Advances in Immunology Volume 34

ADVANCES IN

Immunology V O L U M E 34

CONTRIBUTORS TO THIS VOLUME

MARILYN L. BALTZ ROBERT M. FRIEDMAN A. GONWA THOMAS TED H. HANSEN F. L. OWEN KEIKOOZATO M. B. PEPYS B. MATIJA PETERLIN DAVIDH. SACHS NATHANSHARON JOHN D. STOBO STEFANIEN. VOGEL

ADVANCES IN

Immunology EDlTED

BY

F R A N K J. DIXON

HENRY G. K U N K E L

Scripps Clinic and Research Foundation La Jollo, California

The Rockefeller University N e w York, New York

V O L U M E 34

1983

ACADEMIC PRESS A Subridictry of Horcovrt Bvace Jovanovich, Publishen

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83 84 85 86

9 8 76 5 43 2 1

CONTENTS CONTRIBUTORS ............................................................. PREFACE...................................................................

T Cell

Alloantigens Encoded

by

vii ix

the IgT-C Region of Chromosome 12

in the Mouse

F. L. OWEN I. Introduction ............................. ............. 11. Preparation of Conventional Anti-Tsu Serum ............................ 111. Preparation of Monoclonal Antibodies Specific for Tpre, Tthy, Tind, and Tsu .............................................................. IV. Genetic Characterization of the IgT-C Linkage Group ................... V. Products of the IgT-C Region Define a Unique T Cell Differentiation Pathway ............................................... VI. Evidence That Tpre, Tthy, Tind, and Tsu Are Excluded from Developing B Cells and B Cell Products .... ...................... VII. Cross-Reactive Determinants Shared by T Cell Alloantigens in This Linkage Croup and Soluble T Cell Factors ............................. VIII. In Vitro Functional Role of Cells Expressing Tpre, Tthy, Tind, and Tsu . . IX. I n Viuo Studies on the Function of Tsu and Tind Bearing Cells .......... X. Preliminary Immunochemical Characterization of Tsu and Ti XI. Concluding Remarks ..................................... References . . . . . . . . .........................................

1

3 4

9 14 22 24 27 32

35

Heterogeneity of H-2D Region Associated Genes and Gene Products

TED H. HANSEN,KEIKO

OZATO, AND

DAVIDH. SACHS

I. Introduction .......................................................... 11. Antigenic Heterogeneity of Gene Products Encoded in the Dd Region .... 111. Chemical Heterogeneity of Gene Products Encoded in the Dd Region . . , . IV. Quantitative Comparisons of Gene Products Encoded in the Dd Region ... V. Functional Studies of H-2L" Gene Products ............................. VI. Searches for Allelic Products of H-2Ld in Other Haplotypes .............. VII. Studies Using Genomic Clones of H-2D Region Loci .................... VIII. Evolutionary Models and Future Approaches ........................... References ...........................................................

39 41 46

50 52 54

58 64 67

Human Ir Genes: Structure and Function

THOMAS A. GONWA,B. MATIJA PETERLIN,AND JOHND. STOBO I. Introduction .......................................................... 11. Structure of Ir Gene Products in Mice and Humans ..................... V

71 71

vi

CONTENTS

111. Ir Gene Function in Humans .......................................... IV. Conclusions .......................................................... References ...........................................................

80 92 92

Interferons with Special Emphasis on the Immune System

ROBERTM . FRIEDMAN AND STEFANIE N . VOGEL I . Introduction .......................................................... I1 . Interferon Production ................................................. I11. Actions of Interferons ................................................. IV Interferons and Defense against Viral Infections ......................... V. Interferons and Other Mechanisms Related to Immunity and Inflammation ..................................................... VI Antitumor Effects of Interferons in Animal Systems ..................... VII . Clinical Studies with Human Interferons ............................... References ...........................................................

.

.

97 99 101 128 129 130 132 133

Acute Phase Proteins with Special Reference to C-Reactive Protein a n d Related Proteins (Pentaxins) a n d Serum Amyloid A Protein

.

M . B PEPYS AND MARILYNL. BALTZ I . Introduction .......................................................... I1. Induction and Control of Synthesis of Acute Phase Proteins .............. I11. C-Reactive Protein. Serum Amyloid P Component (SAP). and Related Proteins (Pentaxins): Definition and Nomenclature ...................... IV. C-Reactive Protein .................................................... V. Serum Amyloid P Component ......................................... VI . Serum Amyloid A Protein ............................................. VII Summary ............................................................. References ........................................................... Note Added in Proof ..................................................

.

141 145 151 156 183 190 198 199 211

Lectin Receptors as Lymphocyte Surface Markers

NATHANSHARON I . Introduction

..........................................................

IV. Human Lymphocyte Subpopulations ................................... V. Lymphocytes of Other Animals ........................................ VI Concluding Remarks .................................................. References ...........................................................

213 223 230 265 281 287 291

INDEX..................................................................... CONTENTS OF PREVIOUS VOLUMES ..........................................

299 303

I1. Methodology ......................................................... I11. Murine Lymphocyte Subpopulations ...................................

.

CONTRIBUTORS

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

MARILYNL. BALTZ (141),Immunological Medicine Unit, Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London W12 OHS, England ROBERT M. FRIEDMAN (97), Department of Pathology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814

THOMAS A. GONWA (71), The Howard Hughes Medical lnstitute and the Department of Medicine, University of California, Sun Francisco, California 94143 TEDH. HANSEN(39),Department of Genetics, Washington University School of Medicine, S t . Louis, Missouri 63130

F. L. OWEN (l),Department of Pathology and Cancer Research Center, Tufts Medical School, Boston, Massachusetts 02111 KEIKOOZATO(39),Laboratory of Developmental and Molecular lmmunity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20205 M. B . PEWS (141), Immunological Medicine Unit, Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London W12 OHS, England B. MATIJAPETERLIN(71),The Howard Hughes Medical lnstitute and the Department of Medicine, University of California, Sun Francisco, California 94143 DAVIDH. SACHS(39), Transplantation Biology Section, Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205 NATHAN SHARON (213), Department of Biophysics, The Weizmann lnstitute of Science, Rehovoth, lsrael vii

viii

CONTRIBUTORS

D. STOBO(71), The Howard Hughes Medical Znstitute and the Department of Medicine, University of California, Sun Francisco, California 94143

JOHN

STEFANIE N. VOGEL (97), Departments of Pathology and Microbiology, Unij'ormed Services University of the Health Sciences, Bethesda, Maryland 20814

PREFACE

The selection of subjects presented in this volume reflects the broad scope of immunologic interest. Most of the progress in our field depends upon elucidation of the genetic basis underlying the immune system’s structure and function, and three important genetic areas are represented. They include presentation of a new group of T cell alloantigens with many similarities to the immunoglobulin isotype markers for B cells, discussion of the recently recognized heterogeneity of Class I MHC antigens, and a review of the structure and function of human Ir genes. Additional areas of expanding interest are indicated by three reviews that derive in part from neighboring fields of science but deal with matters of considerable immunologic importance. These are a discussion of interferon, particularly as it relates to and influences immunologic events, a review of the acute phase response to injury which has many paraIlels to and interfaces with the immune response, and, finally, a description of the lectin receptor markers of immunocytes and the imaginative lectin technology that has contributed significantly to the identification of the various functionally heterogeneous lymphocyte populations. A new group of T cell alloantigens encoded by a cluster of tightly linked genes on murine chromosome 12 is described in the first article by Dr. F. L. Owen. Drawing heavily on his and his associates’ work, he defines the gene cluster, designated IgT-C because of its proximity to the immunoglobulin genes, and its four recognized structural genes Tpre, Tthy, Tend, Tsu. The products of these genes appear on T cells at characteristic points during their maturational pathway in the order just listed. Although these antigens are distinct from the Lyt series of markers, their presence is related to T cell regulatory function. Cells bearing three (thy, end, and su) of these markers appear to have distinct suppressing and/or delaying effects on immunologic responses in uitro, and the pre-marker appears to be associated with a nonregulatory, perhaps precursor cell. The maturational pathway defined by these markers is presented in detail with its functional and anatomical correlates and its relationship to other T cell markers. The apparent ,function of these gene products is discussed with special emphasis on the possibility that they represent constant regions of T cell antigen receptors distributed differentially on various T cell subsets in the same way that immunoglobulin isotypes serve as differentiation markers for B cells. ix

X

PREFACE

In the second article, Drs. Hansen, Ozato, and Sachs present recent research that is revealing a newly appreciated serologic, molecular, genetic, and functional heterogeneity of Class I H-2 antigens. The focus of this review is on the H-2D region associated genes and their products, a subject to which the authors have been major contributors. Exactly how many genes exist in each region is not yet certain; however, it is clear that the past dogma citing only one gene product for each H-2K or H-2D region is incorrect, at least for some haplotypes. The emerging picture is one of Class I genes as multigene families in which certain members undergo continuous evolutionary expansion and contraction. Finally, the contribution of the concept of Class I multigene families to our understanding of the evolution of these genes and to the roles played by recombination, duplication, and gene conversion in the process is presented and clearly related to appropriate experimental data. A timely view of the structure and possible function of human Ir genes appears in Article 3 by Drs. Gonwa, Peterlin, and Stobo. The genetic basis and chemical characterization of human Ia molecules, HLA-DR, and related HLA-DC and HLA-SB are described and compared to those of their less complex murine counterparts. The possible mechanisms by which Ir gene products might regulate immunologic responsiveness are reviewed along with examples of such apparent regulation. Particularly pertinent is the authors’ work on the immune response of humans to collagen indicating the HLA-DR4 relationship, the genetic characteristics, and the cellular events involved. Current knowledge about the several varieties of interferons such as their cells of origin, modes of induction, control of synthesis, and numerous actions, particularly those related to the immune system, is presented by Drs. Friedman and Vogel in the fourth article. Although most of this information on the actions of interferon comes from studies employing naturally derived and therefore limited amounts of interferon, it provides an essential background for intelligent exploitation of the large amounts of interferon now being made available by recombinant DNA technology. Apparently, all the interferons, a, p, and y , can either modulate immunologic mechanisms directly and/or retard the growth of pathogens-the targets of immune responses. Gamma interferon, the product of stimulated T cells, is quite properly considered an immunoregulatory lymphokine which can enhance macrophage function, suppress responding B cells, and inhibit T suppressor activity. Another striking immunologic effect of interferon is its stimulation of natural killer (NK) cells presumably via the accelerated differentiation of pre-NK cells to fully cytolytic forms. One of the least

PREFACE

xi

well understood yet most challenging aspects of interferon is its apparent antitumor activity. This complex area is thoroughly discussed, and the several mechanisms of antitumor action, immunologic and nonimmunologic, elicited by interferon are analyzed and evaluated. The acute phase response is the name given to a characteristic increase in concentrations of numerous serum proteins following a wide variety of infections, inflammations, or other tissue injuries and constitutes a significant component of the overall systemic reaction to injury. Although this paraimmunologic event has been well recognized since the identification of C-reactive protein, one of its major constituents, some 50 years ago, its precise role in host defense is poorly understood. However, the fact that many components of the acute phase response have enjoyed evolutionary conservation throughout the vertebrate kingdom would suggest that they subserve a beneficial function. In the fifth article, Drs. Pepys and Baltz review this subject covering the factors initiating and controlling the response, the chemistry of its more prominent components, their biologic properties and functions, and, finally, their role in the diagnosis and monitoring of human disease. From initiation of the acute phase response via injury-induced activation of macrophages and interleukin-1 formation, which then stimulates synthesis of most of the acute phase reactants by hepatocytes, to the interaction of these reactants with microbial or endogenous molecules that may result in complement activation and modulation of inflammation, the parallelism between the acute phase and immune responses is evident. The former is a relatively nonspecific, extremely rapid defense in contrast to the latter specific but delayed reaction. With recognition of the great functional heterogeneity and extensive cooperative interactions that mark cells of the immune system comes the need for means to identify and isolate the separate and distinct cellular entities. Two major tools to achieve this end have been developed: antibodies reactive with lymphocyte surface antigens and lectins reactive with surface saccharides. I n the final article, Dr. Sharon discusses lectin receptors as lymphocyte surface markers and draws on his extensive experience in detailing the use of lectins in the recognition and purification of lymphocyte subpopulations. Cell surface lectin receptors are carbohydrates that reside in the oligosaccharide sequences of membrane glycoproteins or glycolipids as secondary gene products, just as ABO blood group determinants do. Lectins, which are largely of plant origin, are oligomeric proteins with several sugarbinding sites per molecule, and these sites interact with their target noncovalently primarily via hydrophobic and hydrogen bonds. Al-

xii

PREFACE

though the functions of lectin receptors on lymphocyte and other cell surfaces are not known, a large number of such markers have been identified and correlated with cell surface antigens as well as with maturational and functional characteristics of cells. Techniques capable of recognizing lectin receptors on cells in situ and of separating and purifying specific cellular populations have been developed by using a variety of lectins. The use of this technology in diverse experimental situations as well as its potential clinical application in the preparation of non-graft-versus-host reactive bone marrow transplants are also presented.

FRANKJ. DIXON HENRYG. KUNKEL

ADVANCES IN

Immunology V O L U M E 34

This Page Intentionally Left Blank

ADVANCES IN IMMUKOLOGY, VOL. 34

T Cell Alloantigens Encoded by the IgT-C Region of Chromosome 12 in the Mouse F. L. OWEN Department of Pathology and Cancer Research Center, Tufis Medical School, Boston, Massachusetts

I. Introduction ............................... ............. 11. Preparation of Conventional Anti-Tsu Serum ............................ 111. Preparation of Monoclonal Antibodies Specific for Tpre, Tthy, Tind, and Tsu . . . . . . . . ..................................... IV. Genetic Characterizatio C Linkage Group ......... V. Products of the IgT-C Region Define a Unique T Cell Differentiation Pathway ............................................... VI. Evidence That Tpre, Tthy, Tind, and Tsu Are Excluded from Developing B Cells and B Cell Products ............................... VII. Cross-Reactive Determinants Shared by T Cell Alloantigens in This Linkage Group and Soluble T Cell Factors ........................ VIII. I n Vitro Functional Role of Cells Expressing Tpre, Tthy, Tind, and Tsu .............................. ...................... IX. I n Vivo Studies on the Function of Tsu and Ti Bearing Cells ......................................................... X. Preliminary Immunochemical Characterization of Tsu and Tind ....... ........................................... XI. Concluding Remarks ..................................... .......

.............................

1 3 4 14 22 24 27

32 33 34 35

I . Introduction

A new group of T cell alloantigens is encoded by a cluster of tightly linked genes on the murine chromosome 12. This gene cluster, designated IgT-C because of it8 close proximity to the immunoglobulin genes (diagrammed in Fig. l),includes at least four structural genes which code for Tpre, Tthy, Tind, and Tsu. Classical in vivo animal genetic studies predict the gene cluster lies no more than 3 map units distal to alpha, and is therefore spatially closer to the immunoglobulin constant region genes than are the most loosely linked variable region genes (V,nase, 8 map units). The close physical location of these two gene clusters, one specific for B cells (immunoglobulins) and a second apparently specific for T cells (IgT-C),raises questions about a possible evolutionary relationship between the two groups of genes. It has been proposed that these genes may code for constant regions on T cell antigen receptors and may, in fact, have arisen evolutionarily from duplication of a primordial gene coding for both T and B cell antigen 1 Copyright 0 1983 by Academic Press, Inc.

All rights of reproduction in any form reserved. ISBN 0-12-023434-8

2

F. L. OWEN

I

Igh-V

Ly 18,19

I

1

1

Iqh-C

I 1

IpT-C

i

b

2 5 H Rscombinolion

2% Recombinofion 5% Recombinalion

FIG. 1. Map of genes linked to the immunoglobulin locus on chromosome 12 in the mouse. The region proposed to be named IgT-C is distal to alpha and includes four gene products named Tpre, Tthy, Tind, and Tsu. The subscript m (Tpre,) indicates monoclonal antibodies, presumably epitope specific, were used to map these genes. Although Trpe, is more distal than Tsu, the order with respect to the centromere of Tthy,, Tind,, and Tsu, have not been determined.

.

receptors. Some support for this hypothesis is found in the serological cross-reactivity of a monoclonal antibody recognizing Tind and an antigen-specific T augmenting factor, TaF (Section VII). Earlier studies with antiserum had shown stearic blockade of T cell antigen binding. These suggestive data are encouraging. However, the conclusion that products of IgT-C are constant region genes for T cells is critically dependent upon the cloning of a segment of DNA which maps to this region. Use of that genetic material to produce a synthetic polypeptide which shows biological properties of antigen binding and T cell functional replacement and also expresses determinants recognized by one of our monoclonal antibodies would be acceptable evidence that one of these gene products is indeed the elusive C, (Owen and Spurll, 1981; Kronenberg et al., 1980; Schrader, 1979). Similar information for each of the four gene products in this region is required to assume that all four represent isotypes of C.rl-4.Although this is our working hypothesis, it is possible that several different types of structural products may be encoded in this segment of the chromosome, possessing unrelated functional roles. This work was initiated based on the assumption that T cell receptors are unique antigen binding structures and that fine specificity for antigen is a finite property of T cells endowed solely through surface receptor recognition of nominal or self antigens. Difficulties in demonstrating direct antigen binding by helper T cells in vitro or on clones of antigen-specific proliferating cells have led to speculation that T cells may play a physiological, non-antigen binding role in regulating immunoglobulin synthesis. The serological approach outlined below describes an attempt to produce antiserum specific for “constant determinants” on T cell antigen binding structures. A cluster of immunoglobulin genes on chromosome 12 (Honjo and Katavka,

T CELL ALLOANTIGENS

3

1978) specify variable region and constant region genes; despite the fact that extensive descriptions of serological cross-reactivity between anti-VHreagents and T cells have been published (Eichmann, 1978, review; Lonai et aZ., 1978), attempts to demonstrate immunoglobulin constant region genes on T cells have led to negative evidence (Krawinkel et aZ., 1978). If one assumes the serological cross-reactivity of T cell receptors and Igh-V implies at least an evolutionary relationship, if not an identity between the genes encoding immunoglobulin and T cell antigen recognizing structures, then it is biologically most conservative to assume a T cell gene cluster will lie close to the Igh-V genes. If immunoglobulin constant region genes do not encode T cell receptors, then it is assumed that a unique bank of Igh-1 linked T cell genes must exist. Antiidiotype recognizing T cells have been described in mice immunized with rabbit antiidiotypic antiserum and KLH * Ars (Owen et aZ., 1977b) as well as other systems (Bona and Paul, 1979). Surprisingly, the T cells of A/J animals (Igh-le) and C.AL-20 (Igh-Id on a BALB/c congenic background) exhibit the same T cell antiidiotypic specificity in contrast to BALBlc T cells (Owen et aE., 1977a). This finding prompted the choice of strains and serological approach to produce antiallotypic reagents specific for constant region gene products (Owen et al., 1979; Tokuhisa and Taniguchi, 1982a; Aihara et al., 1983). The experiments summarized in the following sections describe attempts to identify target tissues expressing the T cell antigens encoded by the IgT-C region, to identify functional role for cells expressing these antigens, and to look for serological relationships with antigen binding factors. The genetic work at present is confined to identification of surface antigens in recombinant inbred lines of mice. Genetic analysis at a more molecular level must await amino acid sequence data on these antigens and/or good biochemical data with peptide map analysis of possible polymorphisms only suggested by the serological data. II. Preparation of Conventional Anti-Tru Serum

The strains selected for production of polyclonal anti-Tsu were based on the fact that the recombination events in and around the immunoglobulin gene complex in C.AL-20 animals, congenic with BALB/c, is well documented. BALB/c animals accept first set C.AL-20 grafts (Riblet and Congelton, 1977; Owen et al., 1979). Therefore, the antigenic determinants [H(I,)] presumably encoded by the minor histocompatibility locus on chromosome 12, between alpha and preal-

4

F. L. OWEN

bumin, present a favorable unidirectional graft barrier in this strain combination. In addition, the accessibility of a T cell-specific antigen binding system in the C.AL-20 animal (Owen et al., 1976) made this an attractive strain for production for T cell receptor probes. The reagents produced in this effort consequently react with antigens linked to either the Igh-ld or Igh-le immunoglobulin alleles, which are found in strains less often used by cellular immunologists. Briefly, BALB/c AnN animals were injected with C.AL-20 spleen cells grown for 24 hours in 10% FCS containing media with 5 pg/ml concanavalin A. Intraperitoneal injections (5x at 1 week intervals) of cells fractionated on discontinuous BSA gradients (Steinman et al., 1978) and selected for approximately 20% of surviving blast cells resulted in production of an antiserum reacting preferentially with C.AL-20 and not BALB/c cells. Positive antiserum was selected from individual BALB/c mice by testing in visual surface immunofluorescence assays on C.AL-20 spleen cells eluted from nylon wool (Julius et al., 1973). Indirect staining with FITC-goat anti-IgG was used as a screening assay. Serum samples were not adsorbed before testing. Serum titers were typically 1-10 or 1-20 and not greater than 20% of immunized mice in any group were sero-positive. Antiserum from pooled positive samples was aliquoted and frozen at -70°C. Freezethawing resulted in loss of activity. A typical tissue distribution of the antigen(s) detected by surface fluorescence is shown in Table I. Because 10 donor C.AL-20 mice were required for every 1 BALB/c immune animal and only 1 mouse in 5 produced antiserum, this procedure was costly and inefficient. Practical considerations encouraged attempts to produce monoclonal antibodies specific for Tsu. The identification of other gene products encoded in this region was fortuitous and a by-product of our first attempts to produce monoclonal anti-Tsu. Two independent attempts to produce an antiserum specific for T cells by immunization with concanavalin A activated T cell blasts have been reported. Both utilize BALB/c anti-CB.20 serum. Antiserum was screened by antibody and complement-mediated 51Cr release from Con A activated cells. Similar results were obtained, suggesting that the polymorphism of T cell “allotypes” may involve at least three alleles: BALB/ca, CB.20b, and C.AL-20d (Tokuhisa and Taniguchi, 1982b; Aihira et al., 1983). Ill. Preparation of Monoclonal Antibodies Specific for Tpre, Tthy, Tind, and Tsu

Efforts to produce anti-Tsu resulted in the incidental production of monoclonal IgGIK anti-Tind. Continued screening, using the same ap-

5

T CELL ALLOANTIGENS

TABLE I DISTRIBUTION OF REACTIVITYO F ANTI-TS~ ANTISERUM O N CAL.20 LYMPHOID TISSUE Tsd bearing cells Tissue

(%)

Spleen Splenic T cells" Splenic B cellsb Ly 2+ spleen cells Ly 1+ spleen cells Thymocytes (unfractionated) Mature thymocytesC Lymph node Bone marrow Con A blastsd LPS blasts

2-6 5- 12 <1 25-30


T cells were prepared by elution of spleen cells from nylon wool and found to contain fewer than 5% B cells by fluorescence staining methods. Spleen cells were treated with rabbit anti-mouse brain serum, complement, and low-ionic strength buffer. Mature thymocytes were prepared by BSA density gradient fractionation of normal thymus. Blast populations were purified on BSA gradients. Data taken from Owen et al. (1979).

proach, resulted in an anti-Tsu secreting IgG,K line. A screening assay was designed which employed fixing blast cells, activated with concanavalin A, to polyvinyl chloride 96-well plates (Spurll et al., 1983). Plates were blocked with horse serum and media from hybrid lines was incubated with individual wells of the tray. 12sI-labeled goat anti-mouse IgG, or *251-labeledgoat anti-mouse IgM was added as a developing agent. All antibodies which bound to C.AL-20 but no BALB/c cells were expanded and subcloned by limiting dilution analysis. Because our screen employed anti-IgG, and anti-IgM, it is not surprising that the lines selected all release IgM or IgG, antibodies (Table 11).The immunization protocol for the original lines involved injecting C.AL-20 spleen cells, grown for 48 hours in the presence of Con A and media containing fetal calf serum, into BALB/c animals using a variety of immunization models. None was documented to be more successful than others and our frequency of positive lines was 11/12,000 clones tested (Spurll et aZ., 1983). The cross-reactivity of several monoclonals with more than one antigenic determinant could be due to shared specificities, possibly suggesting some homology be-

6

F. L. OWEN

TABLE I1 MONOCLONALANTIBODIES DIRECTED AGAINST PRODUCTS OF THE IgT-C REGION T cell antigen

Antibody secreting clone

Immunoglobulin isotype

Tindd Tindd Tindd Tthyd,Tindd Tsud Tthyd Thyd,Tindd,Tsud Tthyd ? Tsud Tindd Tindd,Tthyd Tpre

9IIIA2 9IIIC 11 9IIF11 23IIIFll 104IC4 17IIC6 102IIIA11 208IE6 9IIIG7 13IIIB4 18IC4 18IG3 F.6.9.1

Y1 P K

PA P P P K P K iK

Y1 Y1 Y1

P P K

A110 (A) or framework (F) determinant A A A A A A A A A A A A A

tween closely related products. Shared carbohydrate groups could explain this relationship, although the more interesting interpretation is that conserved determinants may have been retained in evolution. Structural data are necessary to better interpret these data. Anti-Tindd was so named because the antibody recognizes the d allele of a T cell alloantigen expressed preferentially on Lyt 1+2-3cells (Spurll and Owen, 1981). In vivo administration of anti-Tind monoclonal antibody induces either an amplification or a reduction of the immune response to SRBCs, depending upon the time of administration of antibody (Spurll and Owen, 1981). The choice of nomenclature for the antigens in this series has led to confusion with a functional T cell type named an inducer (Eardley et al., 1980). This cell type expresses the Lyt 1+2-3- phenotype and was originally called an inducer of the suppressor (Eardley et al., 1978, 1980). Cloned inducer cells (Nabel et al., 1981a) are known to support growth of mast cells (Nabel et al., 198lb) and T and B cells and may, in fact, regulate the immune response through a secondary physiological effect (Fresno et al., 1982). It is not inconceivable that the cell type we reported to bear Tind may overlap cells in the physiologically active group named inducer T cells. Although functional studies on the cell which expresses Tindd have been initiated and the preliminary, unpublished data are described in Section VIII, further characterization is essential.

