,118.
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AUTHOR INDEX Numbers in italics show the page on which the complete reference is listed.
A
Axelrod, A. E., 217, 265 Awdeh, Z., 68, 79
Ada, G . L., 68, 85, 264,271 Adler, F. L., 261, 262, 267, 288, 321 Aisenberg, A. C.,281,321 B Bach, F. H., 233, 240, 247, 248, 265, Al-Askari, S., 236,266 268, 269, 297, 298, 321 Albright, J. F., 196, 266 Alescio, Zonta, L., 35, 37, 38, 79 Bachniann, R., 11, 82 Baglioni, C., 29, 35, 38, 43, 48, 49, 70, Alexandre, G. P. J., 280, 321 Algire, G . H., 92, 144, 190, 265, 272 75, 79, 80 Bain, B., 239, 246, 247, 256, 258, 265, Alican, F., 311, 322 Allen, J. C., 19, 21, 83, 97, 144 297, 321 Allison, A. C., 186, 187, 245, 265 Bakemeier, R. F., 34, 84 Amend, J. R., 286,287,323 Balazs, V., 66, 79 Amiel, J. L., 280, 317, 318, 319, 322, 324 Baldeschwieler, J. D., 66, 82 Amos, D. B., 297, 298, 299, 300, 321, Ball, M. R., 291, 325 Ballantyne, D. L., Jr., 278, 319, 325 326 Ballieux, R. E., 14, 15, 79 Anderson, H. C., 171, 187 Anderson, N. F., 284, 286,327 Barabas, A. Z., 17, 79 Barker, B. E., 240, 241, 265, 267 Anderson, W. F., 195, 265 Barker, C. F., 201, 202, 203, 265 Andre, J. A., 197, 265 Barkulis, S. S. 162, 187 Andrews, G. A. 317, 323 Barnes, A. D. 295, 321 Antoine, B., 280, 322 Appella, E., 43, 47, 77, 86, 92, 93, 95, Barnes, B. A., 286, 301, 305, 325 97, 99, 130, 137, 142, 144, 145 Barnes, D. W. H., 315, 321 Barth, W. F., 11, 16, 86, 89, 93, 143 Arbouys, S., 286, 327 Bartholomay, A., 299, 325 Armstrong, J. S., 181,188 Bassett, C. A. L., 314, 321 Arnason, B. G., 286, 327 Bassett, E. W., 51, 79 Amaud, C., 293,325 Bauer, D. C., 63, 84 Arnon, R., 27, 28, 80 Beaumont, J. L., 66, 79 Ashley, F. L., 217, 265 Askonas, B. A., 67, 68, 79, 89, 263, 265 Bechet, J. M., 319, 324 Becker, E. L., 12, 89 Asofsky, R. M., 13, 84, 93, 97, 144 Becker, M. J., 67, 79 Atkins, L., 316, 324 Beiser, S. M., 51, 79 Atkinson, J., 286, 301, 305, 325 Belko, J. S., 291, 325 Attar&, G., 225, 265 Benacerraf, B., 12, 13, 15, 16, 17, 28, 30, Auerbach, R., 260, 261, 267 34, 64, 65, 69, 79, 80, 82, 84, 85, Aust, J. B., 278, 322 88, 220, 270 Austin, C. M., 68,85 Bennet, J. C., 35, 80 Auvert, J., 280, 322 Avery, 0. T., 318, 321 Bennett, B., 213, 226, 236, 237, 238, 244, 265, 271 Avogardo, L., 35, 88 329
330
AUTHOR INDEX
Bennett, C. J., 75, 78, 81 Berken, A,, 15, 80 Berman, I., 191, 271 Bernard, J., 317, 324 Bernhard, W., 240, 265 Bernhisel, H., 16, 83 Bernier, G. M., 9, 14, 15, 29, 68, 79, 80 Bernstein, D. S . , 293,327 Berrian, J. H., 200,270 Bersack, S. R., 220, 266 Bessis, M., 206, 268 Beutler, E., 68, 80 Billingham, R. E., 190, 194, 195, 196, 198, 199, 200, 201, 202, 203, 205, 206, 207, 209, 211, 212, 214, 219, 220, 221, 223, 226, 235, 248, 249, 265, 268, 273, 295, 296, 326 Billington, W. D., 248, 265 Binaghi, R. A., 12, 15, 80, 85 Binet, J. L., 197, 265 Birbari, A. E., 289, 321 Birtch, A. G., 306, 325 Black, M. M., 317, 321 Blakely, J. G., 92, 144 Blanden, R. V., 152, 188 Blau, S., 149, 187 Blaufox, M. D., 289, 304, 321 Blaylock, K., 198, 272 Bloch, J. H., 311, 324 Bloch, K. J., 12, 15, 80, 85 Bloom, B. R., 213, 217, 220, 222, 236, 237,238, 244, 265, 266 Blurnenstock, D. A,, 311, 321 Blumenthal, G., 224, 269 Boag, J. W., 211,267 Boak, J. L., 193, 269 Borjeson, I., 240, 241, 245, 266 Bogoraz, N. A., 312, 321 Boksenbaum, M., 292,321 Bolande, R. P., 293, 323 Boltralik, J. J., 162, 187 Bond, V. P., 262, 267 Bornstein, P., 25, 80, 85 Borsos, T.,93, 140, 143 Boughton, B., 224,272 Bower, J. D., 284, 294, 323 Boyce, C. R., 92, 145 Boyden, S. V., 222, 228, 266 Boyse, E. A., 226,271
Bradley, J., 11, 87 Bradley, S. E., 288, 321 Brands, W. C., 152, 188 Bravo, J. F., 294, 321 Brecher, G. K., 260,266 Brecher, P., 199, 206, 266 Brenner, S., 77, 80 Brent, L., 190, 200, 201, 203, 205, 207, 208, 209, 210, 212, 226, 231 265, 266, 297, 321 Bridges, J. M., 297, 321 Brill, J., 154, 187 Brittain, R. S., 306, 326 Broberger, O., 232, 271 Brockmann, H. 154, 187 Brondz, B. D., 226, 227,266 Broner, H. P., 260, 266 Brooks, J. R., 213, 223, 267, 312, 314, 321
Broster, L. R., 313, 321 Brown, J. B., 207, 208, 266 Brown, R. A., 196,266 Brownhill, L. E., 240, 241, 267 Brunner, K. T., 230,266 Buckley, C. E., 3, 27, 30, 33, 64, 80, 85, 97, 98, 144 Buckton, K. E., 192, 266 Buffe, D., 68, 80 Burch, P. R. J., 75, 80, 191, 266 Burnap, T. K., 291, 325 Burnet, D., 196, 266 Burnet, F. M., 59, 76, 80, 196, 266 Burtin, P., 68, 80 Burwell, R. G., 75, 80, 191, 197, 261, 266, 267 Bussard, A. E., 260 Butler, H. E., Jr., 292, 321 Byron, J. W., 240, 266
C Caffrey, R. W., 193, 266 Cahnmann, H. J., 27,28,80 Calne, R. Y., 218, 221, 271, 280, 281, 289, 307, 321, 325 Camey, M., 280, 323 Camphell, C. H., 11, 83 Campbell, J. B., 314, 321 Cannon, J. H., 316,327 Caravano, R., 180, 187
331.