T CELL ALLOANTIGENS

7

Anti-Tsu was so named because this monoclonal antibody most closely mimics the original antiserum. The antigen is preferentially expressed on Lyt 2.2 bearing peripheral lymphoid cells (Spur11 et al., 1983). The distribution on cells in the lymphoid system (Owen, 1982a) closely parallels that reported for the conventional antiserum (Owen et al., 1979) and the FACS profile of anti-Tsu monoclonal and serum antibodies on unfractionated spleen are analogous (R. Hardy, personal communication). Immunoprecipitation experiments (Section IX) indicate the original serum, anti-Tsu and the monoclonal anti-Tsu precipitate a 68,000 MW species. The monoclonal does induce suppression in vivo (unpublished data). The original antiserum was raised in a strain combination which could have resulted in production of antibodies specific for Tpre, Tthy, Tind, and Tsu. It is puzzling that the major antibody produced was anti-Tsu and that the low titer serum antibody was so predominantly IgG,K. Discovery of anti-Tthy (17IIC6) resulted from fusion of BALB/c spleen cells which were hyperimmunized with peripheral splenic C.AL-20 lymphoid cells and then fused to P3U1, but resulting clones were screened on glutaraldehyde-fixed thymocytes using the assay described above. In addition to this procedure, BALB/c “related” nude (nu/nu) mice (Charles River Laboratories, Wilmington, MA) were injected twice intraveneously with C.AL-20 thymocytes and immune nulnu spleen cells were fused with P3U1 cells. Secreting lines were screened on C.AL-20 versus BALB/c glutaraldehyde-fixed thymocytes. One resulting line (IgG,K, 208IE6) appears to recognize a specificity similar to prototype anti-Thy, 17IIC6, p~ secreting line. The Tthy antigen differs from Tind and Tsu in that it has been difficult to directly demonstrate the presence of this antigen on peripheral cells using either quantitative adsorption or direct lysis. It must be present, however, in either very low densitylcell or frequencykell population because (1)an occasional hybrid line from peripheral immunization expresses Tthy, (2) this antibody arose by immunization with peripheral cells before fusion with P3U1, and (3) preliminary functional tests suggest a role for a Tthy bearing cell in immune spleen (Section VIII). These three prototype antigens appear to be structurally unrelated in mature cell populations. Blocking and co-capping studies illustrate they are three separate, nonlinked specificities on the cell surface. We now have multiple lines specific for each antigen [maintained under laboratory conditions for over 2 years (Table 1I)I. Each of these prototype antigens was studied in ontogenetic evaluation of the developing neonate. Tthy, Tind, and Tsu show postnatal

8

F. L. OWEN

expression as surface antigens and none of these is expressed in nude

AKR nustr/nustranimals, Since committed antigen-specific T cells are known to exist in fetal animals (Owen et al., 1976) and can be activated in nude animals with TCGF containing tissue culture supernatants (Hunig and Bevan, 1982), it seems highly probable that Tthy, Tind, and Tsu are late developmental antigens for T cells. With this basic assumption in mind, a search for a developmental marker on early, antigen committed T cells was initiated. This effort resulted in production of monoclonal anti-Tpre, a fourth antigenic specificity in the same linkage group. The name Tpre was selected because this antigen shows prethymic expression in nulnu mice and in fetal liver at day 13 prior to the appearance of theta in the thymus. Whether it is a “pre T cell” determinant or a marker for a multipotential stem cell overlapping T cells is currently being explored. Anti-Tpre (F.6.9.1, P K ) was seIected from a fusion of “BALBlc related nulnu” spleen cells from an animal immunized twice with C.AL-20 day 17 fetal thymocytes. “BALBlc related nulnu” animals resulted from a commercial attempt to breed the nulnu gene onto the BALBlc genetic background and are known to differ with respect to at least five isoenzyme markers from the BALB/c phenotype (personal communication, veterinarian in residence, Charles River Laboratories, Wilmington, MA). These animals were marketed as BALBlc nulnu. The rationale for this choice was the fact that Tthy, Tind, and Tsu are not present in the nude. It was hoped that a new specificity would be found, even if it was not one expressed on pre-T cells, which are believed to be present in nudes. Clones of cells which grew in HAT selective media were screened on 51Cr-labeled fetal somatic cell hybrids of BW5147 and fetal thymocytes or fetal liver from embryos at days 12, 17, and 19 of gestation. Tissue culture supernatants were incubated with fetal lines in the presence of selected rabbit complement. Clones which bound to one or more of the first five T cell hybrids screened were subcloned by limiting dilution analysis and screened on adult thymocytes for lysis of adult as well as fetal tissue. Nine of the ten positive lines also recognized adult tissue. Only one line, F.6.9.1, recognized an antigen mapping to the region named IgT-C when BALBlc, C.AL-20, and C.B.AL-1 thymocytes were used as the targets in cytotoxicity assays (Owen, 1983). Tpre antigen differs from Tthy, Tind, and Tsu in the fact that it is an antigen expressed in AKR nustr/nustranimals. It is ontogenetically expressed on 13 day fetal liver, although expression overlaps adult thymocytes (see differentiation studies in Section V). Whether Tpre is a

T CELL ALLOANTIGENS

9

part of the same developmental lineage as Tthy, Tind, and Tsu is not clear. It is possible that this is another marker closely linked to the first three, but with an unrelated functional role.

IV. Genetic Characterization of the IgT-C Linkage Group

A short segment of chromosome 12 codes for four T cell structural products, Tpre, Tthy, Tind, and Tsu. These genes are distally linked to the immunoglobulin genes and lie between alpha and a histocompatibility gene (HIg). Four separate gene products have been identified with monoclonal antibodies and the genes which c d e for these have been mapped to a very tight linkage group in the region of chromosome 12 we have proposed to name IgT-C. The naming of the gene cluster was based on an attempt to maintain consistency with previously characterized genes in this region (Fig. 1) and does not imply a relationship between antigens encoded in this region and those recognized by rabbit (Marchalonis et al., 1972) or chicken (Marchalonis et al., 1979; Layton, 1980) antiimmunoglobulin reagents. Although serological cross-reactivity between immunoglobulins and T cell receptors may exist, no obvious cross-reactivity between our reagents and immunoglobulins has been documented. The genes in this region were mapped using a serological approach (Owen and Riblet, 1983).Antigens encoded by these gene products are expressed as surface alloantigens on T cells (Owen et al., 1979) and can be readiIy detected by antibody and complement-mediated cell lysis. Briefly, aTpre and aTthy monoclonals were modified by light haptenization and used, with anti-hapten polyvalent mouse antibodies, to lyse adult (6- to 10-week-old male) thymocytes. Anti-Tind and anti-Tsu antibodies, similarly modified, lyse in vivo stimulated antigen specific lymph node cells (Owen, 1982b). Mice from 25 recombinant inbred lines on 4 different parental backgrounds were typed for surface expression of these antigens. All the mice selected for study had documented recombination events between alpha and prealbumin and were selected in hopes of finding a strain which would show recombination between the genes coding for Tthy, Tind, Tsu, and Tpre. One such animal was found, CAB 13632, which shows recombination between Tpre and the other three known genes in this linkage group, Tthy, Tind, and Tsu. Because the genetic information on this strain documents a previously known recombination event between alpha and prealbumin, this strain suggests Tpre is a gene closer

10

F. L. OWEN

to prealbumin than is Tthy, Tind, or Tsu, which are ordered randomly on the map: CAB 13632: VHDex+ Igh-la

(Tthy-Tind-Tsu-)

Tpred PreO

The alleles preo and Tpred are typical of the A/Icr parental strain and the VHDexIgh-la immunoglobulin genes are typical of the BALB/c Icr parent. The recombination in the IgT-C linkage group is indicated by a cross above. Any other position for Tpre would require multiple recombination events between alpha and prealbumin, which are possible but statistically less likely. A summary of the recombinant inbred data show the map position of the genes in this region (Fig. 1).The approximate map distance between alpha and Tsu is based on a recombination frequency of 3-4% in these strains and assumes random recombination with no evidence for hotspots. These antigens represent a very tight linkage group with the distance between Tthy, Tind, and Tsu too small to document and that for Tsu and Tpre less than 1%. That these antigens are products of separate genes is more obvious in looking at the allelic expression patterns in commercially available inbred strains of mice which were separated genetically some time ago. Table I11 shows 15 strains of mice, some of which show recombinations between allelic forms of these antigens. C.AL-20, AKWJ, and NZB and A/J animals share the same presumed alleles for all these genes. However, mice in the Igh-l" group show recombination between genes, C3H/HeJW expresses Tindd and Tthy" but not Tsu or Tpre; and NIH Swiss mice express Tindd and Tthyd. The allelic forms of Tsu and Tpre can clearly be separately inherited from one another and from Tind and Tthy. No genetic documentation for recombination between Tthy and Tind is available, and it is still formally possible that these antigens could be structurally modified products of the same gene. Independent expression of these antigen in T cell hybrids does not support identity of the genes encoding these antigens but cannot exclude this possibility (Section V). Tsu, the fmt gene in this linkage group, was described using alloantisera and functional assays. Antiserum was raised in BALB/c mice against C.AL-20 cells stimulated with Con A and separated on discontinuous BSA density gradients. Multiple intraperitoneal immunizations resulted in an antiserum which recognized C.AL-20 T cells but not BALB/c (Owen et al., 1979). Surface visual fluorescence was used to screen the antiserum for reactivity with various lymphoid tissues.

11

T CELL ALLOANTIGENS

TABLE 111 EXPRESSION OF ALLELIC FORMS OF TPRE,TTHY,TIND, INBRED STRAINS OF MICE

AND TSU IN

IgT-C Strain C.AL-20 AKWJ

Igh-la

Tsub TindC Tthyd

d d

d d

d d

d d

d d

d d d

d d d

NZB

NJ BALB/c C, H/H eJW C,H/HeJ NIH Swiss CBNTufts

.i

C57BU6J CB.20

b b

SWWJ HRS/J MRULPR MRJJ++

C

Tpree

Allotype was assigned from published data (Staats, 1976). Anti-Tsu (monoclonal antibody from clone 13IIIB4) was used to type peripheral nodes from KLH . TNP immunized mice. Anti-Tind (monoclonal antibody from clone 9IIIA2) was used to type peripheral node cells from KLH . TNP immunized mice. Anti-Thyd (17IIC6) or T h y a (CB.13.10) was used to type thymocytes from adult mice. Anti-Tpre (F.6.9.1) was used to type thymocytes from adult mice. Table is adapted from Owen and Riblet (1983). a

Con A stimulated T cells, a less dense fraction of BSA separated thymocytes, and Lyt 2+ splenic cells show optimum reactivity with the antiserum. Bone marrow and splenic B cells show very little reactivity. When the antiserum was injected into mice in limiting dilutions, the suppressor cells of virgin mice could be triggered polyclonally (Owen, 1980b).This resulted in 50-80% inhibition of in oiuo plaque-forming cell assays (Owen, 1980a). Induction of suppression in vivo was used as a test, concurrently with visual fluorescence microscopy to test a panel of 48 recombinant inbred strains of mice for expression of

12

F. L. OWEN

fluorescent antibody reactivity or functional reactivity. The study resulted in identification of a gene(s) which mapped between Igh-1 and prealbumin. Strains used were as summarized in Fig. 2. The Tsu gene clearly maps outside the immunoglobulin gene complex and lies from 0.6 to 6 map units distal to alpha (Owen et al., 1981). This estimate is based on a recombination frequency of 2 strains in 48 tested where Tsu and Igh-1 types differ. The findings of our first genetic study using a functional assay are entirely consistent with data from the monoclonal antibodies. Typing with monoclonals using a different assay, cell surface cytotoxicity (Owen and Riblet, 1983), confirms the map position and limits the distance between Igh-1 and Tsu to not greater than 4 map units. It is difficult to extrapolate directly from recombination frequencies to distance on the chromosome since hot-spots for recombination could cause large discrepancies between the number of bases between genes and the estimates obtained from classical recombination studies. Attempts to genetically characterize antigens which may be similar to Tpre, Tthy, Tind, and Tsu have been made. Rabbit antiserum 6036 reacts with an allotypic determinant on Lyt I+ C57BL/6 (Igh-lb) T cells. Genetic linkage studies show this antigen to be encoded by a gene(s) located between Ig-V, and prealbumin on chromosome 12 (Suzan et al., 1982). This conclusion is based on 4 recombinant inbred strains of mice developed by 0. Makela (Helsinki, Finland) originating from (C57BL/6 X BALB/c)F, x C57BL16. Frequency of linkage and approximation of the map distance from Ig-VH was not possible from the small statistical sampling size (4 recombinant strains). It seems likely these antigens, linked in expression to Igh-lb, may belong to the IgT-C linkage group. The preparation of rabbit anti-T cell alTsud. A POSSIBLE T CELL RECEPTOR ALLOTYPE Number of Strains I()

7

Iqh-IIex

_--_--_______ X

Igh-C

7-$U"

Pre- I

x .............................. x ---------___--_-______________ .-----____-----____.----------x ___ x ..............................

__ - -

- X

2

. .

1 1

__----___-_----_____-..--------

-.

X

FIG.2. Mapping Tsudwith Igh recombinant strains and RI strains. The Igh to Pre-1 regions of the recombinant chromosomes which were analyzed in the tables are schematically represented. The solid line represents the portion of the recombinant chromosome derived from one parent and the dotted line indicates the segment from the other parent. The crossover point is shown as X.

T CELL ALLOANTICENS

13

lotypic determinants was achieved by immunizing mice with “factor” from the supernatant of B6 anti-CBA MLC cultures and absorbed with MRBC, EL-4 cells, FCS-Sepharose and CBA-NMS-Sepharose. Despite the H-2 restricted origin of the factors, no influence of H-2 on the expression of the allotypic determinants was seen in contrast to earlier studies (Krammer and Eichman, 1978) which had suggested T cell receptors on MHC reactive T cells may be genetically encoded by both MHC and Igh-1 linked genes. The fact that there are T cell alloantigens encoded by this region of chromosome 12 is strengthened by the fact that Owen and Riblet (1983) and Rubin’s group (Suzan et al., 1982) have mapped these antigens to the same region using two unrelated series of mice which share neither genetic origins, breeding facilities, nor, presumably, endemic viral pathogens. Monoclonal mouse antibodies specific for T cell antigen specific factors have been produced (Tokuhisa et al., 1982). Monoclonal 7C5 [BALB/c anti-CB.20 T suppressor factor (TsF)] recognizes an antigen which may be on the constant part of a T cell factor. This reagent contrasts with monoclonal 6A4 which recognizes a determininant on T augmenting factor (TaF), 7C5 anti-TsF recognizes a gene product linked to Igh-lh. Immunoglobulin congenic mice (C3H * SW, Igh-lj, CWB, Igh-lb, and BAB/14 Igh-lh) were used to draw this conclusion. The use of BAB/14 shows the genes which encode the 7C5 specificity could be in a short segment of the variable region near Igh-1, within Igh-1, or to the right of the immunoglobulin genes in the region from alpha to prealbumin. Although it is attractive to speculate that the genes encoded within IgT-C may be responsible for the anti-7C5 and anti-6A4 specificities, formal genetic analysis is required before a definitive conclusion can be drawn. Monoclonal anti-T cell allotypic antibodies have been raised which react with NP specific Tsl T cell hybridomas or Con A blast cells. The surface antigenic determinants recognized map to the right of VH-NP and to the left of prealbumin. Cell lines CT08 and CT91 release antibody reactive with suppressor cell lines while CT25 releases antibody reactive with only Con A blasts and Lyt lf2-3- resting lymphoid cells. No association with T cell factors has been documented. It is possible that these antibodies are directed against products of genes in the IgT-C cluster (Aihara et al., 1983). Other antigens linked to the immunoglobulin gene cluster are illustrated on the map in Fig. 1. Lyb 7, loosely linked to Igh-1, is a B cell differentiation antigen (Subarro et al., 1979). The strain distribution and 25% recombination frequency with Igh-1 clearly exclude the pos-

14

F. L. OWEN

TABLE IV C.B.AL STRAINS

Parentals BABl14 C.AL-9 Recombinant strains C.B.ALI1" C B.AL/2 C.B.ALI3 C.B.ALl4 C.B.ALI5 C.B.AIJ6

.

Dex

Igh-1

IgT-C

Pre

+

b d

d

0 a

-

x

b

-

-

x x

b b

-

x

b

x x

b b

-

x

d

x

a 0 0 0 0 a

sibility that Tpre, Tthy, Tind, and Tsu could be related to this antigen. Ly-18 is a T cell differentiation antigen linked to Igh-1. It is expressed on cytotoxic T cells but not MLR reactive T cells specific for the same antigen (Finnegan and Owen, 1981a). Ly-19 is a differentiation antigen expressed on a wide spectrum of lymphoid targets (Finnegan and Owen, 1981b). H(Ig), a minor histocompatibility antigen (Riblet and Congelton, 1977; Rolink et al., 1978) and Pre, prealbumin (Me0 et al., 1980) have been described. The work of Owen and Riblet has defined a panel of approximately 60 recombinant inbred animals for which recombination events in and around the immunoglobulin genes and IgT-C have been mapped. The genetic background of all these mice is either Igh-la x Igh-ld, Igh-la x Igh-l', or Igh-lb x Igh-ld.The C.B.AL series is particularly well suited to analysis of probable alleles using antisera which recognized Igh-1" and Igh-ld T cell allotypes (Table IV). The complete panel of recombinant mice (generated by R. Riblet, Fox Chase Cancer Research Institute) have been summarized in primary communications (Owen et al., 1981; Owen and Riblet, 1983).

V. Products of the IgT-C Region Define a Unique T Cell Differentiation Pathway

The products of the genes encoded in the IgT-C region are distributed on T cells at characteristic points in a maturational pathway. However, it is unlikely that the primary role of these structural prod-

T CELL ALLOANTIGENS

15

ucts is to serve as cell differentiation markers; it seems biologically wasteful to propose any cell produces nonfunctional luxury products. However, the unique distribution pattern of these antigens has been a useful tool in defining a differentiation pathway for T cells which may not duplicate that of the Lyt antigenic system. Tpre, Tthy, Tind, and Tsu are expressed ontologically in sequence. Using quantitative adsorption of monoclonal antibodies and scoring the residual antibody on known positive target cells, it has been shown that Tpre is a detectable surface antigen as early in fetal hematopoiesis as day 13 fetal liver, prior to development of the thymic rudiment at 13-14 days gestation (Rugh, 1963). However, the limits of sensitivity of this assay may allow cells in earlier developmental stages to escape detection. Expression of Tpre may in fact precede expression of Thy 1.2 (theta antigen at day 13-14 (Owen and R&, 1970; Owen et al., studies in progress). Peak expression of Tpre (density/cell in adsorption studies) is days 15-17 of fetal thymocyte gestation, although Tpre is expressed in adult thymus and in secondary immune lymph nodes, late after antigen driven differentiation (Owen et al., 1983). Tthy, in contrast, is expressed in greatest densitylcell in adult thymus after 3 weeks of neonatal life. It cannot, however, be detected by adsorption in day 19 fetal thymus at 1 0 higher ~ cell numbers than those required to adsorb Tpre. Recent studies showing Thy 1.2 is expressed in hematopoiesis on some nonlymphoid precursors (Schrader et al., 1982; Basch and Berman, 1982) raise the possibility that monoclonal anti-Tpre and antiTthy could be expressed on some myeloid precursors, a problem currently being pursued experimentally. Hybrid cells originating from fusion of BW5147 cells (AKR, Igh-ld, HAT-sensitive thymoma) and fetal thymus and fetal liver have been typed by cytolytic antibodies for expression of Tpre, Tthy, Tind, Tsu, Lyt 1,Lyt 2, Thy 1.2, and surface immunoglobulin (Owen, 1983). Tpre is expressed on day 12 fetal liver hybrids, and day 15 and 17 thymocytes. There is no correlation, however, between Thy 1.2 and Lyt expression in these hybrids and the reported gestational times for appearance of the Lyt antigens in normal ontogeny (Owen et al., 1982; Mathieson et al., 1981; Scollay, 1982). Since an adult thymoma was used as the fusion partner, transcomplementation for expression of gene products in the AKR parental line, could contribute to the precocious appearance of Tthy, Tind, and Tsu in fetal hybrids. One would like to examine products of hybrids generated from BALWc, BAL 5, or LS1784 thymomas and C.AL-20 fetal tissue (Ruddle, 1981). Unfortu-

16

F. L. OWEN

nately, our frequency of successful clones from such fetal fusion attempts was so low that this was not possible. The Tind and Tsu antigens are not readily detected in either fetal or adult thymus, but can be detected in neonatal spleen or lymph node. Both antigens seem to be characteristic of cells which appear late in hematopoiesis, clearly after the fetal divergence of Lyt 1 and Lyt 2 types at day 19 of gestation (Mathieson et al., 1981), in contrast to Tthy which appears parallel to that divergence, but after Thy 1.2 expression. It is not surprising, therefore, that Tthy is expressed on both Lyt 1+2+3+cells in thymus and Lyt 1+2-3- and Lyt 1-2+3+T cell hybrids originating from adult tissue. In contrast, Tind seems to be preferentially associated with Lyt 1+2-3- mature T cells while Tsu is preferentially expressed on Lyt 1-2+3+ mature cells at the whole population level. Tind can be detected at days 1-2 of neonatal life, while Tsu is not detected until days 5-6 of neonatal life. The sensitivity of our assay systems may allow earlier cells to escape detection, but the relative order of expression is clearly Tpre, Tthy, Tind, and Tsu. It seems clear from the ontological studies that Tind and Tsu are acquired after functional divergence of T cells. It is known that hematopoietic precursors for T cells are in fetal liver. Organ culture studies of fetal liver show precursors for suppressor T cells at 19 days gestation (Mosier and Johnson, 1975). Precursors for cytotoxic T cells are in 19 day thymus (Ceredig et al., 1982), and organ cultures of fetal thymus cells can be sensitized to antigen specific alloreactivity by 15 days of gestation (Owen et al., 1976). These studies imply antigenspecific recognition structures and cells with functional capability appear in fetal development. These findings argue strongly against a roIe for Tthy, Tind, or Tsu in determining functional capability of T cells. Rather the expression of Thy, Tind, and Tsu may parallel some functional compartmentalization, because these antigens were acquired in developmental parallel with functional subpopulations. Receptors for alloreactive cells may be the most primitive and are certainly those acquired earliest in evolution. The ability to distinguish self and non-self at a cellular level was acquired evolutionarily as early as the protochordates (Hildemaan et al., 1979; Scofield et al., 1982). This system of allo-recognition clearly precedes the development of immunoglobulin, which is first documented in vertebrates (Clem, 1971). The ability to reject allografts has also been well documented in hagfish (Finsted and Good, 1966). Evolutionarily and ontologically alloreactive cells have been documented to precede other T cell functions. The developmental pathway for T cell antigen recognition may be branched into a path-

T CELL ALLOANTIGENS

17

way marked by receptors on allo-reactive cells and a pathway of receptors for regulatory cells which may not be allo-restricted. This prediction is in marked contrast to what we know about the acquisition of B cell antigen recognizing structures. The earliest immunoglobulin expressed in evolution (Clem, 1971) or in ontological development (Kearney et al., 1977; Kincaide, 1981, a review) is IgM. All cells in the B cell lineage appear to develop from the same functional pre-B cell, in a relatively linear pathway (Lawton et al., 1972), with the IgG, bearing cell the possible exception to that paradigm (Slack et al., 1980). However, in contrast to functional B cells, T cells show quite distinct, nonoverlapping functional compartmentalization (Cantor and Boyse, 1976). The existence of separate groups of genes for antigen receptors on allo-reactive and “imrnunoglobulin-like” T cell recognizing structures could explain this compartmentalization. T helper cell clones do not have cytotoxic T cell function even when the nominal antigenic specificities of the two clones are similar. Many T lymphocyte subsets are H-2 or Ia restricted in antigenic recognition; these cells perform their function by effector cell membrane-target antigen contact. In contrast, the evidence that regulatory cells for immunoglobulin synthesis are H-2 restricted is less compelling. Soluble antigen-specific factors can replace T cell function in many cases (Kapp et al., 1980; Taniguchi and Tokuhisa, 1980). Since there are clearly two separable classes of antigen receptor recognition requirements, one might predict that there are multiple genes for T cell receptors; at a minimum, H-2-restricted and non-H-2-restricted cells should utilize separate gene products. A branched pathway would explain why we fail to see Tthy, Tind, and Tsu expressed on allo-reactive T cells (Spur11 et al., 1983). The question of whether Tpre is the precursor for all T cells or whether it is a precursor for only the regulatory cells for immunoglobulin synthesis is being experimentally tested in a fetal animal model. If Tpre is a precursor for all T cells, then it seems likely that additional markers for allo-reactive T cells may eventually be detected in the same genetic linkage group, The failure to detect these antigens in our initial screen may be due to a biased screening technique which favored regulatory cells. Alternatively, if allo-receptors are more primitive, then polymorphisms in this genetic locus could be more restricted, preventing the production of alloantibodies in the strain combinations we have tested. Only two allelic forms of p have been documented for mouse immunoglobulin in contrast to the 13 known alleles for IgG,, (Green, 1978). Binz and Wigzell (1975a,b) succeeded in producing auto-antiidiotypic antibodies in mice and rats against T

18

F. L. OWEN

cell receptors; the antigen recognized by these antibodies was linked to the immunoglobulin gene locus (Binz et al., 1976).The cells used as the target of the alloantibodies were either MLR responding, Ia restricted cells, or cytotoxic effector cells which were H-2 restricted. These studies would predict that at least a part of the antigen receptor on alloreactive T cells is encoded by a gene linked to the immunoglobulin locus. It is possible that the primitive gene product for alloreactivity was the first antigen recognizing structure. As the immune system developed, this gene could have been duplicated to give rise to the immunoglobulin genes. Under pressures for diverse functions the immunoglobulin isotopes could have evolved. Only after the existence of functional immunoglobulin genes would there have been a need for regulation of expression of those gene products. The genes for T cell receptors which regulate immunoglobulin synthesis could have then evolved further from the Ig genes. Under these conditions allo-reactive T cell receptors could be coded for by a gene bank linked to Ig but distinct from the immunoglobulin genes which would, in turn, be distinct from a third gene pool coding for receptors for regulatory T cells. Considering the evolutionary and functional disparity between T cells which recognize alloantigens and those which regulate immunoglobulin synthesis, one would predict that the acquisition of diverse T cell receptors or “isotypes” would represent a bitruncated pathway of lymphoid development, One cannot eliminate the possibility that helper cells and/or H-2 restricted proliferating cells may have receptors encoded by additional unlinked segments of the genome. Since variable and constant region genes for K light chain (chromosome 6) (Hengartner et al., 1978), lambda light chain (chromosome 16), and heavy chain (chromosome 12) (D’Eustachio e t al., 1980) apparently evolved separately, it is not unreasonable to anticipate some “redudant” gene banks for T cell antigen recognition. Using chromosomal analysis of antigen specific, H-2-restricted T cell hybrids, it has been shown that chromosomes 6, 16, 4, and 17 are not required for antigen recognition. The authors interpret the data to imply chromosomes 8 and 12 cannot be excluded from coding for a T cell antigen-specific structure, although additional gene products are required as well (Marrack and Kappler, 1983). Since T cells are heterogeneous in function, concluding that one particular functional subtype utilizes a gene pool from one particular chromosome cannot be extrapolated to an assumption that all cells use the same genes for antigen specificity. The existence of functional antigen-specific pre-T cells in nude animals is documented. It is known that marrow cells from nude animals

T CELL ALLOANTIGENS

19

can reconstitute the thymus of lethally irradiated animals (Wortis et al., 1971), and that nude cells can be driven in vitro with T cell growth factors, including IL-2, to express cytolytic function (Hunig and Bevan, 1982). There is little evidence for the existence, however, of functional antigen-specific regulatory cells, although presuppressor cells may exist (Jacobsen, 1978). AKR nustr/nust’and +/nust’ animals were examined for the existence of cells expressing Tpre, Tthy, Tind, and Tsu. Only cells expressing Tpre can be detected in homozygous nudes, in contrast to heterozygous animals which express all four antigens (Owen, 1983).Marrow of the nude animal and spleen both show Tpre expression. There is no evidence showing Tpre is a direct cellular precursor for the cells expressing Tthy, Tind, or Tsu, although the data are not inconsistent with that possibility. The close genetic linkage and coexpression of this antigen on some early thymocytes suggests this would be a fruitful line of exploration. The implication that Tpre may be a prethymic T cell marker, overlapping the adult population of cells, stems from the existence of Tpre in marrow of the nude, expression of Tpre ontologically on fetal liver cells and depletion of thymic repopulating cell by anti-Tpre pretreatment of marrow (Owen, 1983). Coexpression of Tpre with other putative markers for pre-T cells has not been demonstrated. Although rabbit anti-brain associated T cell antigen (Golub, 1971) has been used as a marker for pre-T cells, it has been shown that this reagent frequently contains multiple specificities, many of which recognize myeloid precursors (Sato et al., 1980). Terminal deoxynucleotidyl transferase, Tdt, an enzymatic marker for pre-T cells, requires mega-cell preparations for analysis (Silverstone e t al., 1980). A putative rat anti-mouse pre-T cell monoclonal reagent (Lipski et al., 1982) is no longer accessible, frustrating our attempts at comparative studies of somatic cell hybrids of BW5147 and homozygous and heterozygous nude marrow. In contrast to developmental studies with Tpre, Tthy has been shown to be a probable precursor for the cells which express Tind and Tsu. Briefly, a neonatal animal model for modulation of expression of Tthy alloantigen was developed. Monoclonal anti-Thyd (17IIC6) or anti-Tthya (CB.13.10), was injected (10 pg/50 /.&neonate) into newborn C.AL-20 mice at day 0 and at 48 hours thereafter until animals reached 6 weeks of age. The appearance of Tthy, Tind, or Tsu was examined by quantitative adsorption of monoclonal antibody, and tested for residual antibody activity by cytotoxicity. Administration of antiThy” but not anti-Tthya prevents the appearance of the Tthy expressing cell. In addition, the Tind and Tsu bearing cells also fail to appear. Although it is possible that the Tthy-bearing cell is one which is

20

F. L. OWEN

required for maturation of the cells expressing Tind and Tsu, it is equally plausible that the Tthy bearing cell is the direct hematopoietic precursor for the cells expressing Tind and Tsu. In contrast, Tthy could not b e a pre-T cell marker because loss of these three cell populations (Tthy, Tind, and Tsu) does not prevent the appearance of Thy 1.2, Lyt 1, and Lyt 2 bearing cells in thymus and spleen. The number of thymocytes, frequency of Thy 1.2, Lyt 1.2, Lyt 2.2, and sIg surface positive cells, and the cytofluorograph pattern of stained T cells from anti-Tthya treated and age-matched cells from virgin animals are indistinguishable. This implies that T cell compartment is moldable and that depression of a cell population(s) is compensated for by an increase in other cell populations. A similar compensation is well documented for B cell subsets (Eig et al., 1977; Metcalf et al., 1982; Fultz et al., 1982). Functional studies in this animal model have only recently been initiated, but our preliminary data suggest the functional distortion in the T cell lineage is a subtle one. These data clearly imply that many functional T cells exist, including helper cells, which do not express Tthy, Tind, and Tsu and suggest our search for alloantigens encoded in this region has just begun. The Thy, Tind, and Tsu antigens are expressed at characteristic points in immunocompetence as well as ontogeny. This distribution has been determined largely by surface cytotoxicity tests or quantitative adsorption of monoclonal antibodies. Tthy is expressed on both the cortisone-resistant and sensitive thymocyte populations of adult animals (Owen, 1982a) in contrast to Tind and Tsu which are selectively expressed on the cortisone-resistant T cells. Tpre is expressed on both cortisone-resistant and sensitive cells as well. Low density T cells in thymus, separable on discontinuous density gradients, however, have lost Tthy and acquired Tsu. After cells exit the thymus, it is difficult to show surface expression of Tpre or Tthy in spleen or node of virgin animals. Tind and Tsu are both present on the resting spleen and in immunized animals, the Tind and Tsu frequency and/or density/cell are greatly elevated. Thy is not present in detectable levels on antigen-primed node cells in contrast to Tpre, which does reappear as a marker among secondary mouse T cells, although this could be due to a late driven differentiation event. Both Tpre and Tthy occur in marrow of normal animals. The cell expressing Tthy is Tpre+, BAT+, and Thy 1.2+,in contrast to a second cell which appears to be Tpre+, Tthy-, Thy 1%. The latter cell occurs in nude and is the only cell expressing an antigen in this linkage group to be documented in nudes (see Fig. 3). The lineage of cells which appear to develop from the Tthy cell may

NORMAL f C AL-PO j Virgin

STEM

+

-C

N u / N u (AKR STREAKER)

.

r

.

,

/

, c

/-

_----_ - . -.

.

-

Tprs

t

Virgin

ARREST

FIG.3. Schemata of distribution of antigens in this linkage group on developing lymphoid cells.

4NTIOEN 4CTlVATE

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be branched into nonoverlapping groups of cells expressing Tsu or Tind, but not both simultaneously. Analysis of unfractionated cell populations shows that Tind is expressed on Lyt 1 cells but Tsu is expressed on Lyt 2 cells. Closer examination of cells which arise from fusion of BW5147 and antigen-primed lymph nodes show, in a panel of 28 lines examined, none coexpress Tind and Tsu, in contrast to 15% which coexpress Tthy with either Tind or Tsu. It seems likely that Tind and Tsu are expressed on nonoverlapping cells in situ. In summary, there is a conserved order of appearance of the antigens Thy, Tind, and Tsu in ontogeny, in maturation of immunocompetent T cells, and in the functional effectiveness of regulatory T cells in vitro. These antigens seem to be part of a direct developmental pathway of cells, all excluded from alloreactive T cells. In contrast, Tpre is less convincingly restricted to this same developmental pathway, although it is coded for in the same linkage group and clearly overlaps the Tthy developmental lineage. VI. Evidence That Tpre, Tthy, Tind, and Tsu Are Excluded from Developing B Cells and B Cell Products

The T cell alloantigens described here are markers for discrete stages in T cell development. Therefore, in examining the question of cross-reactivity with B cells or B cell products, it has been necessary to examine cells at a spectrum of developmental stages ranging from fetal liver to secreting plasma cells. It can be concluded from the data summarized below that there is no apparent cross reactivity between Tpre, Tthy, Tind, and Tsu and any of the B cell targets examined to date. Fetal B cell hybrids of P3U1 and fetal liver cells at days 12 and 19 of gestation were screened for expression of Tpre, Tthy, Tind, and Tsu. Most lines (8 of 9) examined failed to express any of these antigens; an exceptional ninth line (day 12 gestation) expressed all 4 antigens and Thy 1.2, a T lineage marker (Owen, 1983). It is possible that the lymphoid compartment in day 12 fetal liver is not fully differentiated. This line was not tested for cytoplasmic p or for any B cell differentiation markers. It seems possible that T cells may fuse with P3U1 in a rare occasion. Since interspecies somatic cell hybrids can be made (Ephrussi and Sorieul, 1962), it is not unlikely that an occasional hybrid containing both T and B markers may be identified. C.AL-20 marrow cells transformed with Abelson virus have been examined for expression of Tpre, Tthy, Tind, and Tsu by quantitative adsorption of cytolytic antibodies. Although only three lines were available for study, none expressed any of these antigens. Since these

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virally transformed lines are believed to be pre-B cells, it seems unlikely that any of these antigens is expressed on all B cell precursors at this stage in development (Owen and Rosenberg, unpublished observations). An extension of these studies is planned. Unfractionated bone marrow from adult C.AL-20 cells adsorbs all lytic activity from anti-Tpre and anti-Tthy, but pretreatment of those cells with monoclonal anti-Thy 1.2 antibodies depletes the adsorbing cells. These results suggest the marrow cell is not an early B cell, but a recirculating T cell trafficking through the marrow (Owen, 198213). Pretreatment of thymocytes with monoclonal anti-Lyt I+ antibodies or anti-Lyt 2 depletes a cell which adsorbs anti-Tthy. This suggests the positive cell in thymus is not a B cell. Mature spleen cells from unimmunized animals fail to adsorb anti-Tthy even though 70% of these cells are mature, functionally competent B cells. I n contrast, anti-Tind or anti-Tsu can be adsorbed with resting spleen cells. However, pretreatment with monoclonal anti-Lyt 1 depletes the cell which adsorbs anti-Tind; pretreatment with anti-Lyt 2 depletes the cell which adsorbs anti-Tsu. B cells in spleen would not be expected to be perturbed by either of these reagents (Spurll et al., 1983). In functional assays, pretreatment of spleen cells with FITC monoclonal antibodies specific for Tpre, Tthy, Tind, and Tsu followed by polyclonal affinity purified anti-FITC antibody and complement does not deplete a cell which responds to LPS over a wide dose response range by incorporation of [3H]thymidine (S. Keesee, unpublished observation). An in vivo model for depletion of the T cells bearing Tthy, Tind, and Tsu has been developed. Briefly, injection of monoclonal anti-Tthy in neonatal mice and maintaining them on microgram amounts of antibody until 6-8 weeks of age depletes the Tthy cell as well as those expressing Tind and Tsu. In contrast, neither the frequency nor the surface Ig density of splenic B cells was disturbed b y anti-Tthy pretreatment on analysis in the orthocytofluorograph (Keesee and Owen, 1983). Adult B cell hybrids of P3U1 and antibody secreting lines were tested for surface expression of an antigen recognized by Tpre, Tthy, Tind, and Tsu by cytotoxicity. Neither P3U1 (BALB/c Igh-la), 71A7 (C.AL-20 * P3U1, anti-Con A), nor 103IIIF11 (C.AL-20 P3U1, antiBSA) expresses any of these antigens (unpublished data). Attempts to immunoprecipitate radiolabeled antigens from spleen with monoclonal anti-Tind and anti-Tsu serum (Spurll and Owen, 1981) resulted in identification of three major polypeptides which coprecipitate with each antibody. Preclearing that radiolabeled prepara-

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tion with polyvalent antibodies specific for each of the known B cell isotypes, including IgG, fails to deplete these polypeptides; anti-GP70, anti-Lyt 1, or anti-Lyt 2 also fail to preclear these antigens (Spur11 and Owen, 1981). Monoclonal anti-Tpre, Tthy, Tind, and Tsu were used to lyse spleen cells before stimulating them with LPS * TNP to produce TNPspecific T-independent plaque-forming cells, an event unaltered by our monoclonal reagents (M. Frye, Master’s thesis, Tufts University). This differs from direct lysis of resting B cells in visual assays described above because the frequency of PFC precursors could be too few to see visually. Monoclonal antibodies specific for azobenzene arsonate have been coated onto polyvinyl chloride trays. A second stage label of anti-Tthy, Tind, Tsu, or Tpre, radiolabeled with 1251,was used to evaluate possible cross-reactivity of the T cell alloantigens and secreted products of B cells (studies in progress). It will be necessary to evaluate a large panel of monoclonals, including a spectrum of isotypes, in order to conclude there are no shared determinants on immunoglobulin gene products and known products of IgT-C. However, unusual crossreactions have occurred with monoclonal antibodies in the past. One laboratory (Pillemar and Weissman, 1981) has shown a monoclonal anti-phosphorylcholine antibody reacts with the polypeptide backbone of Thy 1.2. The possible evolutionary relationship between genes encoded in the IgT-C region and immunoglobulin genes, implied by their close proximity on chromosome 12, might lead one to speculate some shared structural homology may exist. This approach may lead to a prediction of one or more shared domains. VII. Cross-Reactive Determinants Shared by T Cell Alloantigens in This Linkage Group a n d Soluble T Cell Factors

Monoclonal antibody specific for Tind (9IIIA2) has been shown to recognize a cross-reactive determinant on an antigen-specific T augmenting factor. A monoclonal T cell line (FLlO), arising from fusion of BW5147 and immune A/J spleen cells, releases an antigen-specific soluble factor which binds to KLH (Hiramatsu et al., 1981) and regulates the hapten-specific response to KLH TNP i n uitro. The cells display Lyt 1.2 and Thy 1.2 (Hiramatsu et al., 1981) and also express Tind as a surface determinant on a high frequency of cells (Nakajima et at., 1983). The regulatory factor derived from extracts of these cells can be affinity chromatographed on KLH Sepharose columns and/or monoclonal anti-I-ATk(line FL10, Hiramatsu et at., 1982). It has been shown that the biological activity, 3-fold amplification of the IgG PFC

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secondary response, requires both the KLH binding component and the I-ATk bearing component. It has been proposed that the active factor is composed of two chains, one binding nominal antigen, and a second bearing I-ATk (Miyatani et al., 1983) and that cell extracts contain the two chains both in associated and nonassociated forms. In a group of experiments using anti-Tind (9IIIA2) insolubilized on Sepharose, KLH-Sepharose, and anti-I-ATk-Sepharose,it has been found that biologically active factor binds to anti-Tind, KLH, and anti-I-ATk. Column effluents are devoid of augmenting activity. Elution of these columns occurs with low pH buffer, although all deplete it. When anti-Tind effluent and KLH effluent are combined, no activity is found. When anti-Tind effluent and anti-I-ATkeffluent are combined, activity can be restored. These data suggest that T augmenting factor from the FLlO line is composed of two chains, one bearing both anti-Tind cross-reactive determinants and a binding site for KLH and a second chain with determinants encoded by the I-A region of chromosome 17 (Nakajima et al., 1983). Characterization of the size of the molecules in the biologically active material extracted from FLlO has been attempted. Affinity purification of [35Slmethionine-labeled KLH binding material from FLlO results in identification of a polypeptide(s) of 67,000 and 33,000 MW. Under reducing conditions only one peak of 33,000 MW was observed. Affinity purification of [35S]methionine-labeled reduced material on KLH or anti-1-ATkcoupled petri dishes results in identification of 33,000 MW polypeptides. Preclearing with anti-Tind Sepharose depletes the KLH binding but not the anti-I-ATkbinding material (Miyatani et al., 1983). These data imply that Tind determinants are expressed on the KLH binding polypeptide of apparent MW 33,000, and that the I-A determinant is present on a second chain of the same molecular weight. This contrasts with an earlier report from our laboratory suggesting the molecular weight of Tind from spleen cells was 62,000 (Spur11 and Owen, 1981). It will be necessary to examine many cell lines and to achieve more in depth biochemical data before a viable picture of the structure of these determinants can be obtained. Posttranslational modification of products of the same gene could occur as a routine mechanism. Although the above data are consistent with a model in which Tind is associated with an antigen-specific T cell factor, this serological cross-reactivity does not prove identity. In the same experiments, anti-Tsu and anti-Tthy did not react with FLlO or with products of FL10. At present only one of the T cell alloantigens in this region has been associated with a T cell factor. The close genetic linkage of all four antigens in this group implicates the other gene products in this

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region, but very indirectly. Caution must be used in drawing a conclusion based on only one cell line. Many investigators have reported that rabbit antiidiotype reagents cross-react with murine T cells (Eichmann, 1978, review), and T cell regulatory products (Krawinkel et al., 1978; Bach et al., 1979; Germaine et d., 1979). Some monoclonal antiidiotypic reagents show cross-reactivity as well (Reth et al., 1981; Cerny et al., 1982). These studies suggest that genes encoding the T cell-specific antigen binding molecules may bear an evolutionary relationship close to immunoglobulin genes, if indeed identical minigene segments are not shared. However, when monoclonal T cell lines, expressing surface determinants cross-reactive with idiotypic determinants on immunoglobulins, have been tested for cross-hybridization with cDNA probes for B cell immunoglobulin idiotypes, the results have been disappointing. In fact, when functional T cell lines have been examined for rearrangement of V-J segments, no rearrangement has been seen, and no deletion of the p gene is apparent (Kronenberg et al., 1980; Kurosawa et al., 1981). However, one exceptional report has shown V-Jjoining in T cell hybrids (Zuniga et al., 1982). This may be nonproductive activity on the twelfth chromosome. One group has reported producing a cDNA probe by cross-hybridizing monoclonal B cell immunoglobulin cDNA with T cells specific for the same hapten. This resulted in cloning a transcription product ubiquitous to T cells and some B cell lymphomas (H. Kurosawa and S. Tonegawa, personal communication, Seminar, MIT, 1982), but has not resulted in cloning a functional T cell receptor molecule. Studies in progress in other laboratories have shown monoclonal reagents specific for the “constant portion” of T suppressor cell factor may recognize products of genes mapping to the twelfth chromosome. It has been shown that the gene(s) encoding this determinant lie between a recombination event in the variable region and prealbumin (see Fig. 1) (Tokuhisa et al., 1982).This segment of the chromosome is inclusive of the region we have named IgT-C. Attempts to produce cDNA probes are active; preliminary data showing identification of active components translated in vitro in oocytes suggest complementation for function by genes encoded on chromosome 17 and chromosome 12 is essential (Taniguchi et al., 1982). These data are in marked contrast to studies showing a single chain factor has determinants cross-reactive with monoclonal anti-1-J reagents and those binding to the hapten, GAT (Krupen et al., 1982). In vitro translation studies indicate a product of 19,000 MW mediates suppressor function (Weider

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et al., 1982). There is currently no implication of determinants encoded on chromosome 12. The studies outlined above suggest two separate gene groups may encode antigen-specific factors. A subset of factors may require two separate polypeptides for function, one encoded within H-2, while the second may be encoded by a region of chromosome 12. VIII. In Vifro Functional Role of Cells Expressing Tpre, Tthy, Tind, a n d Tsu