AUTHOR INDEX
Carbonara, A., 35, 38, 79 Carbone, P. P., 198, 272 Carey, T. A., 302, 326 Carlton, B. C., 92, 145 Carpenter, C. B., 278, 287, 297, 304, 321, 322 Carpenter, C. M., 236, 268 Carr, D. H., 239,268 Carrel, A., 308, 321 Cattan, A., 317, 318, 324 Cavelti, P., 154, 187 Cebra, J. J., 9, 11, 25, 27, 31, 32, 63, 68, SO, 83, 94, 143 Celada, F., 298, 321 Ceppellini, R., 298, 321 Cerilli, G. J., 286, 326 Chanana, A. D., 199, 206, 266, 285, 322 Chang, W. W. Y.,12,86 Chaplin, H., 9, 13, 29, 66, 80, 81, 85, 86
Chapman, J. A., 240, 267 Chapman, N. D., 244, 246, 251, 255, 257, 266 Chase, M. W., 217, 220, 222, 265, 266 Chase, R. M., 184, 185, 188 Chasis, H., 288, 321 Chavez, C. M., 310, 323 Chessin, L. N., 240, 241, 245, 266 Chiappino, G. 68, 80, 86 Childs, B., 68, 81 Chir, M., 311, 326 Choppin, P. W., 186, 187 Choules, G. L., 33, 80 Christie, G. H., 193, 269 Church, A. B., 150, 187 Churchill, W. H., Jr., 140, 143 Cinader, B., 94, 95, 104, 140, 141, 143 Cioli, D., 29, 35, 38, 43, 49, 70, 79, 80 Clamp, J. R., 32, 34, 55, 80 Clark, D. S., 288, 322 Clarke, F. H. J., 16, 88 Clegg, J. B., 35, 37, 38, 85 Clein, G. P., 11, 83 Clem, L. W., 9, 80 Cleveland, R. H., 284, 285, 294, 323 Coady, B. K., 191, 270 Coburn, J. W., 293, 321 Cohen, E. P., 262, 263,266 Cohen, M. B., 293, 321
Cohen, S., 9, 11, 18, 19, 22, 27, 29, 30, 33, 34, 65, 80, 81, 82, 84 Cohn, M., 140, 143,225,265 Colberg, J. E., 68, 80 Cole, L. J., 311, 325 Collins, F. M., 152, 188 Collins, J. J., 291, 327 Colombani, J., 298, 299, 300, 321, 322 Compans, R. W., 186, 187 Congdon, C. C., 317, 318, 322 Connolly, J. M., 225, 269 Conrad, F. G., 288, 324 Converse, J. M., 217, 269, 277, 278, 296, 319, 325 Cook, F. E., Jr., 288, 294, 324 Coons, A. H., 225, 269 Cooper, D. B., 260,267 Cooper, E. H., 240, 245,266 Cooper, €I. L., 240, 241, 245, 266, 271 Cooper, M. D., 11, 87,93, 145 Coppleson, L. W., 196, 266 Corson, J. M., 278, 294, 297, 321, 327 Costea, N., 34, 81 Couch, N. P., 278, 287, 305, 306, 322, 325 Courtice, F. C., 190, 273 Cowen, D. M., 196, 197, 199, 218, 258, 264, 268, 284, 322 Craig, L. C., 35, 37, 38, 40, 42, 83 Crasnier, J., 280, 284, 294, 322 Cree, I. C., 317, 325 Creech, O., Jr-, 308, 325 Cronkite, E. P., 199, 206, 266, 285, 32-1 Crowley, J. H., 193, 271 Crowther, D., 11, 83 Cudowicz, G., 234, 266 Culling, C. F. A., 226, 239, 230, 272 Cummings, M. M., 220, 266 Cunningham, D. S., 312, 323 Curnen, E. C., 153, 187
D Dalton, M. L., Jr., 311, 322 Dameshek, W., 197, 265 Dammin, G. J., 200, 218, 268 Davidson, R. G., 68, 81 Dawson, C., 32, 34, 55, 80 Defendi, V., 195, 265 Delaney, R., 27, 33, 55, 57, 61, 83
332
AUTHOR INDEX
Delorme, E. J., 195, 265 Dempster, W. J., 211, 221, 267, 266, 269 Deutsch, H. F., 11, 17, 23, 28, 29, 32, 81, 84, 88 Deverill, J., 5, 17, 82 Dintzis, H. M., 66,86 Dixon, F. J., 211,267 Dixon, G. H., 61, 81 Donch, J. J., 64,82 Doolittle, R. F., 9, 52, 58, 60, 61, 62, 63, 65, 76, 77, 81, 87 Dona, G., 124,143 Dozy, A. M., 23, 83 Dray, S., 4, 24, 25, 26, 32, 33, 68, 80, 81, 84, 86, 87, 104, 105, 113, 120, 127, 130, 131, 137, 139, 143, 144, 220, 268 Dresser, A. M., 33, 34, 80 Dreyer, W. J., 13, 35, 37, 38, 40, 43, 48, 49, 51, 70, 75, 77, 78, 80, 81, 82, 83, 86, 92, 99, 130, 143, 144 Dubiski, S., 4, 24, 81, 94, 95, 104, 140, 141, 143 Duke, D. I., 198, 202, 203, 268, 272 Dunn, T. B., 92,143 Dutt, N. R., 217,265 Dutton, R. W., 267 Dvorak, H. F., 204, 207, 209, 211, 212, 213, 223, 267, 269
E Eady, J. D., 244, 251, 267 Easley, C. W., 9, 29, 35, 38, 43, 49, 50, 80, 81, 86 Ebert, R. H., 193, 267 Edelnian, G. M., 2, 4, 9, 11, 35, 61, 81, 84, 87, 95, 143 Egdahl, R. H., 199, 203, 216, 262, 269, 270 Eisens, H. N., 140, 143 Eisner, A., 280, 326 Eldredge, J. H., 315, 323 Elkins, W. L., 197, 215, 267 Ellsworth, B., 220, 268 Elrod, L. M., 242, 267 Elves, M. W., 240, 242, 245, 267 Ende, N., 293, 327 Endicott, H. G., 260, 266 Engle, R. L., 13, 85, 87
Engleman, E. P. 292,323 Epstein, C., 41, 61, 81 Epstein, W. V., 13, 81 Eraslan, S., 310, 311, 322, 323 Evans, R. S., 34, 82 Everett, N. B., 193, 266
F Fabian, L. W., 310, 323 Facon, M., 34, 84 Fagelman, D., 29,81 Fahey, J. L., 11, 13, 14, 15, 16, 19, 20, 81, 84, 86, 87, 88, 89, 93, 94, 97, 99, 117, 123, 136, 143, 144, 145 Falls, N. G., 205, 267 Fanger, H., 240, 241, 26S, 267 Farber, M. B., 226, 269 Faris, T. D., 299, 300, 302, 306, 307, 322, 324, 326, 327 Farnes, P., 240, 241, 265, 267 Farr, R. S., 11, 12, 83, 86 Feder, B. H., 317,323 Feinstein, A., 3, 4, 8, 16, 24, 25, 33, 49, 53, 81, 86 Feinstein, D., 13, 17, 21, 81, 82 Feldman, D., 292, 322 Feldman, J. D., 219, 220, 223, 267, 270 Feldman, M., 260, 267 Fellows, R. E., 27, 33, 55, 57, 61, 83 Fernandes, M. V., 228,269 Ferrebee, J. W., 316, 322, 327 Figueroa, J. E., 289, 322 Finch, C. A,, 317,327 Fingerhut, A. G., 192,270 Finland, M., 30, 84, 153, 187 Finstad, J., 288, 322 Firschein, I. L., 233, 240, 247, 268, 269 Fischer, R. J., 25, 61, 88 Fisher, B., 284, 325 Fisher, E. R., 284, 325 Fishman, M., 261, 263, 267 Fitzgerald, P. H., 193, 238, 267, 271 Fjeldborg, O., 278, 293, 302, 323, 325 Fleischman, J. B., 1, 7, 28, 30, 33, 55, 60, 64, 68, 81, 95, 97, 98, 99, 143 Florey, H. W., 193, 267 Forbes, M., 153, 188 Ford, C. E., 196, 197, 218, 268 Forland, M., 203, 269
AUTHOR INDEX
Fowler, R., 195, 196, 271 Fox, E. M., 157, 187 Fox, W. S., 307, 322 Fox, W. W., 294,326 Frangk, F., 30, 64, 81 Frangione, B., 9, 15, 23, 28, 32, 33, 53, 81, 82, 87 Franklin, E. C., 9, 11, 13, 14, 15, 17, 21, 23, 28, 30, 32, 33, 34, 80, 81, 82, 84, 87 Frauenberger, G., 16, 83 Freedman, M. H., 64, 82 Freeman, T., 11, 80 Frei, E., 247, 256, 271, 281, 323 Freimer, E. H., 155, 156, 161, 162, 164, 168, 187, 188 Frendzell, J., 154, 187 Frey, J. R., 201, 267 Friedman, E. A., 200, 218, 268, 277, 278, 280, 296, 301, 322, 323, 324 Friedman, H., 230, 262, 263, 267 Frohlich, M. M., 66, 79 Fuchs, S., 32, 82 Fudenberg, H. H., 1, 9, 13, 15, 21, 23, 32, 34, 81, 82, 84, 88, 307, 324 Fujimoto, Y.,213, 223, 267
G Gaillard, P. J., 312, 322 Galley, J. A., 35, 81 Gallily, R., 260, 267 Garcia, E. N., 217, 265 Gardiner-Hill, H., 313, 321 Carver, R. M., 311, 325 Gaston, E. O., 315, 323 Geisser, S . , 77, 86, 93, 97, 99, 130, 137, 142, 145 Cell, P. G. H., 17, 24, 25, 26, 68, 69, 79, 81, 82, 83, 86, 243, 271 Gengozian, N., 104, 143 Gerald, L., 24, 25, 81 Gerdes, J. C., 317, 323 Gerlough, T. O., 16, 87 Geruhty, R. M., 262, 267 Gesner, B. M., 195, 196, 268 Gewurz, H., 288,322 Gill, T . J., 111, 304, 321 Ginsberg, H., 232, 267 Gitlin, D., 1, 11, 30, 82, 84, 278, 325