Monoclonal antibodies directed against these cell surface alloantigens have been used in vitro as tools to selectively eliminate cell populations expressing these antigens in an effort to determine functional capabilities of the cells. Prototype monoclonals for each of the antigens were chosen (Tpre, F.6.9.1; Tthy, 17IIC6; Tind, 91IIA2; and Tsu, 13IIIB4). Many of these antibodies were not lytic and were modified with FITC and used with an affinity purified anti-FITC polyclonal mouse antibody and complement. Antibody was used to deplete spleen cells of either C.AL-20 or BALB/c mice before initiation of in vitro culture. Cells were tested for the ability to produce TNP-specific plaque-forming cells. Mice were pretreated with HRBC at day 4, and at day 0 were challenged with HRBC TNP. TNP-specific plaques were evaluated on days 3, 4, 5, and 6 in uitro. Monoclonal anti-Tthy, Tind, and Tsu all eliminate regulatory cells for the PFC response. Antigens in this group may, in fact, be restricted to expression on regulatory cells. There is no evidence for elimination of helper cells, but rather removal of the Tthy bearing cell enhances responses at days 3 and 4 by 6- to 7-fold. In contrast, removal of the Tind bearing cell changes the kinetics of the response but not the total response. Tind is expressed on a cell which controls the peak response at day 5; depletion shifts that peak to day 4. Anti-Tsu depletes a late acting regulatory cell on day 6 (Spur11et al., 1983). The magnitude of that change is 2- to 3-fold and is believed to be important because of the reproductability of this effect. That these cells which regulate the PFC response are entirely discrete has not been established. It is known from our in vivo differentiation studies that the Tthy bearing cell is a probable precursor for the cells expressing Tind and Tsu (Keesee and Owen, 1983). It is possible that antigen driven differentiation may take place in vitro and that the monoclonal reagents simply eliminate the same cells in different states of development, thereby shifting the kinetics of the response. In additional studies, the cells selected for in vitro growth originated

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from virgin animals. Primary direct plaque-forming cells were evaluated after 3, 4, or 5 days of growth in the presence of 10 Fg of KLH TNP (13TNP/KLH). Monoclonal anti-Tpre alters the frequency of the day 5 plaques by reducing the total response Sfold. Anti-Tind and anti-Tthy have no effect on the primary response. The role of the cell expressing Tpre is not known. A passive mechanism in which anti-Tpre eliminates a late differentiating cell which is necessary for optimal helper activity on day 5 is attractive, but transfer of cells treated with anti-Tpre to secondary cultures to rule out active generation of a regulatory cell has not been completed. In contrast, when mice primed on day 10 with KLH - TNP were grown in uitru with 10 pg of KLH * TNP/5 x lo8cells, to evaluate in the secondary assay both direct and indirect PFC, the kinetics of the response after pretreatment with monoclonals anti-Tpre, Tthy, Tind, and Tsu were quite markedly different than the true primary response, described above, or the primary B cell response described earlier. Anti-Tsu eliminates a late acting cell which appears to be a suppressor (2- to 3-fold) at day 6 in culture. The suppressor appears to regulate the IgM response but not the IgG response. Anti-Tthy enhances the IgG response at days 4 and 5 only. Anti-Tind enhances both the IgG and IgM response at days 4, 5, and 6 (Frye and Owen, unpublished data). Anti-Tpre has no obvious role in regulating the secondary response. The magnitude of the changes in the secondary response introduced by elimination of regulatory cells is more modest than those affecting the first culture system described. One must conclude that Tthy, Tind, and Tsu are expressed on cells which may have an intimate codependence and possibly redundant functional capabilities. An equally close relationship to the helper cell may be implied, although it seems clear that the mature helper affector itself does not express any of the specificities recognized by our current panel of monoclonals because none of the PFC responses is initially reduced at early onset of culture. Monoclonal anti-Tthy, Tsu, and Tind pretreatment of B cells,.already treated once with anti-theta and complement, does not alter the response, in agreement with earlier visual studies which indicated the primary specificities for anti-Tthy, Tind, and Tsu were T cells. The cells which express these antigens have a preferential association with Lyt surface phenotype at the whole population level. Tthy is expressed on an Lyt 1+2+3+thymocyte, but the surface phenotype of the splenic T cell has not been determined. In fact, the frequency of the positive cells in the periphery is either too low to be detected or the surface density of antigen is too sparse. It is possible that the Tthy

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bearing cell has a “plasma cell-like” role in mature populations so that Tthy is not solely an integral membrane-bound protein in the antigen activated population. Tind is expressed on an Lyt 1+2-3- cell in spleen and Tsu is expressed on an Lyt 1-2+3+cell in spleen. However, parallel expression with the Lyt subgroups is lost in clones of T cell hybrids. In our hybrid panel (most of which are not functional antigen-specific cells), Tind or Tsu shows random association with Ly surface phenotype. Functional capabilities of cells expressing these antigens at the clonal level have not been determined. Since the BW5147 thymoma cell is the fusion partner for these cells, one must consider that genetic complementation could take place so that Lyt 1or Lyt 2 is expressed as a function of the pooled genetic information of the hybrid and does not reflect the native resting state of the antigen primed lymph node. The fusion of two other T cell lines was attempted, but the clonal success rate of fusions was too low to yield clones expressing Tind, Tsu, and Tthy for study. WEHI 7.1, BAL 5, L51784 lines (BALB/c genetic background) were used unsuccessfully. Additional statistical data from T cell clones are desirable to make a conceptual interpretation about the nature of resting clones. The discovery of Lyt surface phenotypes (Boyse et al., 1968) and the assignment of these antigens to functional subgroups of cells (Cantor and Boyse, 1975a,b; Jandinski et al., 1976; Cantor et al., 1976; Huber et al., 1976) was based on antibody and complement-mediated elimination of whole cell populations. Anti-Lyt 1+2-3- cells were assigned to the helper cell population, and only later extended to include cells which induce growth of other cell types, including mast cells (Nabel et al., 1981) and B cells (Fresno et al.,1982). Cells which respond in the mixed lymphocyte reaction (Cantor and Boyse, 1975a) are also believed to belong to this functional subgroup. In contrast, Lyt 1-2+3+ cells are suppressor effectors for antibody synthesis (Jandinski et al., 1976) or cytotoxic T cells (Cantor and Boyse, 1975b). The function of a major group of cells which is Lyt 1+2+3+is less clear, although it has been hypothesized that these cells must be precursors for both the Lyt 1-2+3+ and Lyt 1+2-3- cells (Cantor and Boyse, 1976). Recent developmental data would argue that the Lyt 1+2+3+subset develops after the Lyt l+2-3- cell at 19 days gestation, arguing that the former could not be the precursor for the later (Van Ewijket al., 1982; Scollay, 1982). Fluorescent-activated cell sorter data using monoclonal anti-Lyt antibodies (Ledbetter et al., 1980) suggest almost all cells are Lyt 1+and differences reported earlier were a result of relative antigen density/ cell and not absolute differences in functional capabilities of Lyt l+2-3- and Lyt 1-2+3+ subsets.

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The revolutionary techniques leading to cloning of functional T cell populations (Watson, 1979; Nabel et al., 198la; Gillis and Watson, 1981) have introduced some contrasts between individual clones of cells and answers obtained with polyclonal cells as the target of antisera. Swain (Swain et al., 1981) observed that the Lyt type of the clone was less associated with its function than with its target antigen. Lyt 1 surface phenotype was often associated with cells which recognize Class I1 MHC rather than Class I MHC determinants. In more recent experiments at the whole population level, this Lyt 1 association with Ia cytotoxic T cell target determinants is not found (Miller and Stutman, 1982). One must speculate that there are strong evolutionary pressures for selection and expansion of appropriate clones showing Lyt phenotype and functional restrictions. The rationale for this apparent selection remains obscure; an elusive key may be the primary functional role of the Lyt structures, apart from markers of cell differentiation. The T cell antigens we have described, Tthy, Tind, and Tsu bearing cell populations could not be identical with the Ly subgroups but, alternatively, may developmentally parallel the functional subsets Lyt 1+2+3+,Lyt l+2-3-, and Lyt 1-2+3+.Evidence eliminating this possibility comes from the finding that neonatal mice maintained on antiTthy monoclonal antibodies for 6 weeks of life lose the cells expressing Tthy, Tind, and Tsu but have normal levels of cells expressing Thy 1.2, Lyt 1, and Lyt 2. We conclude the cells bearing Tthy, Tind, and Tsu may lie within or overlap Lyt differentiation pathways but could not directly parallel these groups. The close relationship and interdependence of the cells in the IgT-C regulatory pathway are reminiscent of the multiple cell types reputed to regulate immunoglobulin synthesis in vitro based on Lyt surface phenotype. Anti Lyt-2 pretreatment of cells is known to eliminate a late acting suppressor T cell for in uitro secondary plaqueforming cell responses (Jandinski et al., 1976).Anti-Lyt 1 eliminates a cell which acts as a helper for the PFC response but also acts as an inducer for the suppressor cell responses (Eardley et al., 1978). This observation led to the concept that “feedback” helper cells induce suppressor T cells to function in an immune response. Looking at clones of Lyt 1+inducer T cells (Nabel et al., 1981a), which fail to function on their own as true helpers, suggests that the interrelationship of Lyt 1+2-3-, Lyt 1-2+3+ cells may be more complex than the original observations suggested. It is possible that T cells may release physiological factors which regulate growth and modify the immune response only as a secondary function.

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The suggestion that the suppressor cell pathway is multicellular is strongly mirrored in studies of idiotype regulation by Tsl, Ts2, Ts3, and pre-Ts cells. Regulation of delayed type hypersensitivity responses has been used to define this circuit of regulatory cells (Benacerraf and Germaine, 1978). The Tsl cell expresses I-J, an antigen cross-reactive with the major serum idiotype of the NPh response, and regulates the efferent phase of the immune response (Weinberger et al., 1979a). This cell is an Lyt 1+2-3- cell, in contrast to the Lyt 1-2+3+ Ts2 cell which can be the effector cell in suppression of DTH in the efferent stages. This cell can utilize a receptor which recognizes the NPh idiotype and can bind polyvinyl chloride dishes coated with idiotype (Weinberger et al., 1979b). The Ts2 cell can be induced by in vivo administration of factor(s) from the Tsl cell which has been affinity purified on an antigen-specific column (Okuda e t al., 1981).The Ts3 cell is again Lyt 1+and appears to be generated vary late in the response (Sherr and Dorf, 1980). It was concluded from studying factors extracted from clones that this I-J- cell can be induced by the Ts2 cell. The Ts3 celI also appears to express surface idiotype but in contrast to the Ts 1 cell cannot be directly induced by administration of the rabbit antiidiotype serum (Minami et al., 1982). Pre-Ts cells differentiate very late as a result of cellular differentiation on earlier, apparently committed precursor cells (Dorf et al., 1982). The circuit of T cells, generated in mice, directly or indirectly, pretreated with rabbit antiidiotype serum seems to complete a loop of idiotypic-antiidiotypic interlocking structures. It is difficult, however, to draw any direct parallels between the pathway of suppressor T cells (at least four discrete cells) observed in idiotype regulation and those described earlier by negative depletion with anti-Ly reagents. The Lyt surface phenotypes of cells involved and their time of appearance relative to antigen administration differ sharply. However, in vivo studies in mice stressed with milligram quantities of antiidiotype could alter the normal balance of Lyt cells and select for quiescent clones. When monoclonal factors, possibly products of minor clones, are used to induce effects, this imbalance may become even more marked. Since the idiotype circuit has never been completed in vitro, using antibody synthesis as the endpoint, the comparison must remain remote. The major contributions from these studies may help to define the potential limits of capabilities of the immune system more completely than the normal response to antigen. A relationship between Tthy, Tind, Tsu, and Tpre relative to the Ly loop of regulatory cells or the idiotypic circuit has not been documented. All three pathways appear to support the concept that the

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regulatory function is multicellular and that complex interdependent relationships exist between these cells. The fact that our monoclonals are specific for alleles on genetic background (Igh-ld and Igh-le), less popular among immunologists, has limited collaborative interactions. These questions remain a pressing priority. IX. In Vivo Studies on the Function of Tsu and Tind Bearing Cells

The Tsu antigen was described using in vivo functional assays, largely because the initial alloantiserum was raised in BALB/c AnN animals against C.AL-20 T cells grown in 10% FCS for 48 hours. Although out antiserum detected allele specific antigens on C.AL-20 cells, it also contained high titers of anti-FCS reactivity. Adsorption of FCS-Sepharose columns removed this activity, but freeze-thawing the antiserum destroyed the fragile activity of the antibody. In addition, the active serum antibody was almost exclusively IgG, which failed to fix complement in vitro. Prior to the development of monoclonal antibodies, our assay systems had to avoid the generation of antigenantibody complexes which are known to regulate immune responses. Since all Mishell-Dutton in vitro antibody formation assays use fetal calf serum, we were forced to use in vivo PFC assays. This approach led to the conclusion that the major antibody specificity in the alloantiserum was directed against a suppressor T cell. Briefly, alloantiserum was administered in vivo by intravenous injection in the tail vein of either C.AL-20 of BALB/c mice. Dilutions equivalent to 2-5 pl of serum caused a reduction in the in vivo plaque-forming cell response of recipient C.AL-20 mice. The magnitude of this reduction was 2- to 5-fold, varying from individual to individual. By adoptive transfer of cells to 200 R y-irradiated mice, it was demonstrated that the suppressive effects were active cell mediated events. By depletion of resting spleen cells with antibody and complement followed by adsorption of the antiserum, it was shown that the primary target of the antiserum was a Lyt 1-2+3+ cell (Owen, 1980a). The induction of suppressor T cells seems to be non-antigen-specific or polyclonal in nature. Administration of anti-Tsu suppresses the response to KLH . TNP, KLH - FITC, or KLH - Ars and to SRBC. It does not suppress the response to T-independent antigens Ficoll * TNP or Ficoll * FITC. Reduction in the antihapten response does not preferentially effect either low or high affinity clones or a major cross-reactive idiotype (Owen, 1980b). Tha generation of suppressor T cells is not restricted by the Igh-1

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type of the animals, but rather the IgT-C allelic type. In contrast, the ability of the T cells to act as suppressors on adoptive transfer to nonirradiated animals is restricted to an Igh-V linked gene. A match at the Igh-V genes is suficient to permit function. It is possible that a match at either the IgT-C genes or the Igh-V genes (not both) is adequate to permit function but the congenic mice which match at IgT-C and mismatch at Igh-V were not available for study (Owen et al., 1981). The first monoclonal in this panel, anti-Tind (9IIIA2), was characterized by injecting mice with limiting dilutions of antibody in tissue culture supernatants. Intravenous administration of antibody in 2 ng quantities/mouse caused cyclic alterations in the immune responses (Spurll and Owen, 1981) depending upon the time prior to antigen injection at which the antibody is administered. When antibody is given on day 0, modest suppression is seen; in contrast, 4 day crests of peaks of enhancement or suppression are seen when the antibody is given on days - 10, -4, or - 1.The cyclic waves of either enhancement or suppression look very much like the pattern of regulation one sees when monoclonal antiidiotype antibodies are administered in vivo (Reth et al., 1981). X. Preliminary lmmunochemical Characterization of Tsu a n d Tind

Immunoprecipitation of [3sSlmethionine-labeled spleen cell preparations of either BALB/c or C.AL-20 animals has led to preliminary estimates of molecular weight differences between two antigens encoded in this linkage group (Spurll and Owen, 1981). Briefly, spleen cells activated with concanavalin A in vitro for 48 hours and then incubated for 4 hours with 100 pCi of [35S]methioninein methioninefree, serum-free media were washed and lysed with a detergent cocktail containing 0.1% SDS, 0.5% deoxycholate, and 1% Triton X- 100. Anti-Tsu polyclonal serum precipitates several species, depending upon the individual bleed or pooled sample. Two major polypeptides were precipitated, MW 68,000 or 62,000. Associated smaller subunits coprecipitated. Monoclonal anti-Tind precipitated 62,000,45,000, and 17,000 MW chain, and precleared this chain from the extract to be precipitated with anti-Tsu serum. Anti-Tind does not preclear the 68,000, 45,000, or 25,000 MW species from extracts precipitated with anti-Tsu. An artifact at 65,000 MW is a common polypeptide precipitated with all antibodies produced in ascites fluids. Strain controls (Owen and Spurll, 1981) and controls with Y ~ K (same isotype as anti-Tind 9IIIA2) anti-Con A do not precipitate

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68,000 MW Tsu or 62,000 MW Tind. Antiimmunoglobulin isotype antibodies do not preclear these polypeptides from radiolabeled cell extracts. These data must be considered preliminary since functional assays on precipitated products are not available. Efforts to characterize these antigens at a molecular level would be aided by development of a cell line expressing an antigen in this series on a functional product. Our efforts have concentrated on production of hybrid cells between immune lymph node and BW5147 thymoma cells. I n our hands the cpm precipitable from hybrid cells are fewer than an equivalent number of unfractionated splenic cells stimulated with Con A, even though hybrid cells do express this antigen as a surface serological determinant. The structural work on immunoglobulin molecules was made possible by the availability of myeloma proteins from naturally occurring tumors and later from somatic cell hybrids of antigen-stimulated B cells and nonsecreting HAT-sensitive myeloma cells (Kohler and Milstein, 1975). At present, neither of these two equivalents exists for T cells. XI. Concluding Remarks

Classical serological and animal genetic studies have led to the prediction that a cluster of tightly linked genes (four or more) lies distal to alpha on chromosome 12 in the mouse. The gene products represent alloantigens on lymphoid cells at characteristic stages in T cell maturation and in T cell immunocompetence. It is possible that they may also represent constant regions of T cell antigen receptors which are distributed differentially on T cell subsets and in development in parallel with the immunoglobulin isotypes which also serve as differentiation markers for B cells. Vertical progress on defining the number of genes and relationships of each gene product to another and to immunoglobulin is critically dependent upon good structural, biochemical approaches which at present have not been applied to this problem. A major limitation has been the apparent low density/cell for antigen expression. Until good structural evidence is obtained, molecular biology and gene cloning attempts may be difficult to initiate. Recombinant inbred strains of mice developed for dissection of the genes in the IgT-C region may be helpful in a great number of other studies. Because there are multiple recombinant strains (-60) available for studying additional genes in and around chromosome 12, it

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35

should be possible to further explore this region and define gene products potentially unrelated, but linked, to IgT-C. The development of monoclonal antibodies specific for gene products, T cell hybrids expressing these antigens, and recombinant strains of mice limiting the map position of the gene cluster suggests that acquisition of structural data is an approachable goal.

ACKNOWLEDGMENTS Collaboration efforts of Roy Riblet, Institute for Cancer Research, Philadelphia, PA has made the genetic work described here possible. Drs. P. Nakajima, K. Miyatani, and T. Tada have examined antigen-specific factors for expression of our antigens. Drs. G. M. Spurll, S. K. Keesee, and M. Frye in my laboratory made valued contributions to our monoclonal reagent and information base. The generosity and support of numerous colleagues in the Tufts and greater Boston community have been sustaining factors in this work. Research has been supported by NIH Grants, AI-15262, AI-14772, and CA 24530.

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ADVANCES IN IMMUNOLOGY, VOL. 34

Heterogeneity of H-2D Region Associated Genes and Gene Products TED H. HANSEN,' KEIKO O Z A T 0 , t AND DAVID H. SACHSS Depodment of Genetics, Washingfon University School of Medicine, St. Louis, Missouri,

t laboratory of Developmental and Molecular Immunity, National Institute of Child Health and Human Develapmenf, National Institutes of Health, Bethesda, Maryland, and

$ Transplantation

Biology

Section, Immunology Branch, National Cancer Institute, National Institutes of Health, Befhesda, Maryland

I. Introduction 11. Antigenic He

.............................

39

Products Encoded in the Dd

....................................... Products Encoded in the Dd

....................................... IV. Quantitative Comparisons of Gene Products Encoded in the D" Region ............................................................... V. Functional Studies of H-2L" Gene Products ....................... aplotypes . . . . . . . . . . . . . . VI. Searches for Allelic Products of H-2Ld in 0 VII. Studies Using Genoniic Clones of H-2D Region Loci . . . . . . . . . . A. Characterization of D Region Genes Using Genoinic Clones B. Characterization of D Region Gene Products Using Genomic Clones . . VIII. Evolutionary Models and Future Approaches ........................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

41 46 50 52 54 58 58 60 64 67

I. Introduction

Class I MHC antigens are cell surface glycoproteins defined by their predominant role in allograft (histocompatibility) responses and distinguished by their high level of genetic polymorphism. These antigens have also been determined to b e involved in physiologic immune responses by controlling T-cell recognition of self and non-self cell surface antigens. Although it was shown more than a decade ago that the class I MHC antigens of the mouse are determined by genes mapping to the segments of chromosome 17 designated the H-2K and H-2D regions (cf. Klein, 1975), the number of genes in each region is still a matter of speculation. It is clear, however, that the once accepted dogma that there is only one gene product for each H-2K or H-2D region (the two loci model) is incorrect at least for certain haplotypes. I n the case of the H-2D region of the d haplotype designated D d ,there is now a preponderence of data demonstrating at least two gene products, Dd and L*, determined by two separate genes H-2Dd and H-2Ld, respectively (cf. Demant et nl., 1981).By several structural (Coligan et 39 Copyright 6 1983 by Academlc Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-022434-8

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TED H. HANSEN ET AL.

al., 1980) and functional (Levy and Hansen, 1980) criteria the Ld molecules have been found to be analogous to K and D antigens. However, the cell surface expression of Ld antigens appears 2 to 3 times lower than that of Kd and Dd antigens (Potter et al., 1981) and H - 2 L gene products have been proposed to have unique antigenic properties rendering them less polymorphic than H-2K and H-2D gene products (Demant et aZ., 1978). Subsequent to the discovery of Ld molecules, studies of the molecular heterogeneity of D region encoded molecules using two mouse strains carrying mutant D region genes have indicated the existence of four new molecules (M, R, L2, Lq) which are expressed in nonmutant mouse strains of the appropriate genotype and two new molecules (p40, gp39) which are unique for their respective mutant strains (Table I). Studies to resolve the molecular heterogeneity of D region encoded products in haplotypes other than H-2d are controversial with certain studies suggesting haplotype-related differences in the number of expressed D region gene products (Melino et al., 1982)and other studies failing to detect such differences (Ivanyi et al., 1979).Among the quesTABLE I DEFINITIONOF Dd REGION ENCODEDMOLECULES Detection in haplotype Molecule

Serological definition

Assay (reference)

d

dml

dm2

Yes

Yesa

Yes

H-2.4+

Yes

Yesa

No

Yes

No

Yes

H-2.4-H-2.28+ H-2.64+, H-2.65+ H-2.4+, H-2.28-

Rd

Cocapping Immunoprecipita tion Cocapping (1)b Immunoprecipitation (2-5) Cocapping (6) Immunoprecipitation (7) Immunoprecipitation (8)

Yes

NT

Yes

Lq

Immunoprecipitation (9)

Yes

NT

No

L2

Cocapping (10)

Yes

No

No

Immunoprecipitation (11) Immunoprecipitation (12)

No No

No Yes

Yes NT

Dd Ld(D') Md

H-2.4-, H-2.64+, H-2.65+ Anti-Qa and H-2.28+ H-2.4-, H-2.28+, H-2.29Xeno-anti-H-2 H-2.4-, H-2.28+

Denotes altered structure. (1) Lemonnier et al. (1975); (2) Hansen et al. (1977); ( 3 ) Neauport-Sautes et al. (1977); (4) Hansen et al. (1978); ( 5 ) Hansen and Sachs (1978); (6) Demant and Ivanyi (1981); (7) Sears and Wilson (1981); (8) Hansen et al. (1981); (9) Demant and Roos (1982); (10) Ivanyi and Demant (1982); (11)Robinson (1982); (12)Wilson et al. (1982). a

HETEROGENEITY OF

H-2D

GENES

41

tions emerging from these studies of D region antigens, are (1)do all haplotypes express the same number of D region encoded molecules? and (2) when multiple D region molecules are detected, what is their genetic, molecular, and evolutionary relationship to each other and to other defined class I antigens? The answers to these questions should impact on more general questions regarding the mechanisms responsible for generating the high level of polymorphism of genes encoding class I MHC antigens. New technical advances promise to provide the tools to address these questions. The availability of monoclonal antibodies to H-2K and H-2D antigens has provided a technical stimulus for the study of class I antigens (Kohler and Milstein, 1975; Lemke et al., 1978). Monoclonal antibodies have facilitated the serologic and molecular resolution of H-2 antigens and have circumvented several of the caveats encountered using complex alloantisera. Techniques exploiting the advantages of monoclonal antibodies include immunoprecipitation, cell surface quantitation, and idiotype recognition of H-2 molecules. Recent applications of molecular genetic techniques have also provided a new impetus for the investigation of class I MHC antigens. Genomic clones ofH-2Ldwere among the first MHC genes characterized (Goodenow e t al., 1982a; Evans et al., 1982a). Recombinant DNA technology not only provides precise information on the genetic organization of H-2 genes but when taken in the context of serologic, structural, and chemical findings, should also yield invaluable information regarding the genetic evolution of H-2 molecules. In this article, recent discoveries of the serologic, molecular, genetic, and functional heterogeneity of H-2 antigens are summarized. Because of the focus of our own research in this field and due to the wealth of new information in this area, we have elected to limit our review to H-2D region associated genes and their products. However, an understanding of the D region makes possible models about general M H C gene evolution and suggests approaches testing the validity of such models. II. Antigenic Heterogeneity of Gene Products Encoded in the Da Region

Serologic characterization of H-2 molecules with alloantisera has contributed numerous important insights into the genetic organization and structural relatedness of class I MHC antigens. For example, pioneering studies by Shremer, Klein, and Snell (cf. Klein, 1975; Snell et al., 1975) demonstrated that alloantisera defined serologic specificities which could be divided into two categories designated

42

TED H. HANSEN ET AL.

private and public. Private specificities were determinants unique for an H-2 haplotype, whereas public specificities were shared between different haplotypes. Using private specificities as markers for H-2 gene products, the analysis of intra-H-2 recombinant haplotypes demonstrated that the mouse MHC could be separated into two genetically distinct regions designated H-2K and H-2D. The sharing of a public specificity by antigens encoded in the H-2K region and antigen encoded in the H-2D region provided the first evidence that these non-allelic gene products were structurally related and inspired the gene-duplication model (Klein and Shreffler, 1971; Snell et al., 1971). Alloantisera were also used to define the number of different molecules determined by each H-2K or H-2D region and to determine whether private and public specificities were borne on the same molecules. Although early investigations using co-capping techniques suggested that there was only a single gene product encoded by each H-2K or H-2D region (Hauptfeld and Klein, 1975; Lengerova et al., 1974), other investigations of the H-2Dd region revealed two separate molecules (Lemonnier et al., 1975).This initial observation of H-2Dd region molecular heterogeneity has now been confirmed by diverse techniques and several reports of additional heterogeneity have appeared in the recent literature (see Table I). Resolution of new antigens determined by genes within the Dd region has been facilitated by the study of the two mutant mouse strains B10.D2-H-2dm1 and BALB/c-H-~~"~. The B10.D2-H-2dm1 strain (synonym dml), a mutant of the B10.D2 congenic line, was discovered by Egorov using skin graft responses in mice that had been mutagenized with diethyl sulfate (Egorov, 1967). The d m l mutation was shown to map genetically to the H-2Dd region and was recognized in serologic and histocompatibility assays to have both a gain and loss of antigenic specificities when compared with BlO.D2 antigens (Morgan et al., 1978; Ivanyi and Demant, 1979; Huang et al., 1979). The BALB/C-H-~"~strain (synonym dm2), a spontaneous mutation of the BALB/cKh inbred line, was discovered by Melvold and Kohn also using skin graft rejection as an assay system (Melvold and Kohn, 1976). Histocompatibility and serologic comparisons of the antigens of dm2 and BALB/cKh showed dm2 to be a loss mutation (McKenzie et al., 1977). The d m l and dm2 mutant strains have been used to determine the number of different molecules encoded by genes within the H-2Dd region. A summary of the D d region molecules defined to date using antibodies to either co-cap or precipitate specific antigens is listed in Table I. The molecule bearing the private serologic specificity H-2.4 is

HETEROGENEITY OF

H-2D

GENES

43

designated Dd.Both d m l and dm2 mice have been shown to express a Dd molecule, but in the d m l mutant its structure has been shown to be different and it is therefore designated Ddm'(see Section 111). A second molecule determined by a D d region gene was originally discovered by co-capping techniques (Lemonnier et al., 1975) and later confirmed by immunoprecipitation studies (Hansen et al., 1977a, Neauport Sautes et aE., 1977).This new molecule, now designated Ld, was detected by certain alloantisera to the public H-2 specificity H-2.28, after prior capping of H-2.4 positive molecules. When dm2 mice were examined they were found not to express a D region encoded molecule, tentatively designated D ', which was detected with alloantisera to public H-2 specificities after removal of Dd molecules (Hansen et al., 197713). Immunizations of dm2 mice with BALB/cKh cells produced antibodies which were specific for D' molecules and dm2 anti-BALB/cKh sera defined two new specificities, H-2.64 and 65, on D' molecules (Hansen and Sachs, 1978). Since one of the alloantisera (D28b) used to define Ld molecules failed to react with dm2 cells (McKenzie et al., 1977), presumably due to the anti-H-2.64 antibodies in this sera, the D' and Ld molecules were assumed identical (Hansen and Sachs, 1978). The loci encoding Ld and Dd molecules have not yet been separated by recombination, suggesting that they are very tightly linked. Therefore, both molecules, although now known to be the products of separate genes H-2Ld and H-2Dd, are considered products of D region loci. Subsequent to the discovery of the Ld molecule, a minimum of four newly reported different molecules have been defined (see Table I ) and each of these is characterized by abnormal expression on cells from either the d m l or dm2 mutant. This latest multiplicity of Dd region molecules is at present incompletely defined and randomly designated. The Md molecule was not detected in d m l mice (Demant and Ivanyi, 1981) whereas the Rd (Hansen et al., 1981), Lq (Demant and Roos, 1982), and L2 (Ivanyi and Demant, 1982) molecules were not detected in dm2 mice. It has yet to be determined whether the Rd, Lq, and L2 molecules are distinct from each other and whether each is the product of a separate gene or alternatively a modification of an H-2Ld gene product. Similarly, the Md molecule may represent a structurally altered H-2Dd gene product (see Section 111). Application of monoclonal antibody technology has greatly enhanced the study of the heterogeneity of H-2 antigens. Several hybridomas secreting antibodies to D d region antigens have been characterized and successfully used to dissect previously poorly defined antigenic determinants. As an example of such an approach, Tables I1

44

TED H. HANSEN ET AL.

SEROLOGICAL TESTING OF

TABLE I1 Dd 'PNTIGENS USING MONOCLONALANTIBODIES

Cytotoxicity titer-' on cells of haplotypes:

-

Monoclonal antibody

34-1-2' 34-2-12' 34-4-20' 34-4-21" 34-54" 34-7-29 15-1-5b 27-11-13' 28-11-5'

d

dml

dm2

Other regions or haplotypes encoding crossreactive antigens

Correspondence with previously defined specificity None

10,000 10,000 10,000 0 5,000 5,000 10,000 10,000 10,000 256 256 512 10,000 10,000 10,000 256 256 356 5,000 5,000 5,000 5,000 5,000 5,000 512 256 256

H-2.4 None

H-2.4 H-2.4 None None None None

'Ozato et a1. (1982). Ozato et al. (1980). Ozato and Sachs (1981).

and I11 show the reactivity on d m l and dm2 cells of a panel of monoclonal antibodies to Dn and L'I antigens, respectively. Two monoclonal antibodies (4-9.1 and 4-9.4) detected Dd region determinants missing on both d m l and dm2 cells (R. Harmon and J. Frelinger, personal communication). Because dm2 mice have a normal Dd molecule (Nairn and Nathenson, 1978) and lack cell surface Ld molecules (Hansen et d.,1977b), this observation suggests that antibodies 4-9.1 and 4-9.4 recognize an Ld antigenic specificity not expressed on Ldmlmolecules. Not only does this provide serologic evidence that Ldmlis strucSEROLOGICAL TESTING OF

TABLE 111 Ld ANTIGENSUSING MONOCLONALANTIBODIES

Cytotoxicity titer-' on cells of haplotype: Monoclonal antibody

30-5-7' 23-10-1" 28-14-8 4-%lb 4-9-40

' Ozato et al.

d

dml

5,000 5,000 20,000 20,000 5,000 5,000 10,000 10,000 -

dm2

-

-

Other regions encoding cross-reactive antigens

Correspondence with previously defined specificity

D' DP D'J,Db DQ

H-2.65 H-2.65 H-2.64 H-2.65 H-2.65

D'

(1980). R. Harmon and J. Frelinger (personal communication).

HETEROGENEITY OF

H-2D GENES

45

turally altered, but also suggests that the 4-9.1 and 4-9.4 antibodies may prove useful in defining the relationship between Ld and the newly defined Rd and L2 molecules (see Table I). Three antibodies (30-5-7, 23-10-1, and 28-14-8) were found to recognize determinants encoded by genes mapping to the Dd region and were reactive with d m l cells but not dm2 cells (Ozato et al., 1981).These antibodies most likely define determinants shared between Ldmland Ld molecules. Comparisons of the precipitates of antibodies 30-5-7 and 28-14-8 revealed a new molecule designated Rd (see Section 111). One anti-Dd antibody (34-2-12) defined a determinant missing on d m l cells but present on dm2 cells. Besides providing serologic evidence that the Ddmlmolecule is structurally altered, the 34-2-12 antibody may prove useful in characterizing the newly defined Md molecule (see Table I). It is also noteworthy that of all the anti-Dd antibodies tested on d m l cells, antibody 34-2-12 is the only one that binds the C2 domain of Dd molecules (see Section VII,B) and since this antibody failed to react with d m l cells this result identifies at least one of the genetic regions affected by the mutation. A few general impressions of the serologic findings summarized in Table I11 are noteworthy. First, the serologic specificities defined by monoclonal antibodies to Dd antigens do not always correspond with determinants previously defined by alloantisera. In contrast, the serologic specificity of the five monoclonal anti-Ld antibodies all correspond with the specificities previously defined with alloantisera, namely, H-2.64 and 65. We are presently investigating whether this latter concordance reflects a lower level of serologic complexity of L* than Dd antigens or that the V h gene repertoire of antibodies to Ld antigens is more restricted (Ozato et al., 1983). None of the monoclonal antibodies to Ld molecules shown in Table 111 recognizes a serologic specificity corresponding with H-2.28. This lack of concordance could be due to (1)the complexity of the H-2.28 specificity as indicated by studies of Snell et al. (1974) which showed that H-2.28 could be subdivided into a group of separate specificities comprising the “H-2.28” family or (2) the observation that several members of the “H-2.28” family are common structures shared between K, D, and L molecules of various haplotypes (Demant et al., 1978; Hansen et al., 1978). A second general impression of these serological findings using monoclonal antibodies is that the antibodies (34-2-12,34-4-21,34-5-8)with a serologic specificity corresponding with H-2.4, the private specificity, recognize Dd and not Ld molecules. Although this apparent lack of a private specificity on Ld molecules is consistent with the methods used that first detected them (Lemonnier et al., 1975), the reason for this

46

TED H. HANSEN ET AL.

observation is unclear. Among the possible explanations are (1) H-2L genes may be less polymorphic than H-2K or H-2D genes (see Section VI) or (2) that the structure of Ld molecules is similar to antigens encoded in the Dq region and therefore H-2.65 may represent the most private Ld specificity that can be serologically detected (Hansen et al., 1979). It should be noted, however, that T cells recognize an Ld determinant not shared with the antigens of other haplotypes (see Section V). A third general observation of the results shown in Table I1 and I11 is that serologic testing using monoclonal antibodies frequently leads to further subdivision of specificities previously defined with alloantisera. For example, the reactivity of these monoclonal antibodies on d m l cells divides specificity H-2.4 (antibodies 34-4-2 and 34-5-8 are reactive, whereas antibody 34-2-12 is nonreactive) and subdivide specificity H-2.65 (antibodies 30-5-7 and 23-10-1 are reactive whereas antibodies 4-9-1 and 4-9-4 are nonreactive). Further characterization of the serologic determinants on H-2 molecules has been attained by comparing the binding of different monoclonal antibodies to the same H-2 molecule (Lemke et at., 1979). Assignment of epitopes is based on the presumption that antibodies binding the same epitope block each other’s binding whereas antibodies binding separate epitopes do not (see Fig. 1).As an example of this type of analysis Potter et al. (1981) showed that both anti-H-2.65 antibodies (30-5-7 and 23-10-1)bound the same epitope on Ld molecules while the anti-H-2.64 antibody (28-14-8)bound a distinct Ld epitope. Using DNA-mediated gene transfer techniques (Evans et al., 1982b), the epitope definition on Ld molecules has been extended by the identification of the Ld molecular domain bound by each of these monoclonal antibodies (see Section VI1,B). Ill. Chemical Heterogeneity of Gene Products Encoded in the Dd Region

Several structural comparisons between Ld molecules and the previous defined Dd and Kd molecules have been published, including comparisons using sequential immunoprecipitation (Hansen et at., 1977; Neauport-Sautes et at., 1977), two-dimensional gel electrophoresis (Krakauer et al., l980), tryptic peptide mapping (Sears and Polizzi, 1980; Rose et al., 1980), and partial amino acid sequence analysis (Coligan et al., 1980). These studies demonstrated that Ld is a unique molecule showing amino acid sequence differences from K d and Dd at about 20% of the positions compared, and that the differences are distributed throughout the molecule. The unique primary structure of each of these molecules implies that Kd, Dd,and Ldare the

HETEROGENEITY OF

H-2D

GENES

47

FluorescenceIntensity FIG.1. Definition of two distinct sites, epitopes, on Ld molecules (from Potter et al., 1981). The blocking of binding of two different, fluorescein-conjugated (F) monoclonal antibodies (30-5-7 and 28-14-8) to L" antigens was tested by preincubation of cells with the same or alternative antibody. Measurements of reduced fluorescence intensity displayed on a FACS IV flow cytometer (Becton Dickinson) were used to quantitate the blocking of binding. (a) F30-5-7 staining of B10.D2 spleen cells after preincubation with 30-5-7 (----) compared with no pretreatment (-); (b) F30-5-7 after preincubation with 23-10-1 (-) or 28-14-8 (----); (c) F28-14-8 after preincubation with 28-14-8 (-) compared with no pretreatment (-); F28-14-8 after pretreatment with 23-10-1 (-) or 30-5-7 (----). These results demonstrated that the two monoclonal antibodies of serologic specificity anti-H-2.65 (30-5-7 and 23-10-1) bind the same Ld epitope, whereas the monoclonal antibody of serologic specificity anti-H-2.64 binds an alternative L" epitope.

products of separate loci, rather than products of a common H-2.28 gene (Demant et al., 1978). These structural comparisons also indicated that L" is no more like Dd than it is like any other H-2 molecule, suggesting that H-2Ld did not result from a recent duplication of the H-2Dd gene. Further structural comparisons of Ld with other H-2 molecules are now possible based on DNA sequence data (see Section VII). The structures of the newly discovered molecules Md, Rd, L2d, and Lqd (Table I) have been only partially characterized, thus their genetic basis and relationship to each other and to the Ld and Dd molecules is unclear. The Md molecule renamed H-2Dd (b28-) by Sears and Wilson (1981) was defined using complex alloantisera to precipitate biosynthetically radiolabeled antigens which were then compared by tryptic

48

TED H a HANSEN ET AL.

peptide mapping. These studies suggested that Md and Dd molecules are closely related structurally since most of their respective tryptic peptides co-eluted. The Rd molecule was initially defined by sequential precipitation experiments comparing the precipitates of H-2d antigens by two different monoclonal antibodies (30-5-7 and 28-14-8) previously thought to be Ld specific based on their reactivity with BALB/c and not dm2 antigens (Ozato et al., 1980). When a BALB/c antigen preparation was precleared with anti-H-2.65 (30-5-7), thus removing Ld molecules, and then tested with anti-H-2.64 (28-14-8), an addition glycoprotein, Rd, was detected (see Fig. 2). Two-dimensional gel and V8 protease peptide map comparisons of the 28-14-8 and 30-5-7 precipitates suggested that Rd and Ldare distinct molecules, sharing structural similarities (Koch et al., 1983). The Lq molecule, like Rd, was not detected in dm2 mice and was distinguished from Ld due to an apparent lower molecular weight of 41,000 (Demant and ROOS,1982). The lower molecular weight of Lq and the fact that it was precipitated with an alloantisera known to contain anti-Qa antibodies suggested that Lq was more Qa-like than H-2-like, thus inspiring its unique designation. Because different techniques and/or reagents were used to define Rd, Lq, and L2 (see Table I) it is difficult to assess their interrelationships and to establish whether or not they represent the products of separate genes. The genetic relationship of the Rd, Lq, and L2 molecules with the H-2Ld gene product has important implications for our understanding of the genetic lesion of the dm2 mutant mouse, which resulted in the failure to express each of these molecules. The observation that dm2 mice mount a strong histocompatibility and serologic response to BALB/cKh antigens, but that no response is seen by BALB/cKh mice to dm2 antigens, has been interpreted as evidence that dm2 mice carry a genetic deletion. Since the structures of the Dd molecules isolated from dm2 and wild-type were indistinguishable (Nairn and Nathenson, 1978) and Ld antigens were undetectable in dm2 mice, it was proposed that dm2 mice represent an H-2Ld gene deletion (Hansen et al., 1977). However, subsequent analysis of dm2 antigens with a xenogenic anti-H-2 serum revealed a cytoplasmic, Ld-like protein of 40,000 daltons designated p4Odmz,which was not detected in the cytoplasm of BALB/c cells (Robinson, 1982). The p40 molecule was found not to be expressed on the cell surface and was not detected with alloantibodies to Ld antigens. Therefore, it was proposed that p40 represents the product of a defective H-2Ld gene in dm2 mice, which is prevented from proper maturation necessary for membrane integration (Robinson, 1982). Histocompatibility and serologic analyses of the d m l mouse strain

HETEROGENEITY OF

H-2D GENES

49

Tested With:

.. .f 9

P c 0

2 +

2

Q

FIG.2. Resolution of three antigenically distinct H-2 molecules determined by genes mapping to the D" region (adapted from Hansen et al., 1981). Radiolabeled B10.AKM [K*(D"LQRq)]antigen was precleared with either normal serum, anti-H-2.30, or monoclonal antibody 30-5-7 (anti-H-2.65) as indicated along the left of the figure. Supernatants were then precipitated with the second antibody shown at the top of the figure. The anti-H-2.30 used for this experiment was LP.RIII x BIO.A anti-B1O.AKM and the anti-H-2.64 was monoclonal antibody 28-14-8. The secondary precipitates were analyzed by SDS-PAGE (Cullen and Schwartz, 1976). By using this gel system, H-2 molecules that have an apparent molecular weight of 45,000, migrate approximately onethird into the gel and a running front which includes &-microglobulin appears as a smaller peak toward the end of the gel. These results indicated that the Dq region determines at least three antigenically distinct H-2 molecules; the Dq molecule (I) detected with anti-H-2.30; the Lq molecule (V) detected with anti-H-2.65 after removal of DQmolecules with anti-H-2.30; and the Rq molecule (IX)detected with anti-H-2.64 after removal of Dq and LQmolecules with anti-H-2.65.

have demonstrated that this mutation represents a gain and loss of antigenic determinants encoded by genes mapping to the H-2Dd region (Egorov, 1967). Structural analyses of the lesion of d m l mice using peptide map comparisons with wild-type B10.D2 antigens, showed differences between Ld and Ldml molecules as well as between Dd and Ddmlmolecules (Brown et at., 1978; Wilson et uZ., 1982). Besides these differences, d m l mice were also found to have a unique

50

TED H. HANSEN ET AL.

glycoprotein, gp39dm1,possibly not expressed on the cell surface but similar antigenically to Ld molecules (Wilson et al., 1982). Therefore, these structural analyses of the d m l antigens implicate an alteration of both the H-2Ddm1and H-2Ldm1 genes. Although the genetic basis of the gp39dm' is currently unknown, it is intriguing that both the d m l and dm2 mutants synthesize Ld-like molecules of lower than expected molecular weight. IV. Quantitative Comparisons of Gene Products Encoded in the

Dd Region

Quantitative studies comparing the synthesis, cell surface expression, and rate of shedding of H-2 molecules have suggested differences between K and D region gene products. Emerson et al., (1980) used alloantisera to immunoprecipitate cell surface radioiodinated or biosynthetically labeled antigens and found that in H-2* haplotype mice Kk antigens are more rapidly turned over and shed compared to Dk antigens, whereas in H-2d haplotype mice Kd antigens were turned over relatively slowly compared to Dd antigens. Analysis of intra-Hi-2 recombinants suggested that different H-2K alleles may influence the rate of metabolic turnover of H-2D region encoded antigens. These findings of Emerson and colleagues correlated precisely with previously published quantitative variations seen in the cytotoxic T-cell responses to chemically modified (Schmitt-Verhulst and Shearer, 1975) or virus-infected syngeneic cells (Doherty et al., 1978). Using a lZsIlabeled protein A radioimmunoassay and alloantisera, O'Neill and McKenzie (1980) reported allele-specific variations in the amount of H-2K or D antigens expressed. These latter findings, however, showed no correlation with those of Emerson and colleagues, possibly because of differences in assays and reagents used. Furthermore, the reliance of both of the above studies on alloantisera raises questions regarding the number of molecules being detected (e.g., whether D and/or L antigens were being compared). To selectively compare the amount of Ld versus Dd antigen on the cell surface, monoclonal antibodies have been used as probes. Immunoglobulin class-matched (yG-2a) monoclonal antibodies were either directly labeled or detected with a facilitating reagent and binding was assessed by measuring either fluorescence intensity on the FACS (Potteretal., 1981)orby radioiodination (Doweretal., inpreparation). When compared under optimal binding conditions, antibodies to Ld antigens were found to show 2 to 3 times lower levels of binding compared to antibodies to Dd antigens (see Fig. 3). These studies therefore indicate that there is significantly less Ld than Dd antigen on

HETEROGENEITY OF

ANTI-Od (34-2-121

L . . . I . . . . I . . . . I . . . . , . . ~ ...,. . ,

H-2D

A N T I - L ~( 2 8 - 1 4 - 8 )

1

. . . . I . . .

, . . . . I . . .

51

GENES

I

I....I.,..I.

..,

I...II,...,..,,,..

I....I1...I....1....I

150

RELATIVE INTENSITY

....I..,. , . . . . , . . . . I . . , ,

NEGATIVE CONTROL

NEGATIVE CONTROL

100

. . , . . . . , L . . .