333
Givol, D., 52, 53, 54, 82, 86, 89, 138, 145 Glassock, R. J., 278, 292, 297, 299, 321, 322, 327 Gleason, R., 278, 297, 299, 321, 325 Glenn, J. F., 299, 300, 326 Globerson, A., 260, 261, 267 Godal, H. C., 17, 82 Gold, M. A., 184, 187 Goldberg, A. F., 241, 245, 269 Goldring, W., 288, 321 Golclstein, G., 102, 126, 143, 145 Goldstein, I., 179, 187 Golstein, P., 319, 324 Conick, H. C., 293, 321 Good, R. A., 11, 32, 61, 82, 83, 87, 93, 145, 278, 288, 322 Goodman, J. W., 28, 33, 55, 64, 82, 86 Gordon, B. D., 151, 188 Gordon, J., 247, 267 Gordon, L. E., 260, 267 Cordon, S., 30, 33, 65, 80, 82 Gorer, P., 276, 322 Cormsen, H., 92, 145 Cough, J., 240, 245, 267 Could, B. S., 67, 84 Govaerts, A., 226, 227, 228, 267 Cowans, J. L., 191, 192, 193, 194, 195, 196, 197, 198, 199, 204, 206, 218, 258, 264, 267, 268, 270, 272, 284, 322 Goyette, D. R., 299, 327 Griisbeck, R., 240, 242, 268, 270 Graff, R. L., 249,268 Graham, J. B., 287, 322 Granboulan, N., 240, 265 Granger, G. A., 226, 268 Granville, N., 316, 324 Gray, H. M . , 97, 144 Gray, J. G., 209, 210, 268, 286, 297, 322 Gray, W. R., 35, 37, 38, 40, 43, 48, 49, 51, 70, 77, 82, 83, 92, 99, 143, 144 Graziani, J. T., 199, 270 Green, F. L., 128, 129, 143 Green, I., 69, 82 Green, M., 37,88 Greenough, W. B., 316,327 Grey, H. M., 5, 13, 14, 15, 19, 21, 26, 29, 65, 82, 83, 87 Griffith, 0. H., 66,-88
334
AUTHOR INDEX
Gross, D. J., 13, 81 Grossberg, A. L., 63, 85 Grubb, R., 17, 19, 82, 83, 97, 144 Gruber, M., 61, 88 Gmber, U.F., 291, 325 Griineberg, H., 128, 144 Gruskin, R., 240, 268 Guest, J. R.,92, 145 Guild, W. R., 301, 324 Gustafsson, B. E., 186, 187 Guttmann, R. D., 287, 304,321,322 Gyorkey, F., 312, 323
H Haber, E., 30, 64, 65, 82 HPIMn, J., 66, 88 Hager, E. B., 289, 294, 301, 316, 322, 324,327 Hager, L. A., 16, 83 Halgrimson, C. G., 306,322 Hall, J. G., 201, 206, 268 Hallenbeck, G. A., 317, 325 Halpern, B., 179, 187 Hamburger, J., 280, 284, 294, 322 Hamers, R., 24, 25, 82 Hamers-Casterman, C., 24, 25, 82 Hamilton, L. D., 222, 266 Hammerman, D., 149,187,188 Hammerstrom, R.A., 262, 267 Hammerstron, S., 186, 188 Hamniond, W. S., 293, 324 Hanaoka, M., 226, 268 Hanshaw, J. B., 291, 323 Hanson, L. A,, 30, 82 Hansson, U.-B., 11, 82 Harboe, M., 5, 17, 34, 66, 82 Hardy, J. D., 247, 256, 257, 270, 310, 311, 322, 323 Hardy, P. H., 154,188 Harris, H., 236, 268 Harris, M., 205, 268 Harris, S., 220, 268 Harris, T. W., 220, 226, 268, 269 Harrison, A. T., 27, 84 Harrison, J. H., 280, 289, 301, 324, 325 Harvin, J. S., 288, 294, 324 Hasegawa, T., 213, 223, 267 Hashem, N., 239,240,247,268 Hathorn, E. M., 12, 83
Haughton, G., 234,235,268 Haugland, R. P., 66, 82 Haupt, H., 11, 82 Hawker, C., 293, 325 Hawkins, R. A., 294, 326 Hechtel, M., 226, 269 Heide, H., 11, 82 Heilman, D. H., 236, 261, 263, 264, 268, 270 Heimburger, N., 11, 82 Heimer, R., 28, 82 Heller, P., 34, 81 Helling, J. W., 35, 49, 86 Hellmann, K.,198, 202, 203, 268, 272 Hellstrom, I., 234, 235, 268 Hellstrom, K. E., 234, 235, 2-55, 268 Helsinki, D., 92, 145 Henning, U., 92, 145 Henry, C., 231,259,269 Henry, L., 200, 218, 268, 277, 278, 296, 322, 323, 327 Heremans, J. F., 9, 11, 16, 84, 87, 88 Herman, E. C., Jr., 316, 327 Herman, J. H., 294, 321 Hermann, G., 306,326 Hermann, T. J., 299, 300, 306, 307, 326, 327 Herzenzerg, L. A., 94, 95, 102, 104, 122, 126, 127, 129, 140, 144, 145 Heston, W. E., 132, 134, 144 Heymann, H., 162, 187 Hickler, R. B., 289, 321 Hildemann, W. H., 195, 201, 206, 218, 249,268 Hildreth, E. A., 214, 272, 298, 326 Hill, R. B., Jr., 291, 292, 323 Hill, R. L., 27,30, 33, 55, 57, 61,83,85 Hill, T. J., 314, 321 Hill, W. C., 63, 83 Hilschmann, N., 35, 37, 38, 40, 42, 83 Hinshaw, D. B., 217,269 Hirsch, J. G., 150, 187 Hirschhorn, K., 233, 240, 241, 245, 247, 265, 268, 269, 297, 321 Hirschhorn, R., 241, 245, 269 Hirst, G. K., 150, 188 Hitchcock, C. R., 308, 303,326 Hlad, C. J., 306, 307, 326 Hobbs, J. R., 11, 83
335
AUTHOR INDEX
Hoehn, R. J., 221,271 Hoffman, H. A., 94, 127, 143 Holden, W. D., 293, 323 Holland, P. V., 289, 324 Hollenberg, N. K., 304, 323 Holm, G., 233,269 Holm, S. E., 183,187 HoImes, K. V., 186, 187 Holmes, N. C., 102, 143 Hong, R., 11, 32, 64,83, 87, 93, 97, 144, 145 Hood, L. E., 35, 37, 38, 40, 43, 48, 49, 51, 70, 77, 80, 82, 83, 92, 99, 143, 144 Hopper, J., 280, 322 Horibata, K., 225, 265 Horn, L., 278,319,325 Hombrook, M. M., 12, 83 Horton, R. W., 153, 188 Hough, L., 32, 34, 55, 80 Howard, D. H., 236, 268 Howard, J. C., 193, 269 Howard, J. M., 284, 326 Howe, J. H., 184, 187 Hubay, C. A., 293, 323 Hudack, S., 201,269 Hudgins, P. C., 220,266 Huisman, T. H. J., 23, 83 Hulley, S. B., 213, 269 Humble, J. C., 317, 325 Hume, D. M., 279, 284, 285, 292, 293, 294, 301, 304, 323, 325, 327 Hume, D. R., 199,203, 269, 273 Hummler, K., 226, 269 Humphrey, J. H., 149, 187 Humphrey, W., Jr., 92, 144 Hungerford, D. A,, 255, 269 Hurez, D., 5, 87 Hurvitz, D., 245, 269 Husband, E. M., 244,245,269 Hutt, M. P., 299, 300, 326 I Ibery, P. L. T., 315, 321 Inderbitzin, T., 224, 269 Trvin, G. L., 198, 272 Isacson, A., 186, 187 Iscaki, S., 64, 84 Ishizaka, K., 12, 83
Ishizaka, T., 12, 83 Isliker, H., 9, 84 Israels, M. C. G., 240, 267 Iwasaki, Y.,286, 287, 307, 323, 326
J Jackson, J. F., 247,256, 257,270 Jacob, S. W., 312, 322 Jacobson, L. O., 315,323 Jacox, €3. F., 291, 323 Jager, B. V., 16, 83 James, A. T., 186, 187 James, D. A., 248, 269 James, K., 240, 243, 270 Janeway, C. A., 278, 325 Janis, B., 12, 86 Janis, R., 149, 187 Jankovic, B. D., 223, 269 Jaquet, H., 27, 83 Jarvis, J. M., 35, 37, 38, 85 Jenkin, C. R., 148, 152, 187, 188 Jenkins, C. C., 15, 89 Jerne, N. K., 231, 259,269 Joel, D., 199, 206, 266 Johnson, E. A., 240, 271 Johnson, G. J., 246, 258, 269 Jolley, W. B., 217, 269 Jones, J. P., Jr., 292, 323 Jordan, G. L., Jr., 312, 323 Joseph, N. H., 221, 271
K Kabat, E. A., 63, 83, 148, 153, 187 Kaliss, N., 203, 242, 269 Kantor, F. S., 151, 188 Kaplan, J. M., 241, 245, 269 Kaplan, M. E., 63, 83 Kaplan, M. H., 154, 155, 157, 164, 165, 168, 169, 187 Kapros, C., 221, 269 Karakawa, W. W., 66, 85, 180, 187 Karush, F., 11, 16, 27, 30, 60, 63, 64, 83, 85, 87, 88 Kasakura, S., 239, 247, 269 Kass, E. H., 150, 187 Kates, M., 186, 187 Katzin, H. M., 314, 323 Kaufman, H. M., Jr., 199, 273, 284, 285, 294, 323, 327