ANTI-Ld (30-5-7)

ANTI-Od ( 3 4 - 5 - 8 )

50

4

200

50

100

150

6

200

RELATIVE INTENSITY

FIG.3. Comparison of the cell surface expression of Ld and JY antigens (Potteret al., in preparation). Spleen cells (BlO.D2) were treated with a saturating amount of the indicated monoclonal antibody (all of which are mouse antibodies of y-2a isotype) followed by a fluorescein-conjugated rabbit anti-mouse Ig (y-specific). The relative fluorescence intensity was quantitated on a FACS flow cytometer (Becton Dickinson). These results indicated that both monoclonal antibodies (34-2-12 and 34-5-8) which recognize determinants on Dd molecules bind cells at two to three times the level as monoclonal antibodies (28-14-8 and 30-5-7)which recognize determinants on Ld molecules. It should also be noted that antibody 28-14-8 reacts with Rd molecules in addition to Ld molecules (see Section 111). Therefore, as previously shown using directly labeled monoclonal antibodies (Potter et al., 1982), the Ld molecules are expressed on the cell surface at two- to threefold lower levels than Dd molecules.

the cell surface. Since multiple antibodies were used to show this difference it appears unlikely that this variation in expression of Ld and Dd antigens is due to differences in the number of binding sites or affinity differences between the antibodies being compared. Consistent with these findings, Sears and Wilson (1981) used alloantisera to

52

TED €3. HANSEN ET AL.

precipitate biosynthetically labeled antigens and detected considerably less Ld then Dd antigen. When cells from the dml and dm2 mutant mouse strains were compared with cells from their respective wild-type strain, the binding of anti-Dd monoclonal antibody 34-5-8 was found to be equivalent (Potter et d., in preparation). In these same studies, when anti-Ld monoclonal antibodies were tested, cells from dm2 mice, as expected, did not show binding; however, cells from d m l bound twice the expected amount. Therefore, in dm2 mice the failure to express Ld does not affect the level of expression of Dd antigens and in d m l mice the amount of Dd antigen expressed appears normal in contrast to Ld antigen which appears unexpectedly high. Although the genetic andlor molecular mechanisms accountable for these quantitative variations in the metabolism or expression of H-2 molecules are unclear, there are known functional implications and perhaps also implications for the genetic evolution of MHC genes. For example, an additional parallel can now be drawn between the gene products of the human third-locus HLA-C and the products of the murine H-2L locus, i.e., lower cell surface expression than the products of the other two class I loci. If not fortuitous, such similarities would favor evolutionary models postulating divergence prior to speciation. V. Functional Studies of H-2Ld Gene Products

The selective study of the function of the Ld molecule was greatly facilitated by the availability of the Ld-loss mutant mouse strain, dm2, and the anti-Ld specific reagents made using dm2 mice. These findings have been summarized in a recent review article (Levy and Hansen, 1980) and are therefore only briefly described here, with emphasis also on newer reports which were not previously reviewed (see Table IV). When cells from dm2 mice were stimulated in vitro with BALB/cKh cells, cytotoxic T cells were generated and shown to recognize Ld antigens (Hansen and Levy, 1978). In cold-target inhibition and antisera blocking experiments, anti-Ld cytotoxic effector cells were found to recognize Ld antigens independently of Kd and Dd antigens. When cytotoxic responses were generated in strain combinations recognizing both Ld and Dd antigens, two equally active T-cell subsets could be detected, one cytotoxic for L" antigens and the other for Dd antigens. The fine specificities of anti-Ld cytotoxic cells were analyzed by measuring their activity on cross-reactive allogenic target cells (Vazquez et al., 1980; Meliefet al., 1981). Using different approaches, both

HETEROGENEITY OF

53

H-2D GENES

TABLE IV ROLEOF Dd REGIONENCODEDANTIGENS IN MODIFIED-SELF,H2-RESTRICTED, CYTOTOXIC RESPONSES Involvement of H-2 region-encoded restriction antigens: H-2-reseicted cytotoxic response tested Chemically-modified self TNP FTCb I-AED‘ Virus-modified self Influenza Ectromelia Vesicular s tomatitis Lymphocytic choriomeningitis Minor histcompatibility antigens

P

L“

Yes Yes Yes

No No Yes

Levy et al. (1978) Arora et al. (1982) Levy and Shearer (1983)

Yes Yes No NT

Yes No Yes Yes

Biddison et al. (1978) Blanden et al. (1977) Ciavarra and Forman (1982) Brayton e t al. (1982)

Yes

No

References

Blanden and Kees (1978) ~

Trinitrophenyl.

* Fluorescein isothiocyanate. N-Iodoacetyl-N’-(5-sulfonic-I-naphthyl)ethylene-diamine.

of these studies showed that anti-Ld effectors cross-reacted with antigens encoded in the H-2Dq,H-2Db, and H-2Kk regions. This pattern of cross-reactivity correlated with the previously described crossreactions detected using dm2 anti-BALB/cKh sera (Hansen et al., 1979) and not with the expression of the determinant H-2.28. However, in addition to these shared determinants, cytotoxic T-cells were found to recognize a serologically undefined determinant unique for Ld molecules, a “private specificity” (Hansen and Levy, 1980; Vazquez et al., 1980; Melief et al., 1981). These data therefore support the hypothesis that Ld antigens are comparable in antigenic properties to products of H-2K and H-2D loci. The role of Ld antigens in cytotoxic responses to chemically modified and virus-infected cells has also bee6 assessed and these findings are summarized in Table 1V. In each of the responses tested, H-2 restriction has been demonstrated and products encoded within the Dd region have been shown to be involved. To determine whether Dd andlor Ld molecules were the restriction elements in these responses, antisera blocking and cold target inhibition experiments employing

54

TED H. HANSEN ET AL.

dm2 cells were performed. In chemically modified self-responses Ld molecules appeared not to be involved when cells were TNP-modified (trinitrophenyl) (Levy et al., 1978) or FITC-modified (fluorescein isothiocyanate) (Arora et al., 1982), whereas both Ld and Dd antigens were found to be involved when cells were treated with I-AED [Niodoacetyl-N’-(5-sulfonic-lnaphthy1)ethylene-diaminel(Levy et al., 1982). In studies of virus-modified self-responses, influenza specific cytotoxicity was shown to be restricted by both Dd and Ld antigens (Biddison et al., 1978), ectromelia-specific cytotoxicity appeared to be restricted Dd and not Ld antigens (Blanden et at., 1977), and vesicular stomatitis virus (VSV)-specificcytotoxicity was shown to be restricted by Ld and not Dd antigens (Ciavarra and Forman, 1982). Using DNAmediated gene transfer techniques (see Section VII,B), Ld molecules were shown to serve as restricting elements in lymphocytic choriomeningitis virus (LCMV) infection. Minor histocompatibility antigens were also found to show no apparent Ld restriction (Blanden and Kees, 1978). Since in several H-2 restricted cytotoxic responses differences have been noted regarding which H-2K andfor H-2D gene products are involved, these studies of Ld antigens are consistent with the conclusion that they are independent class I gene products, comparable to K and D molecules. There is little information available on the role in allogeneic and modified-self cytotoxic responses of the more recently defined D region gene products, M, R, Lq, and L2 (see Table I). Experiments using blocking of target antigens with monoclonal antibodies suggest that the Rq molecule is recognized in allogeneic responses (Hansen et al., 1981) and experiments by Ciavarra and Forman (1982) suggest Rd, Md, and L2 are not involved in VSV-restricted cytotoxicity. VI. Searches for Allelic Products of H-2Ld in Other Haplotypes

Two general approaches have been taken to assess the molecular heterogeneity of D region encoded products of haplotypes other than H-P. Demant and colleagues have focused their investigations on the apparent allelism of the public alloantigenic determinants H-2.28 and H-2.1 (Dbmant, 1981).However, H-2.28 and H-2.1 are complex families of specificities (Snell et al., 1974), for example, members of the “28” family include H-2.6, 27, 28, 29, etc. The apparent allelism of “28” and “1”is therefore perhaps one of definition rather than indicating the existence of a comparable gene product. When cells of different H-2 haplotypes were capped to remove antigens detected by an appropriate antiserum to the private D region specificity and then tested

HETEROGENEITY OF

H-2D GENES

55

with either anti-H-2.1 or anti-H-2.28, residual antigens were always detected (Ivanyi et al., 1979). These findings were interpreted as evidence that each D region encodes two molecules, one bearing the private specificity (the D molecule) and the other bearing either the public specificity H-2.1 or H-2.28 (the L molecule). This interpretation would be greatly strengthened if monoclonal antibodies to H-2.28 or H-2.1 could be selected which recognized determinants shared by H-2L allelic products of several haplotypes. However, given the complex nature of these two public determinants and the extensive crossreactions detected thus far between Ld antigens and the K and D molecules of several haplotypes (Dbmant et al., 1978; Hansen e t al., 1979), discovery of monoclonal antibodies specific only for products of H-2L genes in various haplotypes is probably unlikely. Alternatively, we have taken another approach to assess the heterogeneity of D region encoded molecules. Since alloantisera (dm2 antiBALB/cKh) and monoclonal antibodies (30-5-7 and 28-14-8) detect Ld and not Dd or Kd antigens and since these reagents cross-react with antigens of other haplotypes, we have used anti-Ld antibodies as probes for allelic products. This approach led to several unexpected findings which are summarized in Table IV. As mentioned in Section 111, when Dd-encoded antigens were compared by sequential precipitation using monoclonal antibodies 30-5-7 and 28- 14-8, two antigenically distinct molecules were resolved, Ld and Rd, in addition to the Dd molecules. When these same monoclonal antibodies were used to compare the antigens encoded in the Dq region, two molecules, Lq and Rq,were also defined in addition to the Dq molecule bearing the private determinant (see Fig. 2). In contrast, when D b encoded antigens were subjected to this same kind of analysis all the reagents tested, including two monoclonal antibodies (28-14-8, anti-H.2.64 and B22/249, anti-H2.m2) and two alloantisera (dm2 anti-BALB/c and anti-H-2-28), appeared to detect only one molecule (Hansen et al., 1980; Melino et al., 1981). As shown in Fig. 4, two-dimensional gel analysis of the precipitates of these same reagents on D h region encoded antigens have also failed to reveal molecular heterogeneity. Given the number of reagents detecting D b region encoded antigens that have been compared, we feel it is unlikely, although not formaIly excluded, that there is molecular heterogeneity among these antigens. Therefore, our failure to define H-2L allelic products in haplotypes other than H-2d and H-2q suggests haplotype differences in the number of D region encoded molecules expressed. These studies have also revealed unexpected cross-reactions; for example, as summarized in Table V, the Ld-reactive monoclonal antibody 28-14-8 was found to detect mole-

HETEROGENEITY OF

H-2D

GENES

57

TABLE V MOLECULARRESOLUTION B Y SEQUENTIAL PRECIPITATION OF ANTIGENS ENCODEDIN THE D", Dq,AND D" REGIONS USING MONOCLONALANTIBODIES 30-5-7 AND 28-14-8 ~~

Region

Dd 4.6 Dq a,b Dhc,d.e

30-5-7 (anti-H-2.65) positive molecules

28- 14-8 (anti-H-2.64) positive molecules

Ld DqLo -

Ld and Rd Dq LqR" D b

Antigenic detection of D molecule Alloantisera to H-2.4 Alloantisera to H-2.30 Alloantisera to H-2.2 or H-2.64; monoclonal antibody to H-2.m2

Ozato et a / . (1981).

* Hansen et al. (1981). Hansen et a/. (1980). Melino et al. (1982). Hammerling et a/. (1979).

cules bearing the private, D region alloantigenic determinant of the H-2* and H-2q haplotypes. Based on these findings it is difficult to define which of the molecules listed in Table V are allelic products and their letter designations of D, L, and R merely reflect the order in which they were defined. This extensive serologic cross-reactivities seen between Ld antigens and the K and D antigens of various haplotypes (Demant et al., 1978; Hansen et al., 1979) suggest there is no L-ness, i.e., a common structure shared by all putative products of genes allelic to the H-2Ld gene. This conclusion is consistent with previous findings using monoclonal antibodies to H-2K and H-2D gene products which reported the lack of serologically defined K-ness and D-ness (Ozato e t al., 1980). The difficulty in determining which H-2 molecules are the products of allelic genes is accentuated by comparisons of the primary structure of different products of D-region genes. For example, Maloy and ColFIG. 4. Two-dimensional gel comparison of the precipitates of three different reagents detecting antigens encoded in the D" region (Nichols et al., in preparation). Biosynthetically labeled (36S)splenic antigen was precipitated with (A) monoclonal antibody B22/249 (Hammerling et a/., 1979); (B) monoclonal antibody 28-14-8 (Ozato et a/., 1981); or (C) an alloantiserum specific for Dd encoded private and public antigens. Precipitates were analyzed by two-dimensional gel electrophoresis (Jones, 1977). All three of these precipitates of Dd encoded antigens give an indistinguishable constellation of spots suggesting they all precipate a chemically very similar if not the same H-2 molecule.

58

TED H. HANSEN ET AL.

igan (1982) have determined that Ld and Db molecules have a significantly higher amino acid sequence homology (94%) than D d and Dh molecules (84%). In addition, Db and Ld molecules appear to bind µglobulin very poorly (Maloy et al., 1980; Coligan et al., 1980) compared with Dd molecules, and both have three glycosyl units whereas Dd molecules have only two (Maloy and Coligan, 1982). Therefore, if the term allele is applicable to D region genes, then H-2Ld and H-2Db are more likely to be alleles than are Ddand Db.This conclusion has recently also been supported by comparisons of the DNA structure of the H-2Ld and H-2Db genes (G. Jay, personal communication). VII. Studies Using Genomic Clones of H-2D Region loci

D REGION GENESUSING GENOMIC CLONES Recently genomic DNA fragments containing genes encoding murine class I genes (H-2) have been cloned in a number of laboratories (Steinmetz et al., 1981, 1982; Evans et al., 1982a; Cosman et al., 1982; Mellor et al., 1982; Reyes et al., 1982a,b), allowing gene structure to be studied at the level of the DNA. The H-2D region locus, H-2Ld,was the first to be cloned and identified as a functionally active H-2 gene (Goodenow et al., 1982a; Evans et al., 1982a). The identification of the H-2Ld gene has been achieved both by DNA sequence analysis and by immunological examination of the translated products of the cloned genes, as will be described below. Complete DNA sequencing of the first H-2 genomic clone (Evans et al., 1982a; Moore et al., 1982) showed a sequence corresponding to the 76 amino acids known for the Ld protein sequence (Coligan et al., 1980). However, the DNA sequences reported from the two laboratories for their respective H-2Ld genes revealed minor differences. It is not clear at this time whether these differences are due to (1) the presence of two very similar genes, (2) the origin of the two clones being from slightly different H-2d sources, or (3) represent technical artifacts. Expression studies (see below) suggest that both are complete genes whose products are expressed on the cell surface. The overall structure of the H-2Ld gene is depicted in Fig. 5. The three main coding sequences, or exons, N, C1, and C2 (shown as solid bars) separated by intervening sequences of various length (shown as open bars), correspond precisely with the three distinct globular domains suggested previously by amino acid sequence data (Ploegh et al.,

A.

CHARACTERIZATION OF

HETEROGENEITY OF

H-2D GENES

59

275

G Domains

L

N

c,

Exon 0 lntron

Cz

M 1 3 12 13 0 0.5 1.0

KB

FIG.5. Comparison of the structure of the Ld glycoprotein and the H-215“ gene.

1981). Additional exons encode the leader sequence (L), and the transmembrane (M) and intracellular (I) domains of the Ld molecule. This same basic structure has been found in all genes encoding class I MHC antigens analyzed thus far. The identification of another D region gene, H-2Dd, has also been reported (Margulies et al., 1982; Goodenow et al., 198213). The H-2Dd gene has been sequenced through 854 base pairs, which includes exons L, N, and a part of C 1 (Margulies et al., 1983). The DNA sequence obtained agreed with the sequence of all 126 amino acids previously identified for the Dd molecule (Maloy and Coligan, 1982). Nucleotide sequence comparison between the H-2Ld and H-2Dd genes revealed a homology of 88,87, and 92% in the L, N, and part of the C 1 coding regions, respectively. The nucleotide differences in the compared regions correspond to 34 amino acid differences between Ld and Dd molecules. Reyers et al. (1982b) found a c-DNA clone which apparently encodes the Db antigens by comparing the sequence of DNA and amino acids. Independently a genomic clone contained in a cosmid vector which expressed Db antigens in L cells has been isolated (Mellor et al., unpublished; Townsend et al., 1982). Furthermore, the gene encoding the Dp molecule has been identified by DNA mediated gene transfer techniques among a genomic library prepared from H-2” haplotype (J. A. Frelinger and J. G. Woodward, unpublished). To assess the number and relative chromosomal locations of H-2 genes encoding class I MHC antigens, Southern blots of partially digested DNA from mouse strains with recombinant or congenic H-2 haplotypes were analyzed (Southern, 1975) and the presence of between 10 and 20 different genes encoding class I or class I-like anti-

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gens was demonstrated (Margulies et al., 1982; Pease e t al., 1982). Several of these genes were found to map on the chromosome to the right of the H-2Ld and H-2Dd genes into the Qa-Tla region. To further address questions of gene number and location, Steinmetz et al. (1982) constructed cosmid clones with long DNA inserts containing multiple H-2 genes. Thirteen clusters encompassing 837 kb of DNA were identified, containing 36 H-2 or H-2 like genes. The H-2Ldand H-2Dd genes were located in 2 independent cloned clusters. The cosmid clone containing the H-2Ld gene was found to carry an additional undefined H-2 (or H-2-like) gene in the same segment of DNA, whereas no additional H-2-life gene was found in the cosmid containing the H-2Db gene (Steinmetz et al., 1982). Thus the Dd region appears to have the DNA sequences for at least three H-2 or H-2-like genes. Since no overlapping clones between these clusters were found, the distance, orientation, and precise genomic location in the MHC of these 2 genes have not been elucidated. It is currently not known whether the number and organization of H-2 genes is different among H-2 haplotypes. Differences in the number of D-region gene products synthesized has been suggested using immunoprecipitation techniques (Hansen et al., 1981; Melino et al., 1982). In these studies heterogeneity of D region encoded molecules was only detected in the products of the Dd and Dq regions and not the Dbregion (see Section VI). Consistent with these findings, Steinmetz et al. (1982) used a 5’ flanking sequence of the H-2Ld gene in a Southern blot analysis and reported hybridization with genes of the H-2d and H-2q haplotypes and not the H-2b and H-2k haplotypes. Recently Mellors et d., and Frelinger et al. (submitted) have isolated DNA segments as cosmid or A clones from the H-2b and H-2P haplotypes containing H-2D region genes. It is therefore likely to be determined in the near future whether or not the H-2L genes exist in these H-2 haplotypes.

B. CHARACTERIZATION OF D REGION GENEPRODUCTS USING GENOMICCLONES DNA-mediated gene transfer has been used to introduce cloned mammalian genes into the mouse fibroblast L cell lines along with an appropriate selective gene such as Herpes simplex thymidine kinase gene (Camerini-Otero et al., 1980; Wigler et al., 1979). Selective growth conditions are then employed to permit growth only of transfected cells and these are then examined for gene expression. Successful transfer and expression of both H-2Dd and H-2Ld genes have been reported (Goodenow, 1982a,b; Evans, 1982a; Margulies, 1983; Townsend et al., 1982). L cells transformed with cloned H-2 genes

HETEROGENEITY OF

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GENES

TABLE VI DETERMINATION OF THE FINESPECIFICITY OF MONOCLONAL ANTI-H-gdANTIBODIES BY THE USE O F CELL LINESTRANSFORMED WITH CLONED H-2Dd OR H-2Ld GENES Reactivity with H-2 gene transformants Specificity by conventional serology

Monoclonal antibody

23-5-21 28-8-6 34-1-2 34-2-12 34-4-21 34-5-8 27-11-13 28-11-5 34-4-20 34-7-23 23-10-1 28-14-8 30-5-7

T.lll (Ld)

-

D region of H-2d haplotype (Ddand possible cross-reaction to L" molecules)"

Ld

Ld/RLL L"

-

-

+ + + + + + +

T483 (D")

Untransformed

+ + +

-

+ + + + + + + -

-

Refined specificity

Dd D" Dd

IT

-

Dd

-

D" and Ld D" and L" Dd and Ld D" and L"

-

Dd

-

Ld

-

Ld

Ld

a These monoclonal antibodies (Ozato et al., 1980, 1982; Ozato and Sachs, 1981) had been shown to react with H-2D region products of the H-2dhaplotype by testing a panel of mouse strains of various haplotypes and their recombinants.

express new H-2 antigens on their surface as a result of the activity of the inserted DNA in addition to their own H-2k antigens. Reactivities of the transformed cells with monoclonal antibodies of defined specificities allowed conclusive identification of the transfered genes via their products. Table VI shows a series of monoclonal antibodies reacting with H-2 antigens of the Dd region, including those specific for the Ld antigens. Products of the putative cloned H-2Ld gene reacted with all 3 H-2Ld antibodies, but no reaction was found with anti-K'l or anti-Dd antibodies. Likewise, products of putative H-2Dd gene were reactive with all of the anti-Dd antibodies shown in Table VI, while being completely negative with anti-L' antibodies. These results provided key evidence for the positive identification of the cloned H-2Ld and H-2Dd genes. Goodenow et al. (1982b) identified single H-2Kd,H-2Dd, and H-2Ld genes in three clusters of cosmid clones in which there are a total of 36 H-2-like genes by analyzing transformed cells lines prepared by DNA mediated gene transfer. Of the H-2-like genes and corresponding clusters, several might be mapped to the Qa-TZa region and others remain unmapped, perhaps encoding novel class I MHC antigens (Goodenow

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TED H. HANSEN ET AL.

et al., 1982b; Frelinger et al., unpublished). In light of these findings of only two D region genes, H-2Dd and H-2Ld,questions can be raised concerning the genetic basis of the newly defined D region-associated antigens Md, Rd, and L2 (see Table I). Three possible answers have been proposed (Goodenow et al., 1982b): first, these additional D region encoded antigens may be products of either the H-2Dd or H-2Ld genes, but differ from Dd and Ld molecules due to posttranslational modifications or due to alternative methods of RNA splicing; second, other D region loci may exist but go undetected because their DNA restriction maps are indistinguishable from the H-2Dd or H-2Ld maps; finally, the genes determining the Md, Rd, and L2d molecules may not have been successfully cloned or their gene products may not have been expressed in the transformants in sufficient quantities to be detected by available reagents. Definition of these new molecules is currently based on the sequential treatment of antigens with complex antisera (Md and L2) or monoclonal antibodies (Rd). New monoclonal antibodies which recognize unique determinants on Md, Rd, and L2 molecules would greatly facilitate studies of their genetic characterization. Since the Md, Rd, and L2 antigens were distinguished by their differential expression in either the d m l or dm2 mutant mouse strains (see Section 11), analysis of the D region genes of d m l and dm2 mice may also help elucidate the genetic basis of this antigenic heterogeneity. These transformed cell lines constitute a new MHC environment in which Ld antigens are expressed in the absence of Dd antigens. Thus, these cells allow the further definition of the specificities of some monoclonal antibodies. Because of the lack of recombinant mouse strains separating H-2Dd and H-2Ld loci, classic serological studies did not permit definite assignment of the molecular specificity of antibodies reacting with antigens encoded by the H-2D region except by coprecipitation analysis (Ozato et al., 1980).Previously, the possibility had remained that these antibodies possessed reactivity to H-2Ld in addition to H-2Dd gene products. As shown in Table VI, of the 10 monoclonals, 6 antibodies reacted only with H-2Dd but not with H-2Ld transformants, indicating that these antibodies reacted with determinants unique to Dd molecules. The remaining 4 monoclonals reacted with both H-2Ld and H-2Dd transformants, indicating that they detected cross-reactive determinants shared by Ld and Dd molecules. In addition to serological reactivity, these transformants were susceptible to lysis by alloreactive cytotoxic T cells specific for appropriate Ld or Dd antigens, providing further evidence for functional identity of the gene products (Woodward et al., 1982). Moreover, these transformed cells offered a unique opportunity for defining H-2D re-

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gion encoded molecules were responsible for the H-2 restricted cytotoxic T lymphocytes killing in modified self-responses. Thus, H-2Ld transformed cells devoid of Dd antigen were shown to be effective targets for a number of virus-specific H-2D region restricted cycotoxic T cell responses, including VSV (Brayton et al., 1982), LCM (Forman et al., 1983), and Influenza (Reiss and Burakoff, 1983). These reports extend previous findings (see Section V) by demonstrating that Ld molecules independent of Dd molecules are capable of functioning as H-2 restriction elements. With the aid of H-2Dd and H-2Ld transformed cells, Levy and Shearer (1983) demonstrated that both Dd and Ld antigens were functional restricting molecules in a system of SH reactive AED hapten self cytotoxic responses (Levy et al., 1981). In contrast, in TNP specific cytotoxic responses Dd and not Ld molecules were found to be restriction elements, confirming previous findings (Levy et al., 1978). The availability of D region genes, H-2Dd and H-2Ld,and of in vitro DNA recombination techniques has recently made it possible to construct recombinant H-2 genes in which domains of the two original genes were exchanged (Evans et al., 1982b). Using transformed cells expressing the recombinant H-2 genes the reactivity of various monoclonal antibodies could be mapped to distinct domains of the H-2 molecules (see Fig. 6). In this way some antibodies (28-14-8 for Ld molecules and 34-2-12 for W molecules) were found to react with determinants on the C2 domains which had been regarded as much less polymorphic. In accord with expectations most antibodies reacted S

NH2

H-2Ld

H2Dd

I

S I

N

I 30.5.7 23.10.1 34.5.8 28.8.6 34.1.2 34.4.21 23.5.21

c1

S

S

I

I

2

//A

1 COOH

28.14.8 34.2.12

FIG.6. Localization of the polymorphic determinants on Ddand Ld molecules (taken from Evans et al., 1982b). This diagram summarizes the reactivity of Ld and Dd specific monoclonal antibodies with hybrid H-2 genes constructed by joining together fragments (exons) of the H-2Ld and H-2Dd gene. The reacting monoclonal antibody is listed in relation to the globular subunit of the protein to which it binds. These results indicated that although most antibodies were found to recognize the N-terminal half of the H-2 molecule which was known to be more polymorphic (Coligan et al., 1981), two monoclonal antibodies 28-14-8 and 34-2-12 bound the C-terminal half of the Ld and Dd molecules, respectively.