336
AUTHOR INDEX
Kawahara, K., 64, 88 Kay, G. D., 319,323 Keel, A., 224, 269 Keelev, C. E., 132, 134, 144 Kehn, J. E., 97, 145 Kelly, W. D., 288, 322 Kelus, A. S., 4, 17, 24, 25, 26, 68, 79, 81, 82, 83, 86, 94, 144, 243, 267 Kennedy, J. C., 196,269 Kenyon, J. R., 289, 325 Kern, F., Jr., 292, 323 Khorana, H. G., 7 3 , 8 5 Kidd, J. G., 218, 269 Kiho, Y., 67, 83 Killander, J., 29, 87 Kirby, D. R. S., 248, 269 Kirkpatrick, C. H., 308, 309, 326 Kirschbaum, A,, 205, 267 Kisken, W. A., 298, 321 Kissam, R. S., 307, 323 Kissmeyer-Nielsen, F., 278, 289, 293, 302, 323, 325 Klein, G., 226, 227, 272 Klevit, H. D., 239, 270 Klinman, N. R., 16, 63, 83, 87 Knight, E. J., 192, 193, 198, 267 Knight, P., 291, 325 Knopf, P. M., 67, 68, 85, 88 Knox, W . G., 288,323 Koch, A. E., 186,187 Kohler, P. F., 11, 83 Kolff,W. J., 289, 322 Kolodny, R. L., 240, 247, 268 Koprowski, H., 228, 269 Kornblum, J., 214, 272, 298, 326 Korngold, L., 68, 84 Koshland, M. E., 15, 23, 32, 64,81, 83 Koskimies, O., 226, 227, 272 Kosumen, T. U., 207, 209, 211, 212, 267, 269 Kot’ynek, O., 64, 81 Kountz, S. L., 221, 269 Kourilsky, F. M., 12, 15, 80 Kramer, N. C., 184, 187 Krawse, R. M., 66, 85, 155, 180, 187 Kretchniar, A. L., 317, 323 Kretschmer, R. R., 223, 269 Krieg, A. F., 293, 323 Krisher, J. A,, 306, 325
Kritzman, J., 66, 83 Krohn, P. L., 295, 312, 321, 323 Kuff, E . L., 13, 84, 86, 97, 119, 130, 144, 145 Kulpina, L. M., 67, 85 Kunkel, H. G., 5, 13, 14, 15, 17, 19, 20, 21, 22, 23, 26, 29, 34, 66, 82, 83, 84, 87, 89, 97, 130, 144, 171, 187 Kurnick, N. B., 317, 323 Kurrus, F. D., 310, 323 Kushner, D. S., 181, 188 Kushner, I., 154, 168, 169, 187 Kuss, R., 280, 323 Kuttner, A. G., 172, 188 Kyle, R. A., 317, 325
1 Lahecki, T. D., 310, 323 Lagercrantz, R., 186, 188 Lagnaux, S., 25, 82 Laitha, M. A. G., 240, 266 Lalanne, C. M., 280, 317, 322, 324 Lanim, M . E., 9, 11, 13, 24, 25, 30, 61. 64, 65, 84, 85, 87, 89, 97, 145 Lanckman, M., 33, 84 Landy, M., 281, 323 Lange, C. F., 182, 188 Lange, K., 184, 187 Lapanje, S., 64,88 Larrieu, M . J., 317, 324 Lathrop, A., 133, 134, 144 Laurell, 0.-B., 11, 82 Laurent, T. C., 149, 188 Lavender, A. R., 203, 269 Lawler, S. D., 18, 19, 84 Lawrence, H. S., 217, 236, 266, 269, 277, 278, 296, 325 Lawrence, T. G., 23, 84 Lehovitz, H. E., 27, 33, 55, 57, 61, 83 Leddy, J. P., 34, 84, 291, 323 Leder, P., 92, 144 Lederberg, S., 76, 84 Leduc, E. H., 225,269 Lee, E. H., 12, 83 Lee, H. M . , 284, 285,294, 323 Legrain, M., 280, 323 Lehrfeld, J. W., 200, 272, 319, 323 Lenard, J., 32, 84
AUTHOR INDEX
Lennox, B., 211,267, 319,322 Lennox, E. S., 4, 34, 69, 81, 84, 225, 243 265, 267 Levey, R. H., 242, 269 Levin, R. H., 281, 323 Levy, R. N., 292, 325 Lewis, D., 67, 85 Lewis, J., 247, 269 Lewis, N. R., 236, 271 Lieberman, R., 92, 93, 94, 99, 101, 104, 105, 113, 120, 122, 126, 127, 129, 130, 131, 133, 137, 139, 143, 144, 145 Lillehei, R. C., 311, 324 Lilly, F., 226, 271 Lind, K., 11, 34, 66, 82, 85 Lindqvist, K., 63, 84 Lindquist, R. R., 287, 322 Ling, N. R., 240, 243, 244, 245, 269, 270 Linscott, W. D., 195, 268 Little, J. R., 140, 143 Litwin, S. D., 13, 15, 19, 20, 21, 22, 23, 83, 84, 87, 89 Liu, C. T., 184, 188 Liungman, S., 66, 88 Loeh, L., 133, 134, 144 Lo Grippe, G. A., 193, 271 Longerheam, J. K., 311,324 Lorenz, E., 315, 323 Lostumho, M. M., 289, 324 Lowe, M., 217,265 Lowenstein, L., 239, 246, 247, 256, 258, 265, 269, 297, 321 Lower, R., 310, 326 Lowey, S., 66, 88 Lowtit, J. E., 315, 321 Luck, R. J., 284,327 Lumb, G. A., 293, 326 Lyampert, I. M., 165, 188 Lycette, R. R., 238, 271 Lynch, C. J., 138, 144 M hlcBride, R. A., 194, 196, 270 McCahe, R. E., 288, 323 Macalalag, E. V., Jr., 304, 325 McCardle, R. J., 307, 326 McCarthy, J., 66, 83
337
McCarty, M., 155, 171, 179, 187, 318 321 McCelland, J. D., 298, 326 McCluskey, J. W., 220, 270 McCluskey, R. T., 220, 270 McConahey, P. J., 211, 267 McCracken, B. H. 308,325 McCrory, W. W., 184,188 McCulloch, E. A., 196, 269 McDevitt, H. O., 69, 84, 95, 144 McFarland, W., 261, 263, 264, 270, 316, 324 McGavic, J. D., 291, 325 McCehee, W., 21, 82 McGeown, M. G., 297,321 McGhee, B., 29, 81 McGovern, J. J., Jr., 316, 324 McCregor, D. D., 191, 192, 195, 196, 197, 198, 199, 218, 258, 264, 267, 268, 270, 284, 322 McIntire, K. R., 13, 84, 86, 97, 130, 144 McIntosh, D. A., 288, 293, 294, 324 MacKaness, G . B., 152, 188 McKelvey, E. M., 11, 81 MacKenzie, M. R., 17, 81, 84 McKhann, C. F., 200,270 MacLean, L. D., 247, 265, 267, 311, 326 MacLeod, G. M., 318,321 McMaster, P. D., 201, 269 McNall, E. G., 217, 265 McPhaul, J. M., Jr., 288, 293, 294, 324 Mage, R. G., 26,27,84 Magee, J. H., 284, 294, 323 Magovern, G. J., 311, 324 Maier, P., 15, 89 Main, J. M., 279, 315, 324 Mainland, D., 191, 270 Maisonnet, M., 280, 322 Maize], J. V., 67, 87 Makin, G. S., 284, 326 Makinodan, T., 196, 266 Malucci, L., 245, 265 Manax, W. G., 311,324 Mandel, M. A., 93, 144 Mandy, W. J., 9, 88 Mangalo, R., 64,84 Manner, G., 67, 84 Mannick, J. A., 199, 216, 217, 262, 270
338
AUTHOR INDEX
Mannik, M., 5, 13, 26, 34, 64, 65, 82, 84 Mansa, B., 11,85 Mantel, N., 92, 144 Marchalonis, J., 2, 4, 9, 11, 61, 84 Marchesi, V. T., 192, 270 Marchioro, T. L., 284, 286, 287, 299, 300, 302, 306, 307, 308, 309, 322, 323, 324,326,327 Margoliash, E., 41, 75, 84 Markowitz, A. S., 181, 182, 188 Markowitz, M., 172, 188 Marks, E. X., 315, 323 Marshall, D. C., 200, 218, 268, 277, 278, 296, 322, 323, 324 MarshaI1, S., 11, 313, 325 Marshall, W. K., 240, 270 Mirtensson, L., 18, 19, 21, 23, 83, 84, 97, 144 Masson, P. L., 9, 84 Mathb, G., 197, 265, 280, 317, 318, 319, 323, 324 Matoltsy, M., 236, 272 Mattius, P. L., 298,321 Mauel, J., 230, 266 Mayer, D. J., 284, 324 Mayer, M. M., 153, 187 Medawar, P. B., 190, 200, 202, 203, 205, 207, 208, 209, 210, 211, 212, 221, 226, 231, 242, 265, 266, 269, 270, 276, 297, 321, 324 Melchers, F., 34, 84 Mellman, W. J., 239, 270 Mellors, R. C., 66, 68, 83, 84 Meltzer, M., 23, 82, 84 Mendelson, C. G., 292,321 Merler, E., 30, 84 Merrill, J. P., 200, 218, 268, 277, 278, 279, 280, 287, 289, 292, 294, 296, 297, 299, 301, 304, 320, 321, 322, 323, 324, 325, 327 Merryman, C., 16, 85 Merwin, R. M., 92, 93, 101, 144 Meshaka, G., 5, 26,87 Metchnikoff, E., 286, 324 Metzger, H., 9, 29, 62, 64, 81, 84, 93, 144 Meyer, K., 148, 149, 188 Meyerserian, M., 154, 168, 169, 187 Micheli, A., 9, 84