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TED H. HANSEN ET AL.

with the N and/or,Cl domains. This work exemplifies a new avenue for determining the localization of polymorphic determinants recognized by antibodies and b y T cells (Ozato et al., 1983). Site-specific mutagenesis (Wallace et al., 1981) will allow even more precise localization of polymorphism and the generation of new structural variants in H-2 molecules, leading to a more detailed understanding of the structural correlates of antibody and T-cell recognition of histocompatibility antigens. In conclusion, recombinant DNA technology has begun to provide new information on the structure and evolution of H-2 genes. Further, cells possessing unique MHC environments provide new cellular tools which should be useful for the clarification of structure-function relationships and of the functional significance of class I molecules. VIII. Evolutionary Models a n d Future Approaches

Our evolving understanding of H-2D region genes as a family of genes in a dynamic equilibrium may be helpful in resolving one of the major paradoxes concerning the evolution of class I loci, namely, the temporal relationship between duplication of loci and divergence of species. The existence of more than one expressed class I locus in every mammalian species studied to date has long been interpreted to indicate that the duplication of class I loci occurred prior to speciation. However, other predictions which one might make from such an hypothesis are inconsistent with serologic and structural data. For example, if the H-2K and H-2D loci are derived from separate primordial genes which duplicated before human and murine species diverged, then one might expect that either H-2K or H-2D would be more similar to one of the subloci of the human MHC than it would to the other. This would be similar to what has been observed in the case of light chain loci, for which there is much greater homology between the K products of man and mouse than between K and A light chains in either species (Kabat et al., 1979). However, in the case of class I MHC products, the opposite has been found. Thus, there appears to be much greater homology between the various class I gene products of the mouse than there is between any of these products and their counterparts in other species. At the serologic level, this hierarchy of homology is reflected in numerous public H-2 specificities shared by the products of different alleles of H-2K7 H-2D, and H-2L7 while very few serologic specificities are shared between HLA antigens and H-2 antigens (Lunney et al., 1979). At the protein sequence level, there is an average homology of 60-70% for class I products compared between

HETEROGENEITY OF

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65

species and 80-90% for class I products compared within a species (Nathenson et al., 1981). I n fact, the homology between different alleles at a single H-2 locus or between products of different H - 2 loci is approximately the same, leading to the surprising inability to distinguish “K-ness” from “D-ness” when H-2 sequences have been compared (Vitteta and Capra, 1978). These data therefore pose an evolutionary paradox, suggesting either that there has been some functional reason for which the products of duplicated loci within each species must remain similar to each other, or that the duplications leading to multiple expressed loci in each species occurred subsequent to speciation. Until recently the latter hypothesis seemed untenable since it was thought to imply some cataclysmic event occurring recently in evolution leading to a sudden duplication of the class I genes in each species. However, this interpretation made the tacit assumption that all class I loci were expressed and that the number of class I loci was the same in all members of a species and in all species. It is obvious from the data and discussion presented here for the H-2D region that this assumption is far from correct. Even within the murine species the number of expressed class I genes in the D region appears to differ among strains. In addition, there appear to be numerous genes derived from a common primordial class I gene which are either not expressed or expressed at undetectable levels. Given this apparently very large gene pool and the fact that the number of genes expressed differs even within a single species, it does not seem unlikely that different members of this gene pool could be chosen for expression in different species. Indeed, the class I genes are probably best thought of as multigene families, in which there is continuous expansion and contraction of certain members of the family. Therefore, at any point in evolution a given gene might be caught in a state of expansion or contraction. If expansion of a gene is somehow related to its expression, then different genes could be expressed in different strains and certainly in different species. The multiplicity of expressed D region genes in the H-2d and H-2“ haplotypes could indicate recent expansion of the D region gene pool in these haplotypes. This might also account for the great degree of homology seen between different gene products of D region genes. Conversely, we have seen that the H-2Db allele appears to be more similar to H-2L alleles than to other H-2D alleles (Coligan et al., 1980). This was true at the levels of sharing of serologic specificities (Hansen et al., 1980), degree of association with µglobulin (Maloy et al., 1980; Coligan et al., 1980), protein sequence (Maloy and Coligan, 1982), and, most recently, DNA sequence homologies (G. Jay, personal communication).

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Therefore, H-2b could represent a situation in which the D region has been caught in the process of contracting, retaining expression of the H-2Lb gene, but having lost expression of the H-2Db gene. Another genetic mechanism at the DNA level which could help to account for many of the features of class I genes is the phenomenon of gene conversion (cf. Baltimore, 1981). By this mechanism, stretches of DNA from one gene can be transposed to another homologous gene en bloc, thereby “converting” a portion of the gene to the sequence of another homologous gene in the same genome. When the amino acid sequences of the “mutated” portions of a large number of H-2Kb mutants have been determined, they have been found in almost every case to b e consistent with gene conversion of sequences from H-2L or equivalent genes (Schulze et al., 1983). Thus, these “mutants” could represent gene conversion from the H-2L gene rather than point mutations. Frequent occurrence of such gene conversions between class I genes could explain the high level of homology maintained between the class I genes within a species and the lower degree of homology between all class I loci of one species and class I loci of another species. Such a dynamic evolutionary model in which expansion and contraction of class I genes is occurring and in which gene conversion between duplicated genes is common appears to us to be the most likely explanation for the high level of molecular relatedness observed between different H-2D region genes and their gene products. A more static evolutionary model has been proposed by Demant and colleagues (1978), which hinges on the apparent allelism of two groups or “families” of public H-2 specificities H-2.1 and H-2.28, defined using alloantisera. This model postulates that all haplotypes determine the same number of D region encoded molecules, distinguishable antigenically by their reactivities with different sets of alloantisera (H-2.28 or H-2.1). This model predicts that allelic forms of H-2Ld should be found in all haplotypes, with L molecules sharing structural features based on whether or not they react with H-2.28 or H-2.1 sera. Such a model implies that the separation of D region loci occurred prior to the divergence of the H-2 haplotypes under study. By the dynamic model we are proposing, the number of D region genes and expressed products might be expected to differ in different haplotypes, and the structural relatedness of these products could also vary with haplotype. For example, the Dd region, in which Dd and Ld molecules are not more closely related structurally than are Kd and Ddmolecules, could represent a recombination leading to the introduction of the second D region gene rather than duplication. Recombination, duplication, and gene conversion are all proposed as continuing processes resulting in haplotype differences in the number of D region genes expressed.

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Testing the validity of the above models will depend upon parallel investigations using serologic, molecular, and genetic approaches. New monoclonal antibodies with established specificity for a single D region encoded molecule should be characterized for several haplotypes. Chemical comparisons of the primary structure of D region gene products should also be made in several representative haplotypes and comparisons made with studies of their respective genomic clones. These approaches should yield exciting information concerning the genetic evolution and the genetic mechanism for the establishment and maintenance of the high level of polymorphism of genes encoding class I MHC antigens.

ACKNOWLEDGMENTS The authors are grateful to Dr. Terry Potter and Ms. Elizabeth Nichols for their critiquing of this review, to Drs. R. Harmon and J. Frelinger for sharing their monoclonal antibodies, and to Mrs. Lorri Caffrey for expert secretarial assistance.

REFERENCES Arora, P. K., Levy, R. B., and Shearer, G. M. (1982).J.Immunol. 128, 551. Baltimore, D. (1981). Cell 24, 592. Blanden, R. V., and Kees, U. (1978)./. E x p . Med. 147, 1661. Blanden, R. V., McKenzie, I. F. C., Kees, U., Melvold, R. W., and Kohn, H. I. (1977).J. E x p . Med. 146,869. Biddison, W. E., Hansen, T. H., Levy, R. B., and Doherty, P. C. (1978)./. Exp. Med. 148, 1578. Brayton, P. R., Woodward, J. G., Harmon, R. C., and Frelinger, J. A. (1982). Nature (London) 297,415. Brown, J. L., Naim, R., and Nathenson, S. G. (1978)./. Immunol. 120, 726. Canierini-Otero, R. D., and ZasloK, M. A. (1980).Proc. Natl. Acad. Sci. U.S.A. 77,5079. Ciavarra, R., and Forman, J. (1982)./. E x p . Med. 156, 778. Coligan, J. E., Kindt, T. J., Nairn, R., Nathenson, S. G . , Sachs, D. H., and Hansen, T. H. (1980).Proc. Natl. Acad. Sci. U.S.A. 77, 1134. Coligan, J. E., Kindt, T. J., Uehara, H., Martinko, J., and Nathenson, S. G. (1981).Nature (London) 291,35. Cosman, D., Khoury, G., and Jay, G. (1982). Nature (London) 295,73. Cullen, S. E., and Schwartz, B. D. (1976)./. Immunol. 117, 136. Demant, P., and Ivanyi, D. (1981). Nature (London) 290, 146. Demant, P., and Roos, M. H. (1982). lmmunogenetics 15,461. Demant, P., Ivanyi, D., Neauport-Sautes, C., and Snock, M. (1978).Proc.Nat2.Acad. Sci. U.S.A.75, 4441. Demant, P., Ivanyi, D., Oudshoom-Snoek, M., Calafat, J., and Roos, M. H. (1981). Zmmunol. Reu. 60, 5. Doherty, P. C., Biddison, W. E., Bennink, J. R., and Knowles, B. B. (1978)./. E x p . Med. 148, 534. Egorov, I. K. (1967). Genetika 3, 136. Emerson, S. G., Murphy, D. B., and Cone, R. E. (1980).J.E x p . Med. 152,782.

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Evans, G. A., Margulies, D. H., Camerini-Otero, R. D., Ozato, K., and Seidman, J. G. (1982a). Proc. Natl. Acad. Sci. U.S.A. 79, 1994. Evans, G. A., Margulies, D. H., Shykind, B., Seidman, J. G., and Ozato, K. (198213). Nature (London)300,755. Forman, J., Goodenow, R. S., Hood, L., and Ciavarra, R. (1983).J.E x p . Med. 157,1261. Goodenow, R. S., McMillian, M., Om, A., Nicholson, M., Davidson, N., Frelinger, J., and Hood, L. (1982a). Science 215,677. Goodenow, R. S., McMillan, M., Nicholson, M., Tylor Sher, B., Eakle, K., Davidson, N., and Hood, L. (198213).Nature (London)300,231. Hammerling, G . J., Hammerling, U., and Lemke, H. (1979). Zmmunogenetics 8,433. Hansen, T. H., and Levy, R. B. (1978).J. lmmunol. 120, 1836. Hansen, T.H., and Sachs, D. H. (1978).J. lmmunol. 121, 1469. Hansen, T. H., Cullen, S. E., and Sachs, D. H. (1977a).J . E x p . Med. 145, 438. Hansen, T. H., Cullen, S. E., Melvold, R., Kohn, H., Flaherty, L., and Sachs, D. H. (1977b).J.Exp. Med. 145, 1550. Hansen, T. H., Ivanyi, P., Levy, R. B., and Sachs, D. H. (1979). Transplantation 28,339. Hansen, T. H., Ozato, K., and Sachs, D. H . (1980).Ann. Immunol. 131,327. Hansen, T. H., Ozato, K., Melino, M. R., Coligan, J . E., Kindt, T. J., Jandinski, J. J., and Sachs, D. H. (1981).J.Zmmunol. 126, 1713. Haupfeld, V., and Klein, J. (1975).J. E x p . Med. 142, 288. Huang, C.-M., Huang, H-J. S., and Klein, J. (1979). Zmmunogenetics 9, 173. Ivanyi, P., and Demant, P. (1979). Immunogenetics 8, 539. Ivanyi, D., and Demant, P. (1982). Zmmunogenetics 15,467. Ivanyi, D., Snoek, M., and Demant, P. (1979).Tissue Antigens 14,233. Jones, P. P. (1977).J.E x p . Med. 146, 1261. Kahat, E. A., Wu, T. T., and Bilofsky, H. (1979). In “Sequences of Immunoglobulin Chains.” PHS. Klein, J., and Shreffler, D. C. (1971). Transplant. Reu. 6, 3. Klein, J. (1975). In “Biology of the Mouse Histocompatibility Complex.” SpringerVerlag, Berlin and New York. Koch, S., Robinson, P. J., Koch, N., and Hammerling, G. J. (1983).Zmmunogenetics 17, 215. Kohler, G., and Milstein, C. (1975). Nature (London)256,495. Krakauer, T., Hansen, T. H., Camerini-Otero, R. D., and Sachs, D. H. (1980).]. Zmmunol. 124,2149. Lemke, H., Hammerling, G. J., Hohrnann, C., and Rajewsky (1978). Nature (London) 271, 249. Lemke, H., Hammerling, G. J., and Hammerling, U. (1979).lmmuno2. Reu. 47, 175. Lemonnier, F., Neauport-Sautes, C., Kourilsky, F. M., and Demant, P. (1975). Zmmunogenetics 2, 517. Lengerova, A., Pecknicous, J., and Pokorna, Z. (1974).J.Immunogenet. 1, 239. Levy, R. B., and Hansen, T. H. (1980).lmmunogenetics 10,7. Levy, R. B., and Shearer, G. M. (1983).J. Zmmunol., in press. Levy, R. B., Shearer, G. M., and Hansen, T. H. (1978).J . Immunol. 121, 2263. Levy, R. B., Shearer, G. M., Richardson, J. C., and Henkart, P. A. (198l).J.Zmmunol. 127, 523. Lunney, J. K., Mann, D. L., and Sachs, D. H. (1979). Scand. J. Zmmunol. 10,403. McKenzie, I. F. C., Morgan, G. M., Melvold, R. W., and Kohn, H. I. (1977). Immunogenetics 4,333.

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Maloy, W. L., and Coligan, J. E. (1982).Immunogenetics 16, 11. Maloy, W. L., Hammerling, G., Nathenson, S. G., and Coligan, J. E. (1980)J Zmmunol. M e t . 37, 287. Margulies, D. H., Evans, G. A., Flaherty, L., and Seidman, J. G. (1982).Nature (London) 295, 168. Margulies, D. H., Evans, G. A., Ozato, K., Camerini-Otero, R. D., Tanaka, K., Appella, E., and Seidman, J. G. (1983).J.Immunol. 130, 463. Melief, C. J. M., D e Wad, L. P., vander Meulen, M. Y., Ivanyi, P., and Melvold, R. W. (1981). Zmmunogenetics 12, 75. Melino, M. R., Nichols, E. A., Strausser, H. R., and Hansen, T. H. (1982).J . Immunol. 129,222. Mellor, A. L., Golden, L., Weiss, E., Ballman, H., Hurst, J., Simpson, E., James, R. F. L., Townsend, A. R. M., Taylor, P. M., Schmidt, W., Ferluga, J., Leben, L., Santamaria, M., Atfield, G., Festenstein, H., and Flavell, R. A. (1982).Nature (London)298,529. Melvold, R. W., and Kohn, H. I. (1976). Immunogenetics 3, 185. Moore, K. W., Taylor-Sher, B., Sun, Y. H., Eakle, K A., and Hood, L. (1982).Science 215, 679. Morgan, G. M., McKenzie, I. F. C., and Melvold, R. W. (1978).lmmunogenetics 7,247. Nairn, R., and Nathenson, S. G. (1978).J.Imrnunol. 121,869. Nathenson, S. G., Uehava, H., Ewenstein, B. M., Kindt, T. J . , and Coligan, J. E. (1981). Annu. Rev. Biochem. 50, 1025. Neauport-Sautes, C., Morello, D., Freed, J., Nathenson, S. G., and Demant, P. (1977). E u r . ] . Immunol. 7, 511. O’Neill, H. O., and McKenzie, I. F. C. (1980). lmmunogenetics 11, 225. Ozato, K., and Sachs, D. H. (1981).]. Immunol. 126, 317. Ozato, K., Hansen, T. H., and Sachs, D. H. (1980).J. Immunol. 125, 2473. Ozato, K., Mayer, N. M., and Sachs, D. H. (1982).Trunsplantation 34, 1113. Ozato, K., Epstein, S. L., Bluestone, J. A., Sharrow, S. O., Hansen, T., and Sachs, D. H. (1983a).Eur. J . Zmmunol. 1, 13. Ozato, K., Evans, G., Shykind, B., Margulies, D. H., and Seidman, J. G. (1983).Proc. Nntl. Accid. Sci. U.S.A. 80, 2040. Pease, L. R., Nathenson, S. G., and Leinwand, L. A. (1982). Nature (London) 298,382. Ploegh, H. L., Orr, H. T., and Strominger, J. L. (1981).Cell 24, 287. Potter, T., Hansen, T. H., Habbersett, R., Ozato, K., and Ahmed, A. (1981).J.Zmmunol. 127, 580. Reyes, A. A., Schold, M., Itakura, K., and Wallace, R. B. (1982). Proc. Natl. Acad. Sci. U.S.A. 79, 3270a. Reyes, A. A,, Schold, M., and Wallace, R. B. (1982). Zrnmunogenetics 16, 1. Robinson, P. J. (1982).Zmmunogenetics 15, 333. Rose, S. M., Hansen, T. H., and Cullen, S. E. (1980).I . Immunol. 125, 2044. Schmitt-Verhulst, A. M., and Shearer, G . M. (1975)./. E x p . Med. 142,914. SchuIze, D. H., Pease, L. R., Geier, S. S., Reyes, A. A,, Sarmiento, L. A., Wallace, R. B., and Nathenson, S. C . (1983).Proc. Notl. Acud. Sci. U.S.A. 80, 2007. Sears, D. W., and Polizzi, C. M. (1980). Immunogenetics 10, 67. Sears, D. W., and Wilson, P. H. (1981). Zmmunogenetics 13,275. Snell, G. D., Cherry, M . , and Demant, P. (1971).TranPplant. Proc. 3, 183. Snell, G. D., Demant, P., and Cherry, M. (1974).Folia. Biol. (Prahn)20, 146. Snell, G. D., Dausset, J., and Nathenson, S. G. (1976). “Histocompatibility.” Academic Pregs, New York.

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Southern, E. M. (1975).J.Mol. B i d . 98, 503. Steinmetz, M., Moore, K. W., Frelinger, J. G., Taylor Sher, B., Shen, E. A., Boyse, E., and Hood, L. (1981). Cell 25, 683. Steinmetz, M., Winoto, A., Minard, K., and Hood, L. (1989). Cell 28, 489. Townsend, A. R. M., Taylor, P. M., hlellor, A. L., and Askonas, B. A. (1982). Immunogenetics 17, 283. Vazquez, A., Senik, A., and Neauport-Sautes, C. (1980).J . Zmmunol. 125, 74. Vitteta, E. S., and Capra, J. D. (1978).Ado. Zmmunol. 26, 147. Wallace, R. B., Schold, M., Johnson, M. J., Dembek, P., and Itakura, K. (1981). Nucleic Acid Res. 9, 3647. Wigler, M., Sweet, R., Sim, G. K., Wold, B., Pellicer, A., Lacy, E., Maniatis, T., Silverstein, s., and Axel, R. (1979). Cell 16, 777. Wilson, P. H., Nairn, R., Nathenson, S. G . , and Sears, D. W. (1982).Zmmunogenetics 15, 225. Woodward, J. G., Om, A., Harmon, R. C., Goodenow, R. S., Hood, L., and Frelinger, J. A. (1982).Proc. Natl. Acad. Sci. U.S.A. 79, 3613.

ADVANCES IN IMMUNOLOGY, VOL. 34

Human Ir Genes: Structure a n d Function' THOMAS A . G O N W A , ' B. MATIJA PETERLIN, A N D J O H N

D. STOB03

The Howard Hughes Medical Institute and the Depadrnent of Medicine, Univetsify of California, Son Francisco, California

I. 11. 111. IV.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure of Ir Gene Products in Mice and Humans ...................... Ir Gene Function in Humans ........................................... Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

71 71 80 92 92

I. Introduction

One of the most significant advances in immunology has been the demonstration that immune reactivity is controlled b y immune response (Ir) genes in the major histocompatibility complex (MHC). This finding has not only increased a basic understanding of cellular interactions required for immune responses, but has also provided clues as to the role specific immune responses may play in the pathogenesis of certain clinicaI disorders. This article will focus on existing data which implicate the existence of Ir genes in humans. The text will begin with a brief review of the genetic and molecular structure of Ir genes and their products and end with a discussion of select studies indicating how Ir genes function in humans. II. Structure of Ir Gene Products in Mice a n d Humans

The murine Ir region occupies a 0.2 cM region on the short arm of chromosome 17 (Fig. 1). Products of this region, termed immune response associated or Ia molecules, consist of a bimolecular complex (a chain with a molecular weight of 35,000 and p chain with a molecular weight of 2S,OOO) noncovalently associated on the surface of B cells, macrophages, some T cells, dendritic cells, and Langerhan's cells (Cullen et al., 1976; Klein, 1979; Uhr et al., 1979; McMillan et al., 1981). Both chains are inserted through the plasma membrane, and possess three main regions: (1)a cytoplasmic carboxy terminus, (2) a hydrophobic intramembranous region, and (3) an extracellular amino Supported in part by USPHS Grant A1 14104. of AmericdGiannini Foundation. Investigator-Howard Hughes Medical Institute.

* Fellow-Bank

71 Copyright Q 1983 by Academic Press, Inc. All rights of reproduction in any form resewed. ISBN 0-12-022434-8

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Mouse chromosome 17: a. genetic map

-

0.03cM I 0.2194 I 0.2 CM I - I1-

I

d K 1

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Human chromosome 6: genetic map

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FIG. 1. Genetic map of the murine MHC compared to the molecular map of the I region and the genetic map of the human MHC. At present it is not possible to construct the molecular map of the human I region equivalent.

terminus. The extracellular region of both chains can be further subdivided into two domains. The first is near the amino terminus while the second, near the membrane, has homology with mouse immunoglobulin (McNicholas et ul., 1982).The structure ofa chains from different strains of mice appears relatively constant while the polymorphism among strains is carried on the /3 chains (Cullen et al., 1976; Klein, 1979; Cook et al., 1979; Silver and Ferrone, 1979; Silver, 1981). There are at least two loci in the I region coding for murine Ia antigens: I-A and I-E (Cullen et al., 1976; Uhr et al., 1979; Silver, 1981). Genetic studies with recombinant strains demonstrate that the I-A locus codes for three molecules, &, AD,and EP (or AE) while the I-E locus codes for one molecule, E,. These products combine to form the I-E (E,E,) and I-A (A,A,) molecules on the cell surface (Jones et al., 1978a,b; Murphy et al., 1980; McMillan et al., 1981). More recent studies done at the molecular level demonstrate that the I-A and I-E regions are contiguous (Steinmetz et al., 1982). Therefore, the I-A region may only code for two molecules, %, and while both E, and EP are coded for by genes in the I-E locus. A comparison of the genetic and molecular maps of the murine MHC is given in Fig. 1.