Michie, D., 141, 144, 196, 266 Mickey, M. R., 277, 299, 327 Migdalska, Z., 316, 324 Migita, S., 35, 86 Mihaesco, C., 5, 26, 87 Miller, B. F., 279, 292, 301, 323 Miller, C. P., 260, 267 Miller, E. J., 66, 85 Miller, F., 29, 84, 93, 144 Miller, J. F. A. P., 283, 324 Mills, J. A,, 244, 270 Mills, S. D., 317, 325 Milstein, C., 29, 35, 37, 38, 39, 40, 41, 42, 43, 49, 50, 51, 59, 60,70, 76, 77, 78, 84, 85, 144 Minna, J. D., 122, 127, 144 Minden, P., 12, 86 Mintz, B., 235,270 Mishell, R., 93, 94, 99, 122, 143, 144 Mishell, R. I., 259, 270 Misra, D. K., 316, 324 Mitchell, R. M., 221, 270 Mitchison, N. A., 190, 201, 226, 265, 270 Mitus, W. J., 197, 265 Moller, E., 234, 235, 270 Moller, G., 234, 235, 255, 268, 270 Moen, J. K., 236,270 Monaco, A. P., 277, 286, 295, 322, 325 Mongan, E. S., 291, 323 Montano, A., 317, 323 Moon, H. D., 226, 228, 229, 262, 267, 271 Moore, A. E., 294, 326 Moore, F. D., 291, 293, 306, 325, 327 Moore, R. D., 261, 263, 271 Moorhead, J. F., 261, 270 Moorhead, P. S., 239,270 Moor-Jankowski, J. K., 17, 83, 94, 144 Moreno, G . D., 206, 268 Morgan, A. R., 73, 85 Morgan, J. M., 292, 321 Morlino, M. J., 195, 268 Morns, B., 201, 260, 268 Morton, J. I., 29, 81 Moscona, A. A., 235, 270 Moseley, R., 291, 325 Mosely, R. V., 221, 270 Mota, I., 12, 85
339
AUTHOR INDEX
Motulsky, A. G., 41, 61, 81 Mowbray, J. F., 289, 325 Moynihan, P. C., 247, 256, 257, 270 Mozes, E., 34, 87 Miiller-Eberhard, H. J., 140, 144 Mulholland, J. H., 277, 278, 296, 319, 325 Muller, P., 24, 81 Mumaw, V. R., 261, 263, 271 Munn, E. A., 4, 8, 81 Munro, A. J., 68, 85 Murray, J. E., 221, 270, 278, 280, 281, 284, 285, 286, 287, 289, 291, 293, 294, 299, 301, 304, 305, 312, 321, 322, 323, 324, 325, 327 Muschel, L. H., 153, 188
N Nachman, R.L., 13,85,87 Nagel, B., 247, 269 Najarian, J. S., 219, 220, 223, 267, 270, 292, 308, 323, 325 Nakamoto, S., 289, 322 Nathan, H. C., 281,325 Nathan, P., 203, 270 Natvig, J. B., 19, 21, 85, 130, 144 Nay, H. R., 288,323 Nedey, R., 280, 323 Neely, W. A,, 310, 323 Nelp, W. B., 291, 325 Nelson, C. A., 3, 27, 28, 30, 33, 85, 97, 98, 144 Nelson, R. A., Jr., 288, 325 Nelson, S. D., 297, 321 Newsome, J., 243, 270 Nezlin, R. S., 67, 85 Nicholson, W. F., 293, 326 Nicks, P. J., 297,321 Nigogosyn, G., 287, 322 Nilsson, H. R., 140, 144 Nirenberg, M., 92,144 Nisonoff, A., 25, 26, 64,81, 83, 86, 97, 144 Nitowsky, H. M., 88,81 Noelken, M. E., 3, 27, 30, 33, 64, 85, 97, 98, 144 Noll, H., 67, 87 Nordin, A. A., 231, 259, 269 Nordman, C. T., 240, 242, 268, 270
Norman, A,, 192, 270 Norton, W. L., 67, 85 Nossal, C. J. V., 68, 85, 264, 271 Notake, K., 226, 268 Notani, G. W., 68, 85 Nowell, P. C., 192, 238, 240, 245, 248, 253, 255, 264, 269, 271, 273 Noyes, W. D., 317, 325 Nussenzweig, R. S., 16, 85 Nussenzweig, V., 13, 15, 28, 30, 34, 64, 65, 84, 85
0 O’Brien, J. P., 293, 325 O’Connor, M., 320, 327 Oettgen, H. F., 12, 15, 80,85 O’Folghludha, F. T., 199, 273, 285, 327 Ogden, D. A., 299, 300, 308, 309, 326 Ogsten, A. C., 149,188 Old, L. J., 226, 271 Olesen, H., 11, 85 Oliner, H., 316, 324 Olsen, S., 278, 289, 293, 302, 323, 325 Onoue, K., 9, 63, 85 Oppenheim, J. J., 247, 256, 269, 271 Order, S. E., 306, 325 Ortiz, J., 151, 188 Osawa, E., 153, 188 Osgood, E. E., 239,271 Osler, A. E., 154, 188 Osserrnan, E. F., 15, 28, 88 Osterland, C. K., 13, 66, 85, 140, 143, 180, 187 Ottenson, J., 192, 271 Ottoman, R. E., 192, 270 Oudin, J., 4, 17, 18, 24, 25, 26, 80, 85, 243, 267 Oudin, J. J., 94, 144 Oura, H., 67, 87 Ovary, Z., 11, 15, 16, 80, 82, 85, 86, 88 Owen, K., 289, 325 Ozer, F. L., 66, 86 P Pain, R. H., 11, 30, 81, 86, 95, 97, 143 Palm, J. 249, 250, 271 Palmer, J. W., 148, 188 Papermaster, B. W., 61, 82 Pappenheimer, A. k.,29,86
340
AUTHOR INDEX
Paraskevas, F., 33, 86 Park, 0. K., 293, 324 Parker, S. J., 203, 272 Parkhouse, R. M . E., 244, 266 Parks, J. J., 262, 266 Parrish, A. E., 184, 187 Patnode, R. A., 220,266 Patterson, R., 12, 86 Paul, W. E., 69,82 Pauling, L., 41, 89 Peacocke, N., 297, 321 Pearce, J. D., 244, 251, 267 Pearl, M. A., 308, 325 Pearmain, G., 238, 271 Peart, W. S., 289, 325 Pease, G. L., 317,325 PArez-Tamayo, R., 223, 269 Perham, R., 43, 47, 86, 92, 95, 99, 144 Perlmann, H., 226, 227, 272 Perlmann, P., 186, 188, 226, 227, 232, 233, 269, 271, 272 Pemis, B., 68, 80,86 Perper, R. J., 308, 325 Perrin, J,, 308, 325 Perry, S.,247, 269 Persky, L., 293, 323 Petermann, M. L., 29, 86 Peters, G. N., 306, 308, 309, 322, 326 Peters, R. N., 201, 208, 268 Peterson, E . W., 294, 324 Peterson, M., 217, 269 Peterson, N., 287, 322 Petz, A. J., 193, 271 Phillips, J. H., 317, 327 Phillips, W. D., 67, 88 Pickren, J. W., 287, 322 Pierce, A. E., 16, 86 Pierce, J. C., 280, 325 Pietruszkiewica, A., 149, 188 Piggot, P. J., 28, 52, 53, 54, 58, 59, 60, 62,86, 138,145 Pike, M. C., 192, 266 Pilgrim, H. I., 93, 145 Pink, J. R. L., 41, 59, 60,86 Pinkerton, W., 218, 268 Pinnell, S. R., 29, 89 Poljak, R. J., 66, 86 Pollara, B., 11, 32, 83 Polmar, S. H., 18, 19, 86, 88
Porter, K. A,, 218, 221, 271, 286, 287, 289, 299, 300, 302, 306, 307, 308, 309, 322, 323, 324, 325, 326, 327 Porter, R. R., 2, 16, 22, 28, 29, 30, 33, 52, 53, 54, 55, 58, 59, 60, 63, 64, 81, 82, 86, 88, 89, 95, 97, 98, 99, 138, 144, 145 Posborg-Petersen, V., 278, 289, 293, 302, 323, 325 Potter, M., 13, 35, 43, 47, 77, 80, 84, 86, 92, 93, 94, 95, 97, 99, 101, 102, 104, 105, 113, 119, 120, 122, 126, 129, 130, 131, 133, 137, 139, 142, 143, 144, 145 Poulik, M. D., 27, 28, 86, 95, 143 Poutasse, E. F., 289, 322 Prahl, J. W., 54, 86 Preaux, J., 299, 300, 321 Prehn, R. T., 190, 265, 272, 279, 315, 324 Prelli, F., 32, 33, 81 Prendergast, R. A., 9, 12, 17, 83, 88, 93, 145, 219, 271 Press, E. M., 9, 28, 52, 53, 54, 55, 58, 59, 60, 62, 64, 80, 81, 86, 89, 95, 98, 99, 138, 143, 145 Pressman, D., 9, 15, 34, 63, 65, 85, 87, 89 Priest, R. J., 193, 271 Prout, G . R., Jr., 284, 294, 304, 323, 325 Pruzansky, J. J., 12, 86 Putnam, F. W., 9, 14, 15, 29, 35, 37, 38, 40, 42, 43, 48, 49, 50, 63, 77, 79, 80, 81, 86, 88, 89
Q Quinn, R. W., 149, 188
R Radzimski, G., 64, 65, 87 Rams, J. J., 203, 269 Ramseier, H., 207, 208, 209, 212, 213, 214, 271, 272, 298, 326 Rapaport, F. T., 184, 188, 217, 269, 277, 278, 296, 299, 319, 322, 325 Rapp, H. J., 93, 140, 143 Rask-Nielson, R., 92, 145 Rasmusen, B. A., 17,86 Rasmussen, H., 293, 325
341
AUTHOR INDEX
Raynaud, M., 64,84 Razavil, L., 240, 271 Rebuck, J. W., 193,271 Redmon, L. W., 92, 93, 101, 144 Reemtsma, K., 308, 309, 325 Reid, R. T., 12, 86 Reid, T. R., 315, 323 Reisfeld, R., 240, 241, 266 Reisfeld, R. A., 26, 32, 33, 34, 84, 86, 87 Remington, J. S., 30, 84 Rendall, J. M., 221, 271 Retan, J. W., 277, 296, 322 Retik, A., 304, 323 Reynolds, C. A., 23, 83 Reynolds, E. S., 292, 322 Rice, E., 236, 268 Rich, A , , 67, 79, 83, 88 Rich, A. R., 236, ,071 Richards, F. F., 30, 64, 65, S2 Richardson, A., 130, 145 Riddle, J. M., 193, 271 Rieke, W. O., 193, 257, 266, 271 Rifkind, D., 291, 307, 308, 309, 323, 324, 326 Rigas, D. A., 239, 240, 271 Rhodes, J. M., 263, 265 Robbins, J. B., 25, 80 Robbins, J. H., 240, 271 Robbins, M. M., 317, 325 Robert, L., 179, 187 Roberts, K. B., 240, 270 Roberts, M. S., 17,88 Robertyon, C. L., 92, 145 Robinson, R. R., 299, 300,326 Robson, M. J., 315, 323 Rockey, J. H., 12, 16, 63, 83, 87 Rogentine, G. N., 11, 87 Roholt, 0. A,, 34, 64,65, 87 Rosen, F. S., 11, 82, 247, 268, 278, 325 Rosen, S. M., 304, 323 Rosenau, W., 226, 227, 228, 229, 262, 267, 271 Rosenberg, L. T., 95, 140, 144, 145 Rothbard, S., 150, 164, 188 Rothfield, N. F., 9, 87 Rowe, A. J., 3, 81, 87 Rowe, D. S., 11, 13, 30,68, 86, 87, 88 Rowlands, D. T., Jr., 291, 307, 323, 324 Rowley, D., 152,188
Rubin, A. D., 240, 241, 245, 266, 271 Rubin, A. L., 68, 88 Rubini, M. E., 293, 321 Ruohani, J., 292, 325 Russell, P. S., 209, 210, 218, 220, 246, 258, 268, 269, 272, 277, 286, 295, 297, 316, 322, 324,325 Ryan, M., 149,188 Rytel, M., 151, 188
5 Sabesin, S. M., 238, 271 Sachs, L., 232, 267 Sahler, 0. D., 316, 327 Salmon, C., 280,322 Samuelson, J. S., 284, 325 Sanderson, P. H., 313, 325 Sandor, G., 9, 87 Sandson, J., 149, 187, 188 Santos, G. W., 311, 325 Sasaki, M. S., 192, 270 Saubier, E., 308, 325 Savitsky, A., 292,325 Scanlon, E. F., 294, 326 Scharff, M. D., 67,87,8& Schejter, A,, 75, 84 Schindler, R., 230, 266 Schlegal, J. U., 308, 325 Schmidt, P. J., 289, 324 Schmidt, W. C., 161, 180,188 Schnappauf, H. P., 199, 206, 266, 285, 322 Schneider, M., 317, 318, 324 Schoenberg, M. D., 261,263,271 Schooley, J. C., 191, 271 Schreibman, R. R., 240, 268 Schrek, R., 238, 271 Schubothe, H., 34, 82 Schultze, H. E., 9, 11, 16, 82, 87 Schuniacher, W. C., 291, 325 Schwartz, J. H., 35,87 Schwartz, R. S., 197, 265, 280, 316, 324, 326 Schwartzman, R., 12, 87 Schwarz, E., 132, 145 Schwarz, H. K., 132, 145 Schwarz, M. R., 257, 271 Schwarzenberg, L., 317, 318, 319, 324 Scothome, R. J., 197, 200, 271
342
AUTHOR INDEX
Scott, J. T., 11, 83 Seastone, C. V., 149, 150, 151, 187, 188 Seijen, H. G., 61, 88 Sekiguchi, M., 319, 324 Sela, M., 27, 32, 34, 64, 69, 80, 82, 84, 87, 95,144 Seligman, M., 5, 26, 87 Sell, S., 25, 69, 87, 93, 117, 136, 143, 243,271 Shapiro, A. L., 67, 87 Sharp, J. A., 261,271 Sharp, J. T., 154, 188 Shbil, A. G. R., 221, 270, 291, 325 Shelp, W. D., 290, 326 Shelton, E., 315, 323 Sheppard, C., 260,273 Shinoda, T.,35, 37, 38, 48, 89 Shumcart, W. A., 306, 325 Shumway, N. E., 310,326 Sieker, H. O., 297, 321 Silvers, W. K., 195, 198, 199, 200, 202, 205, 206, 219, 221, 223, 235, 249, 250, 252, 255, 264, 265, 268, 271, 273, 295, 296, 326 Simek, L., 64, 81 Siminovitch, L., 196, 269 Simmons, E. L., 315, 323 Simms, E. S., 140, 143 Simon, V., 184, 187 Simons, M. J., 195, 196, 271 Simonsen, M., 195, 211, 272, 298, 326 Singer, S. J., 9, 32, 33, 58, 61, 62, 63, 65, 76, 77, 80, 81, 84, 87 Singh, K. P., 149, 188 Singh, L. M., 284, 326 Sitterson, B. W., 317, 323 Sjoquist, J., 33, 34, 66, 87, 89 Skalba, D., 17, 87 Slapak, M., 311, 326 Slayter, H. S., 67, 84 Sloan, R. F., 217, 265 Slot, G. M. J., 11, 83 Small, P. A., Jr., 9, 11, 25, 26, 30, 31, 32, 33, 34, 61, 80, 84, 86, 87, 94, 97, 143, 145 Smith, C., 162,187 Smith, E. L., 16, 83, 87 Smith, H. W., 288,321 Smith, J. R.,288, 294, 324
Smith, L. F., 75, 87 Smith, L. L., 291,325 Smith, W. S., 294,326 Smithies, O., 77, 87 Smyth, C. J., 294,321 Smythe, C., McC., 292,321 Snell, G. D., 203, 234, 249, 268, 272, 277, 326 Solomon, A., 5, 9, 11, 12, 13, 29, 83, 87, 88, 93,145 Solomon, J. B., 195, 272 Solomon, J. M., 263, 267 Soulier, J. P., 280, 322 South, M. A., 11, 87, 93, 145 Southam, C. M., 294,326 Sparrow, E. M., 184, 188 Spector, W. C., 224, 272 Speer, F. D., 317, 321 Spent, J. F. A., 147,188 Spicer, E., 240, 242, 243, 267, 270 Spiegelberg, H. L., 64, 87 Spiro, D., 218, 272 Springer, G. F., 152, 153, 188 Staats, J., 131, 134, 138, 145 Staehelin, T., 67, 87 Stanbury, S. W., 293,326 Stanier, J. E., 149, 188 Stark, R. B., 288, 323 Starzl, T. E., 281, 284, 2.86, 287, 299, 300, 302, 306, 307, 308, 309, 322, 323,324, 326, 327 Stavitsky, A. B., 283, 267 Steenburg, R. W., 291, 325 Stein, S., 13, 85, 87 Steinberg, A. G., 14, 18, 19, 20, 21, 86, 88, 97, I45 Steiner, L. A., 66, 88 Steinmuller, D., 195, 206, 265 Stemke, G. W., 24, 25, 61, 88 Stengle, T. R., 66, 82 Stenzel, K. H., 67, 88 Stetson, C. A., 218,272 Stickel, D. L., 299, 300, 326 Stiehm, E. R., 23, 29,81, 82 Stimfling,J. H., 130, 145, 203, 235, 272 Stobo, J. D., 9, 88 Stolinski, C., 221, 271 Stollerman, G. H., 151, 175, 188 Stone, M., 140, 144
AUTHOR INDEX
Stone, M. L., 317,321 Storey, R. H., 260, 273 Straffon, R., 302, 326 Streilein, J. W., 208, 213, 214, 271, 272, 295, 296, 298, 326 Strober S., 204, 272 Strong, L. C., 133, 134, 145 Struthers, J. E., Jr., 292, 323 Stryer, L., 66, 82, 88 Stuart, A. E., 232, 272 Sturgis, S. H., 314, 321 Surmont, J., 317, 324 Svec, K. H., 165, 187 Svet-Moldavsky, G., 227, 272 Swan, H. T., 11,83 Swift, H. F., 236, 270 Szenberg, A., 68, 85, 195, 196, 272 Szulman, A. E., 299, 309, 326
T Tachibana, D. K., 95, 140, 144, 145 Takacs, F. J., 301, 324 Takatsuki, K., 15, 28, 88 Taliaferro, L. G., 211, 272 Taliaferro, W. H., 211, 272 Talmadge, D. W., 284,326 Tan, E. M., 9, 12, 30, 82, 88, 93, 145 Tanenbaum, S. W., 51, 79 Tanford, C., 3, 27, 30, 33, 64, 80, 85, 88, 89 Tanford, S., 97, 98, 144 Tao, T. W., 241, 272 Tawde, S., 67, 88 Taylor, A. G., 200, 272, 319, 323 Taylor, H. E., 226, 229, 230, 272 Taylor, P. D., 306, 307, 326 Terasaki, P. I., 195, 272, 277, 298, 299, 300, 326, 327 Terry, W. D., 14, 15, '17, 19, 20, 33, 34, 84, 88, 97, 145 Thaxter, T. H., 201, 206, 268 Thomas, E. D., 316, 317, 322, 327 Thomas, L., 236, 266, 277, 278, 296, 325 Thompson, D. D., 67, 88 Thomson, W. B., 289, 325 Thorhacke, G., 15, 88 Thorn, G. W., 279, 292, 301, 323 Thorpe, N. O., 23, 28, 32, 88 Till, J. E., 196, 269
343
Tillett, W. S., 277, 278, 296, 325 Tillit, W. S., 217, 269 Tilney, N. L., 284, 285, 291, 327 Titani, K., 35, 37, 38, 40, 42, 48, 63, 77, 88,89
Todd, C. W., 25, 61,88 Tomasi, T. B., Jr., 1, 9, 11, 12, 87, 88, 93, 145 Tomassini, N., 226, 269 Tominaga, K., 9, 14, 15, 29, 79, 80 Toolan, H. W., 218, 269 Traeger, J., 308, 325 Trentin, J. J., 315, 327 Trowell, 0. A., 191, 272 Tsubura, Y.,134, 144 Tubiana, M., 280, 322 Tucker, D. F., 202, 268 Tulles, H., 312, 323 Tunis, M., 240, 272 Tunner, W. S., 198, 272 Turk, J. L., 198, 203, 220, 245, 267, 272 Turner, M. D., 310, 311, 322, 323 Turner, M. W., 30, 88 Tyrell, D. A. J., 186, 187
U Uhr, J. W., 67, 87, 88 Uphoff, D., 315, 323 Utsmi, S., 27, 30, 60, 64,88
V Vaerman, C., 9, 88 Vaerman, J. P., 9, 16, 88 Vainio, T., 226, 227, 272 Valentine, R. C., 3, 7, 88 Van Bekkum, D. W., 319,327 van Dalen, A., 61, 88 Vander Meul, V. A., 231, 259,273 van der Scheer, J., 16,88 van Furth, R., 34, 82 Van Leeuwen, A., 298, 327 Van Rood, J. J., 298, 327 Van Twisk, J. M., 231, 259, 273 Varco, R. L., 278, 280, 288, 322, 325 Vas, M., 239, 246, 247, 256, 269 Vasquez, J. J., 154, 188 Vaughan, M. H., 33, 34, 87 Vaysse, J., 280, 322 Vega, R. E., 284,326
344
AUTHOR INDEX
Veith, F. J., 284, 306, 325, 327 Verbos, S., 240, 268 Vinnik, I. E., 292, 323 Viahakis, G., 134,144 Volkman, A., 193, 194,272 Von Kaulla, K. N., 306, 326 Vourch'h, C., 280, 323 Voynow, N. K., 248,265 Vredevoe, D. L., 277, 299, 300, 326, 327 Vvedenskaya, 0. I., 165,188
W Waddell, W. R., 284, 299, 300, 306, 307, 308, 309, 324, 326 Waksman, B. H., 204,207,213,218,232, 236,267,269, 272, 286,327 Waldenstrom, J., 66, 88, 89 Waldmann, T. A., 11, 87, 89 Walker, G. R., Jr., 311, 322 Wallace, M. E., 141, 145 Wardlow, A. C., 140, 141, 143 Warner, J. R., 67, 88 Warner, L. A., 94, 122, 127, 144 Warner, N. L., 102, 126, 143, 145, 195, 196, 272 Watson, C. G., 213, 223, 267 Watson, J. D., 76, 88 Watson, R. F., 164, 188 Watt, M. F., 184, 187 Weaver, J. M., 190,265, 272 Webb, W. R., 311,322 Webster, E. W., 316, 324 Wecker, E. E., 227, 262, 273 Weigle, W. O., 64,87 Weiler, E., 68, 88 Weimer, H. E., 236,268 Weinstein, A. B., 290, 326 Weintraub, R. M., 140, 143 Weir, R. C . , 16, 28, 33, 54, 55, 64, 86, 88,89 Weisberger, A. S., 261, 263, 271 Weissman, G., 241, 245, 269 Weizer, R. S., 226, 268 Wells, R. D., 73, 85 Welsh, P. D., 240, 241, 245, 266 Wen, S.-F., 290, 326 Wenk, P., 201, 267 Werner, B., 233, 269 Wettstein, F. O., 67,87
Whang, J., 247, 256, 263, 271 Wheelock, E. F., 241, 272 White, R. G., 15, 89 Whitley, E., 35, 37, 38, 40, 42, 63, 77, 88 Whitney, P. L., 64,80, 89 Wiener, D., 184, 187 Wiener, J., 218, 272 Wigmore, R. A,, 311, 326 Wikler, M., 35, 37, 38, 48, 89 Wilheim, E., 24, 89 Wilkinson, J. M., 52, 53, 58, 59, 60, 86, 89, 138, 145 Wilkinson, P. C., 15, 89 Williams, M. A., 221, 266, 269 Williams, R. C., 13, 23, 29, 84, 89 Williamson, A. R., 67, 68, 79, 89 Williamson, N., 240, 243, 270 Williamson, P., 152, 188 Willoughby, D. A., 224, 272 Wilson, D. B., 198, 199, 205, 206, 219, 221, 223, 226, 227, 228, 229, 230, 231, 240, 246, 247, 249, 250, 252, 255, 256, 258, 262, 264, 265, 273 Wilson, J. B., 23, 83 Wilson, R. E., 278, 289, 291, 293, 294, 296, 301, 313, 324, 325, 327 Wilson, W. E. C., 308, 309, 326 Winblad, S., 86, 88, 89 Winn, H. J., 203, 226, 272, 273 Wish, L., 260, 273 Wittner, M. K., 157, 187 Wochner, R. D., 11,89 Wolf, J. S., 199, 273, 285, 327 Wollheim, F. A., 11, 87, 93, 145 Wolstenholme, G. E. W., 320, 327 Wood, A. E., 132,145 Woodruff, M. F. A., 195, 199, 265, 273, 277, 284, 286, 312, 313, 314, 315, 327 Wunderlich, J. R., 93, 94, 99, 104, 122, 143, 145 Wyckoff, R. W. G., 16, 88
Y Yagi, Y., 9, 15, 63, 85, 89 Yakulis, V., 34, 81 Yates, A. J., 311, 324 Yoffey, J. M., 190, 273
AUTHOR INDEX
Yonofsky, C., 92, 145 Young, C,. O., 24, 25, 93, 81, 143 Yount, W. J., 15, 19, 20, 22, 83, 89
Z Zaalberg, O., 213, 259, 273 Zabriskie, J. B., 155, 162, 164, 168, 188 Zak, S. J., 278, 322 Zanaloa, A., 298, 321
345
Zettervall, O., 66, 89 Ziff, M., 67, 85 Zikh, J,, 30, 64, 81 Zintel, H. A., 288, 323 Zmijewski, C. M., 299, 300, 326 Zuckerkandl, E., 41, 89 Zuhlke, Y., 286, 287, 323 Zukowski, C. F., 281, 289, 293, 325, 327 Zvaiffer, N. J., 12, 89
SUBJECT INDEX A Adrenal gland, transplantation of, 313314 Allotypic determinants, immunoglobulin, localization of, 101 Antibody, immunoglobulin combining site and, 63-66 Antigen, macrophages and, 236-238 transplantation, streptococci and, 184185 Antilymphocyte sera, tissue transplantation and, 286-287 Antisera, homologous, failure to produce, 126 myeloma-specific, 139 immunoglobulin, general, 103 myeloma immunoglobulin and, 104 normal immunoglobulins and, 103104 testing of, 104-109
Cytopathogenic effects, manner of mediation by lymphocytes, 221-224
D Donor, kidney transplantation and, 303 Drugs, tissue transplantation and, 280283
C Genetics, heavy-chain determinants and, 127131 immunoglobulin structure and, 69-78 Craft-versus-host reactions, local in kidney, 214-215
H
Heart, transplantation of, 310-311 Heavy-chain determinants, distribution in inbred and wild mice, 131-139 genetic studies and, 127-131 yA Heavy-chain determinants, distribution and localization of, 117-119 B y F Heavy-chain determinants, distribuBiological mimicry, tion and localization of, 119-122 enhancement of pathogenicity, y C Heavy-chain determinants, distribuother organisms, 151-154 tion and localization of, 110-113 streptococci and, 148-151 y H Heavy-chain determinants, distribugeneral considerations, 147-148 tion and localization of, 113-117 pathogenesis of disease and, 154-184 Heavy-chain linkage groups, possible Blood vessels, grafts of, 314-315 evolution of, 137-139 Bone, grafting of, 314-315 Homograft, significance of cellular infilBone marrow, transplantation of, 315trate in, 217-221 319 Human tissue transplantation, biology of, 277-279 C complications and, 288-295 Cells, immunologically competent, transdiagnosis of rejection, 303-305 plantation immunity and, 194-196 endocrine glands, 312-314 Complement component, hemolytic, 140general considerations, 276 141 immunosuppressive treatment, Corneas, grafting of, 314 antilymphocyte sera, 286-287 Corticosteroids, tissue transplantation corticosteroids and, 287 and, 287 drugs and, 280-283 Cutaneous inflammatory reactions, transextracorporeal irradiation, 285 plantation immunity and, 207-213 local irradiation, 285-286 346
347
SUBJECT INDEX other, 287-288 splenectomy and, 284 thoracic duct fistula, 284-285 thymectomy and, 283-284 total-body irradiation, 279-280 moral and ethical aspects of, 320 prognosis, renal allograft, 305 terminology and, 276-277 tissue typing and, 295401 visceral organs, 305-311
1 Immune response, in vitro studies, 224-225 blastogenic response of lymphocytes,
238-260 cooperative interaction of lymphocytes and macrophages, 260-264 lymphocytes as cffectors of immunity, 225-236 macrophages and antigen, 236-238 Immunoglobulins, antibody combining site of, 63-66 antigenic declassification of, 4-5 chains, structure of, 30-63 determinants, unassigned, 122-126 enzymatic and chemical fragments of,
27-30 general considerations, 1-2, 78-79 heavy-chain determinants, distribution and localization of, 109-
126 gene linkages and, 127-131 inbred and wild mice and, 131-139 mouse, gene linkages and, 127-131 general considerations, 92-95, 141-
143 heavy chain determinants and, 109-
126, 131-139 hemolytic complernent component and, 140-141 homologous antisera and, 103-109 inhibition of precipitation method and, 126-127 myeloma specific antisera and, 139 structural characteristics of, 95-103 normal, homologous antisera and, 103-
104
papain fragments, similarities between, 131 peptide chains, heterogeneity of, 32-34 separation of, 30-32 sequence studies on, 34-63 synthesis and assembly of, 67-69 structure and properties of, 2-26 chemical identification of chains, 99-
101 general, 95-97 genetic implications of, 69-78 localization of allotypic determinants, 101 myeloma proteins and, 101-103 subunits and, 97-99 C-terminal stretches, genes controlling,
70 N-terminal stretches, genes controlling,
70-78 urinary fragments of, 29-30 variants, allotypic, 17-26 idiotypic, 26 isotypic, 5-17 Immunological reflex, effector side of,
205-224 Immunological status, lymphoid cells, transformation by ribonucleic acid,
216-217 Immunosuppressive treatment, human tissue transplantation and, 279-
287 Irradiated hamster test, tissue typing and,
298 Irradiation, extracorporeal, tissue transplantation and, 285 local, tissue transplantation and, 285-
286 total-body, tissue transplantation and,
279-280
K Kidney, human, transplantation of, 301-305 local graft-versus-host reactions in,
214-215
348
SUBJECT INDEX
Kidney homografts, site of sensitization, 199-205
L Leucocytes, serotyping of, 298-299 Liver, transplantation of, 305-307 Lung, transplantation of, 311 Lymphocytes, blastogenic response in culture, 238260 cooperative interaction with macrophages, 260-264 cytopathogenic effect, mediation of, 221-224 as effectors of immunity, 225-236 mixed cultures, tissue typing and, 297298 nonspecific mitogens and, 239-243 peripheral blood, transplantation immunity and, 190-193 specific mitogens, primary response, 246-260 secondary response, 243-246 transfer, tissue typing and, 297 Lymphocyte reactions, irradiated skin and, 213-214 Lymphoid cells, destructive activity of, 226-232 normal, cell destruction by, 232-236 transformation, ribonucleic acid and, 216-217
M Macrophages, antigen and, 236-238 cooperative interaction with lymphoc y t e , 260-264 transplantation immunity and, 193194 Mimicry, see Biological mimicry Mitogens, nonspecific, lymphocytes and, 239-243 specific, primary response, 246-260 secondary or ‘iecali” response and, 243-246 Mouse, immunoglobulins, gene linkages and, 127-131
general, 92-95, 141-143 heavy-chain determinants and, 109126, 131-139 hemolytic complement component and, 140-141 homologous antisera and, 103-109 inhibition of precipitation method and, 126-127 myeloma-specific antisera and, 139 structural characteristics of, 95-103 inbred, heavy-chain determinants in, 131-133 wild strains, heavy-chain determinants in, 133-137 Myeloma immunoglobulins, allotype antisera and, 104, 139 Myeloma proteins, genetic control of, 101-103
N Nephritis, post-streptococcal, mimicry and, 181-184
biological
0 Organs, preservation of, 311
P Parathyroid glands, transplantation of, 312 Peptide chains, immunoglobulin, heterogeneity of, 32-34 separation of, 30-32 sequence studies on, 34-63 synthesis and assembly of, 67-69 Pituitary gland, transplantation of, 312313 Precipitation, inhibition, comparison of results with, 128-127
R Recipient, kidney transplantation and, 301-303 Red cell antigens, tissue typing and, 299 Rheumatic fever, biological mimicry and, 154-181 Ribonucleic acid, lymphoid cell transformation and, 216-217
349
SUBJECT INDEX
S Sensitization, process of, 196-199 site of, 199-205 Skin, human, transplantation of, 319 irradiated, mixed lymphocyte reactions and, 213-214 Skin grafting, tissue typing and, 296-
297 Skin honiografts, site of sensitization,
199-205 Spleen, transplantation of, 305-307 Splenectomy, tissue transplantation and,
284 Streptococci, enhancement of pathogenicity of, 148-
151 Group A, transplantation antigens and,
184-185
leucocyte serotyping and, 298-299 mixed lymphocyte cultures and, 297-
298 normal lymphocyte transfer and, 297 red cell antigens and, 299 skin grafting and, 296-297 summary of, 299-301 Transplantation immunity, delayed cutaneous inflammatory reactions and, 207-213 historical, 189-190 in uiuo studies, effector side of immunological reflex, 205-224 identification of immunologically competent cells, 194-196 sensitization process, 196-205 macrophages and, 19.3-194 peripheral blood lymphocytes and,
190-193
T Thoracic duct fistula, tissue transplantation and, 284-285 Thyniectomy, tissue transplantation and,
28.3-284 Thyroid gland, transplantation of, 313 Tissue typing, irradiated hamster test and, 298
U Urine, imniunogIobuIin fragments in,
29-30
X Xenografts, feasibility of, 307-310
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