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The F, progeny of inbred strains of mice have I-A and I-E molecules representative of those present in each parent as well as new Ia molecules created by associations between a and @ chains inherited from each parent. For example, an I-A a from parent 1can combine with an I-A p from parent 2, thereby creating in the F, an I-A moIecule not expressed in either parent. (Presently it appears that there is very little if any associa7ion of E,A, or &ED in the Fl.) Th,is process by which parenteral genes interact to produce a new molecule is termed gene complementation. There is a third type of molecule detected in immunoprecipitations of murine Ia molecules, the invarient chain Ii. This glycoprotein is common to all Ia immunoprecipitates, has a molecular weight of 31,000, and appears to be associated with Ia antigens during their cytoplasmic processing. It is not associated with them on the cell surface. The function of Ii is at present unknown (Jones et d., 1978a,b; Moosic et al., 1980; Koch and Hammerling, 1982a,b). The human MHC occupies a 1.5-2.0 cM region of the short arm of chromosome 6. Presently, there are three defined regions (DS or DC, DR, and SB) which encode molecules resembling murine Ia molecules in that they (1) are displayed on M 4 , B cells, some T cells, dendritic cells, and Langerhan’s cells, (2) serve as stimulating determinants in an allogenic mixed lymphocyte reaction, and (3) have a similar structure consisting of a noncovalently linked a and @ chain (Walford et al., 1975; Winchester et al., 1975; van Rood et al., 1976; Hushberg et al., 1976; Billing et al., 1976; Snary et al., 1976; Albrechtsen et al., 1977; Albrechtsen, 1977). Figure l compares the genetic map of the human MHC to the genetic and molecular maps of the murine MHC. The best characterized human Ia molecule (HLA-DR) is a product of genes in the HLA-D region. Many laboratories have isolated cDNA genomic clones for HLA-DR antigens and much of the knowledge concerning structure of these molecules was obtained from molecular biology studies. HLA-DR antigens consist of a 2 chain glycoprotein with molecular weights of 34,000 and 29,000 for the a and @ subunits, respectively (Billing et al., 1976; Snary et al., 1976; Springer et al., 1977a,b; Klareskog et al., 1979; Silver, 1981).Both a and @ subunits are transmembrane proteins with a cytoplasmic, hydrophilic carboxy terminus (Kaufman and Strominger, 1979). The carboxy terminus of the a chain is phosphorylated (Kaufman and Strominger, 1979), but the function of this phosphorylation is unknown. Nucleotide sequencing of the genomic clones coding for the a chain reveals that it is 229 amino acids in length and consists of four domains: (1) a hydrophilic cyto-

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plasmic terminus of 15 amino acids, (2) an intramembranous hydrophobic domain of 22 amino acids, (3) one extramembranous domain near the membrane containing one disulfide loop between cysteines in positions 107 and 163, and an N-linked complex carbohydrate attached to an asparagine at position 188, and (4) an amino terminus domain with an N-linked complex carbohydrate attached to an asparagine at position 78 (Larhammer et al., 1982a; Kaufman and Strominger, 1982). Similar nucleotide sequencing of cDNA clones coding for the HLA-DR /3 chain reveals a protein of 229 amino acids with a four domain structure consisting of (1)a hydrophilic cytoplasmic carboxy terminus 10 amino acids in length, (2) a hydrophobic intramembranous domain of 27 amino acids, (3) an extracytoplasmic domain near the membrane containing one disulfide loop between cysteines at positions 117 and 173, and (4) a polymorphic amino terminus domain containing a disulfide loop between cysteine at positions 15 and 79, and an N-linked complex carbohydrate attached to an asparagine at position 19 (Kaufman and Strominger, 1982; Larhammer et al., 1982b). The structure of HLA-DR a and /3 chains is diagramed in Fig. 2.

CY chain

“i’

N72

chain

Domain I Domain I

Domain 2

Domain 3 Domain 4

COOH

COOH

CYTOPLASM FIG. 2. Diagrammatic representation of the HLA-DR molecule. Refer to text for explanation.

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Immunoprecipitates of HLADR molecules also contain a third protein, the invariant spot (Ii). As noted for the murine Ii, this 31,000 glycoprotein is felt to be intimately associated with HLA-DR in the cytoplasm, but not on the cell surface. The function of this molecule is unknown (Charron and McDevitt, 1980; Kvist et al., 1982). Comparison of the amino acid sequences of HLA-DR a and p chains has revealed some striking homologies. The second, extramembranous domains (see Fig. 1) of both a and p chains have appreciable amino acid homology with the third external domain of class I antigens, p2microglobulin, and the homologous constant region domains of immunoglobulin light and heavy chains (Larhammer et al., 1982a,b; Kaufman and Strominger, 1982). This has led to the speculation that all of these molecules which are involved in immune recognition derive from a common ancestral gene. Studies of the individual subunits of HLA-DR molecules reveal that the polymorphism is carried on the p chain. This has been demonstrated by two-dimensional electrophoresis (Charron and McDevitt, 1979; Shackelford and Strominger, 1979; Charron and McDevitt, 1980), two-dimensional peptide mapping (Corte et a1., 1981), analysis of peptide fragments using high performance liquid chromatography (Silver et al., 1979; Silver and Russel, 1979; Kaufman et al., 1980; Silver and Ferrone, 1980), and direct demonstration that an allospecific monoclonal antibody binds to the p chain (Johnson et al., 1982). Furthermore, studies utilizing the cDNA probes of HLA-DR reveal no restriction endonuclease polymorphism in the HLA-DR a chain except for a BgZII site difference between DNAs from cA-Sc and T5-1 cell lines (Stetler et al., 1982). However, there is restriction endonuclease polymorphism among the p chains from different individuals (Wake et al., 1982a,b). As stated before, a number of laboratories have isolated cDNA and genomic clones for HLA-DR antigens (Stetler et al., 1982; Korman et al., 1982; Wake et al., 1982b; Long et al., 1982a,b; Lee et al., 1982; Das et al., 1983).HLA-DR a chains are encoded by a single copy, 7.5 kb gene. Under hybridization conditions of low stringency, HLA-DR a cDNA clones cross-hybridize with HLA-DS (DC) a and possibly HLA-SB a cDNA clones. A full length cDNA clone for the HLA-DR p chain has also been described (Larhammer et al., 1982b; Wiman et al., 1982; Long et al., 198213). In contrast to the a chain gene, there exists multiple p chain copies in the genome. By limited amino acid sequencing, Kratzin et al. (1981) found at least six separate p chains. By Southern blotting, a similar if not greater gene copy number is revealed. Some of the genes hybridizing with this probe may be

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THOMAS A. GONWA ET AL.

coding for HLA-DS (DC) or HLA-SB /3 chains. An understanding of the generation of diversity in this gene family must await extensive cataloguing of Ia nucleotide and amino acid sequences. Whether point mutation, recombination with unequal crossing over, or gene conversion is the predominant mechanism leading to the polymorphism found in HLA-DR will then be revealed. It is clear, however, that the human Ir gene complex is more complex than the mouse where to date only two a chain genes and four /3 chain genes have been found (Steinmetz et al., 1982). Limited amino acid sequencing of the amino terminus of the a chain of HLA-DR demonstrates striking homology with murine I-E LY chains (Springer et al., 1977a,b; Allison et al., 1978; Silver et al., 1979; Silver and Russel, 1979; Mann et al., 1979, Silver, 1981). In other words, HLA-DR resembles murine I-E. Several observations suggest that humans, like mice, have more than one type of Ia molecule. Serological studies demonstrate that not all HLA-DR typing alloantisera are directed against one determinant (Springer et al., 1977; Suciu-Foca et al., 1979; Tsuji et al., 1979). Several studies demonstrate a dissociation between HLA-D products determined serologically (HLA-DR) and those (HLA-D) determined by mixed lymphocyte reaction. HLA-DR identical individuals can be HLA-D disparate (Reinsmoen et al., 1978; Suciu-Foca et al., 1978; Hartzman et al., 1978). Data obtained from immunoprecipitation studies indicate that there may be more than one type of human Ia molecule (Lampson and Levy, 1980; Quaranta et al., 1980, 1981; Letarte and Falk, 1982; Finn and Levy, 1982). Coincident with these studies, serological screening of pregnancy sera revealed that some sera reacted with groups of DR specificities. This led to the description of new series of antigens, MB (Duquesnoy et al., 1979, 1982), MT (Park et al., 1980), DC (Tosi et al., 1978), LB (Termitjelen et al., 1980), and TE (Park et al., 1978). It has not been discerned yet whether all of these antigens are supertypic determinants shared by distinct HLA-DR molecules or whether they all are present on separate human Ia molecules. However, a few of these antigens have been studied in detail. The best studied molecule is the determinant DC-1, seen in association with either HLA-DR1, HLA-DR2, or HLA-DR6. Early studies demonstrated that the DC-1 molecule differs in papain sensitivity from HLA-DR molecules (Tanigaki et al., 1980). Furthermore, twodimensional electrophoresis and peptide mapping demonstrate that DC-1 is on a molecule distinct from HLA-DR (Shackelford et al., 1981; Corte et al., 1981a,b). Two-dimensional electrophoresis demonstrates that the DC-1 molecule associated with HLA-DR1 is distinct from the

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DC-1 molecule associated with HLA-DR6 although both share a common serologically defined epitope (Shackelford et al., 1981). Therefore, DC-1 is a serologically defined determinant shared by three molecules which are structurally distinct, each of which is distinct from HLA-DR. Recent biochemical studies have demonstrated the clear existence of this Eecond type of human Ia molecule, now termed HLA-DS or HLA-DC. This molecule, like HLA-DR, has a 2 chain structure consisting of an a and p chain having molecular weights similar to those of the a and @ chains of HLA-DR. However, HLA-DR and HLA-DS can be distinguished by two-dimensional electrophoresis. Partial amino acid sequencing of the amino terminus of the a chain of HLA-DS reveals a sequence different than that of the a chain of HLA-DR. The HLA-DS molecule has striking amino acid sequence homology with the murine I-A molecule, thus providing the first direct demonstration of a human equivalent of murine I-A (Goyert and Silver, 1981; Goyert et al., 1982). It appears that the DC-1 specificity resides on HLA-DS molecules. It can be demonstrated that anti-DC-1 sera react with a two chain molecule whose a chain differs in amino acid sequence from the a chain of HLA-DR and which demonstrates homology with murine 1-A. The amino acid sequence of the DC-1 a chain is identical to that described for the a chain of the HLA-DS (Bono and Strominger, 1982). Thus, HLA-DS and HLA-DC are identical molecules which represent the human equivalent of I-A. Preliminary studies indicate that the HLA-DS (DC) locus is closely linked and telomeric to HLA-D (i.e., between HLA-D and HLA-B). With the demonstration of at least two structurally distinct human Ia molecules, a word of caution is needed concerning the use of antibodies with purported specificity for only DR molecules. Many antibodies were produced and reported to have specificity for only HLA-DR based on analysis of immunoprecipitated material analyzed by electrophoresis on one-dimensional SDS-PAGE. However, since HLA-DR and HLA-DS (DC) molecules appear similar when analyzed in this way, careful study of these antibodies may reveal that some of them are directed against HLA-DR and some against HLA-DS. This is demonstrated in Fig. 3 which depicts immunoprecipitates from a B cell line utilizing (1) a monoclonal anti HLA-DR antibody (CA206; Charron and McDevitt, 1980), (2) a rabbit sera directed against all HLA-DS molecules (Rb03; Goyert et al., 1982), (3) a rabbit sera produced against all B cell glycoproteins (R3; Gonwa and Stobo, unpublished studies), and (4)a second monoclonal antibody reported to be

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THOMAS A. GONWA ET AL.

FIG.3. Demonstration of 3 Ia molecules present on a human B cell line. Cells (2 x 10') from a human lymphoblastoid cell line were internally labeled with

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directed against HLA-DR (1.41; Charron and McDevitt, 1980). The B cell line was biosynthetically labeled with [35Slmethionine in the presence of tunicamycin, an antibiotic which prevents glycosylation. Immunoprecipitates were analyzed by two-dimensional gel electrophoresis. Note that the DR and DS immunoprecipitates demonstrate distinctly different p chains with the /3 chain of DS ( p 2 ) being more basic and of lower molecular weight than the p chain of DR (PI).There may be a small amount of cross reaction between DR and DC molecules as both the immunoprecipitates demonstrate a small amount of the other p chains. The rabbit sera produced against B cell glycoproteins (R3) immunoprecipitates DR and DS molecules along with class I transplantation antigens and p2-microglobulin. The second monoclonal “anti HLA-DR” (1.41) also precipitates both DR and DS molecules. Furthermore, both R 3 and 1.41 precipitate what appears to be a third structurally distinct p chain (labeled “?”). Thus, using this B cell line and these experimental conditions, ab 1.41 reacts with three distinct Ia molecules. One must therefore be careful that “anti-HLA-DR’ antibodies are of well-defined specificity. This is particularly important when one attempts to interpret in vitro studies which examine the ability of anti-HLA-DR reagents to modulate immune responsiveness. Figure 3 demonstrates what appears to be a third p chain from B cell line. Others have reported the possible existence of a third structurally distinct human p chain (Karr et al., 1982). This may be encoded by genes in a new histocompatibility locus, HLA-SB (Kavathas et al., 1981; Shaw et al., 1981). Products of this locus stimulate in a 2”but not in a 1” mixed lymphocyte reaction. Products of the SB locus have been characterized as two chain molecules which resemble HLA-DR and HLA-DS when analyzed by one-dimensional SDS-PAGE (Shaw et al., 1981, 1982; Nadler et al., 1981; Hurley et al., 1982). Preliminary structural characterization of the SB molecule indicates that the a chain has some sequence homology with the a chain of HLA-DR (Hurley et al., [9]methionine in the presence of tunicamycin, solubilized, and immunoprecipitated with either control sera, a monoclonal anti-HLA-DR antibody (DR), a rabbit anti-DS antisera (DS), a rabbit anti-B cell glycoprotein antibody (R3), or a putative monoclonal anti-HLA-DR antibody (141). Immunoprecipitates were analyzed by two-dimensional polyacrylamide gel electrophoresis. First dimension is isoelectric focusing pH 3.5 (right) to pH 10 (left). Second dimension is 10% PAGE, nonreducing conditions. Area of gels from 43,000 to 12,000 kd is shown. (“i” refers to invariant spot, “a” refers to a chain of either HLA-DR or HLA-DS, “PI” refers to the p chain of HLA-DS, “T” refers to unknown third type p chains, “clI” refers to class I transplantation antigens, “b2 m” refers to µglobulin.)

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1982). This raises the possibility that SB arose from duplication and transposition of the HLA-DR locus. Both molecules are distinct from HLA-DS molecules. At present, the molecular biology of HLA-DS (DC) and HLA-SB has not been studied as extensively as HLA-DR. Substantial evidence demonstrates that the human genome codes for at least three structurally distinct Ia-like antigens. Two have structural homology with murine I-E (HLA-DR and SB) and one has homology with murine I-A [HLA-DS (DC)]. Further characterization of these molecules and a careful search for new types of human Ia molecules will be necessary before their full role in controlling the human immune response can be defined.

Ill. Ir Gene Function in Humans

I n order to understand how I r genes can influence human immune reactivity, it is necessary to briefly review the participation of Ia molecules in the differentiation and activation of immunocompetent cells. During their development within the thymus, T cells contact Ia bearing stromal cells. This contact reflects a selection process which results in the emigration of only T cells which can recognize self Ia molecules in conjunction with other conventional antigens (Longo et al., 1981). T cells capable of recognizing Ia molecules alone undergo intrathymic death and therefore do not exist in the repertoire of antigen reactive peripheral T cells. Activation of peripheral T cells requires that they not only recognize determinants inherent in conventional antigen, but also determinants inherent in Ia molecules displayed by antigen presenting cells. Interactions occurring among activated T cells or between activated T cells and other immunocytes which are designed to regulate immune reactivity are also governed by cellular recognition of Ia molecules (Benacerraf and Germain, 1979). Therefore products of Ir genes are involved in (1) determining the repertoire of antigen reactive T cells which exit from the thymus, (2) presenting antigen to reactive T cells, and (3) dictating interactions involved in regulating immune reactivity. When considered within this framework, there are three points at which Ir genes can exert their effect. First, they can determine which clones of antigen reactive T cells actually pass into peripheral lymphoid tissue. For example, it has been suggested that I r gene controlled unresponsiveness to a specific antigen reflects the fact that self Ia alone structurally mirrors self Ia plus the specific antigen (Schwartz, 1978). Therefore, clones of T cells potentially reactive to the specific

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antigen would also be potentially autoreactive and would not be allowed to pass into the periphery. Second, Ia molecules can determine whether or not an antigen is presented to T cells in a manner suitable to initiate T cell activation. Here, Ir gene controlled unresponsiveness to a specific antigen would not reflect an absence of antigen reactive T cells. Instead, the antigen cannot be presented to the reactive T cells by self Ia. Third, Ia molecules can dictate the extent to which cellular interactions following activation affect the relative baIance between helper and suppressor cells. In this situation, Ir gene unresponsiveness does not reflect an absence of antigen reactive T cells or failure of self Ia to suitably present antigen. Exposure to antigen results in the activation of both helper and suppressor T cells. Unresponsiveness represents a predominance of immunosuppressive influences. Depending on the antigen and strain of mouse studied, each of these three mechanisms (depletion of antigen reactive T cells, inability of Ia molecules to present antigen, and preferential activation of suppressive influences) has been shown to represent the phenotypic expression of Ir gene controlled unresponsiveness. Based on the demonstration that products of HLA-D genes, HLA-DR determinants, are structurally homologous to murine Ia molecules, several studies have attempted to demonstrate that these genes and molecules function to control immune reactivity in humans. The earliest of these studies attempted to abrogate antigen-induced T cell activation, usually measured by proliferation, by blocking interactions between accessory and T cells with anti-DR antibodies (Bergholtz and Thorsby, 1978; Breard et al., 1979; Engleman et uZ., 1980). Many of these studies yielded positive results, thus implicating HLA-DR as a restriction element in the interactions between antigen-reactive T cells and antigen presenting accessory cells. However, three other possibilities must also be considered. First, it has been demonstrated that coating of cells with antibodies directed against MHC molecules can arm these cells for destruction by antibody-dependent, cell-mediated cytotoxicity (Geier, 1978). Therefore, inhibition of T cell activation by anti-HLA-DR antibodies may also be consistent with the conclusion that the HLA-DR bearing accessory cells are required for this reactivity and not that HLA-DR itself is involved in antigen presentation. A second consideration is that many of the HLA-DR antisera used in early studies were poorly characterized and may have specificity for other human Ia molecules in addition to HLA-DR (see Fig. 3). A third consideration is that by binding to surface HLA-DR molecules on accessory cells, antibodies can sterically block antigen presentation by an adjacent, but structurally distinct molecule. When considered to-

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gether, the results of blocking studies suggest, but do not prove, that HLA-D gene products function as antigen-presenting molecules. Another approach to implicate HLA-DR molecules as antigen presenting units is to determine if antigen primed T cells can be activated only by antigen presented along with the same HLA-DR molecules displayed by accessory cells present during the priming. For example, it is possible to determine if T cells from an HLA-DR4+ individual reactive to purified protein derivative can be activated, in uitro, by PPD seen in conjunction with HLA-DR4+ vs HLA-DR7+ accessory cells, The most elegant of such studies are represented b y those reported by Sredni et al. (1981). These investigators developed established clones of T cells reactive to keyhole limpet hemocyanin (KLH) from a KLH immune HLA-DR2+, 6+ individual. They then tested the ability of these clones to be activated by KLH presented by macrophages bearing a variety of HLA-DR determinants. The results of these studies demonstrated that T cells could be activated only when the accessory macrophages displayed one HLA-DR determinant (i.e., HLA-DR2) shared b y the responding T cell clone. These studies strongly implicate HLA-DR molecules in determinant presentation. However, they are also consistent with the possibility that some other determinant encoded by genes closely linked to, but distinct from, HLA-DR actually represent the true antigen presenting molecule. It should be emphasized that the majority of these studies were performed prior to the demonstration of human Ia molecules which are distinct from HLA-DR. As indicated, HLA-DS or HLA-DC are Ia molecules which demonstrate a structure distinct from HLA-DR and which display an amino acid sequence homologous to murine I-A molecules. The amino acid sequence of HLA-DR on the other hand is homologous to murine I-E molecules. In mice, responsiveness or unresponsiveness to the majority of the antigens tested is associated with specific I-A and not I-E molecules (Klein et al., 1981; Dorf, 1981). Based on this, it might be predicted that HLA-DS (DC) rather than HLA-DR molecules would serve as the most common restricting elements in humans. Although this remains to be proven, two observations indirectly support a role for HLA-DS molecules in antigen presentation. The first derives from studies initiated by Gonwa et al. which were designed to investigate the display of distinct Ia molecules by subpopulations of human macrophages (Gonwa et al., 1983). The results of these studies indicated that while >90% of human, adherent peripheral blood macrophages display HLA-DR molecules, approximately only 50% also display HLA-DS (DC). Most importantly, the HLA-DS+, HLA-DR+, but not the HLA-DS-, HLA-DR+ population is effective in reconstituting antigen induced proliferation among T en-

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GENES

TABLE I RELATIVE ABILITYOF HLA-DR+, HLA-DS+ vs HLA-DR+, HLA-DS- MACROPHAGES TO SUPPORT ANTIGEN-INDUCED T CELL PROLIFERATION" M 4 added

70 M 4 added

HLA-DR+, HLA-DS+

HLA-DR+, HLA-DS-

0 0.1 1.0 10.0

210 4016 6002 9934

210 652 755 8 16

a The indicated final % M 4 was added to autologous T enriched M$J depleted cells and reactivity to three concentrations of C. alhicans tested. Results are presented as maximal Acpm.

riched, macrophage depleted cells (Table I). The HLA-DS-, HLADR+ population of macrophages does not suppress antigen induced proliferation and cannot reconstitute reactivity even when exogenous interleukin I is added. This suggests that the difference in the two populations to support antigen-induced T cell proliferation represents differences in their ability to suitably present antigen. This in turn suggests that HLA-DS is more important than HLA-DR in restricting T cell-macrophage interactions in this model of reactivity to C. albicans. However, it is possible that the display of HLA-DS simply correlates with some other property or metabolic function of macrophages required for antigen presentation by HLA-DR. A second observation which implicates HLA-DS molecules in antigen presentation is the observation that antigen induced T cell proliferation can be blocked by anti-HLA-DS reagents (Table 11). The fact that the anti-DS reagent did not completely block reactivity is consistent with the possibility that with a complex antigen such as C . albicans, some determinants would be restricted by HLA-DS and others by HLA-DR. These blocking studies, however, are subject to the same alternative explanations as those outlined for blocking with antiHLA-DR reagents. The results of the blocking studies implicate both HLA-DR and HLA-DS in the T dependent proliferation to complex antigens. The relative role that each plays in this function remains to be established. Another approach to establishing HLA-D linked genes in controlling immune reactivity has been to link specific immune reactivity in families or the general population with given HLA-D linked gene products. Scher et al. (1975) immunized 61 normal volunteers with the

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THOMAS A. GONWA ET AL.

TABLE I1 ABILITY OF ANTI-HLA-DSTO BLOCK ANTIGEN-INDUCEDT CELL PROLIFERATION" Dilution of serum used

Expt. 1 NRS Anti-HLA-DS

1/200

1/400

1/800

8,982 481

13,093 3,002

10,544 5,160

25,045 9,268

26,479 10,549

29,018 13,355

Expt. 2

NRS Anti-HLA-DS

"Normal rabbit serum (NRS) or anti-HLA-DS was added to peripheral blood mononuclear cells to achieve a dilution of sera as indicated; 1 x los cells were tested for responsiveness to C . albicans. Results are expressed as maximal Acpm. Acpm in cultures with no antisera added were 9,018 and 36,749 in Expt. 1 and 2, respectively.

synthetic terpolymer, L-glutamic acid L-lysine L-tyrosine (GLT). Thirty-four of these individuals demonstrated a positive delayed hypersensitivity skin test in response to challenge with GLT. This responsiveness showed no association with known HLA-A or HLA-B determinants. HLA-DR typing was not performed. Three different groups of investigators have demonstrated that a substantial portion of normal, nonimmunized individuals acquire immune reactivity to several synthetic polypeptides. Young and Engleman (1980) demonstrated that 57, 85, and 71% of normal volunteers demonstrate reactivity to the synthetic polypeptides glutamic acid : tyrosine (GT), glutamic acid : alanine : tyrosine (GAT), and poly(L-tyrosine, L-glutamic acid)-poly(DL-alanine) poly(L-lysine) (T,G)-AL, respectively. Responsiveness, as measured by in vitro proliferation, occurred despite the fact that none of the individuals was purposely immunized with the antigens and responsiveness was not noted using cord blood lymphocytes. Hsu et a2. (1981) similarly demonstrated a naturally occurring T proliferative response to poly(Lhistidine, L-glutamic acid)-poly(DL-alanine)-poly(L-lysine), (H,G)-AL and (T,G)-AL in 64 and 54% of normal volunteers, respectively. Katz and colleagues (1981) demonstrated that T cells from 50% of normal donors could be induced to secrete an antigen-specific, T cell replacing factor upon challenge with (T,G)-AL. The precise nature of the naturally occurring immunogen which leads to this reactivity is unknown. Nonetheless, the presence of reactivity to simple, defined anti-

HUMAN

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85

gens provides an opportunity to determine if it is controlled by HLA-D linked genes. In a study of nine families, Young and Engleman (1980) could find no association between a specific haplotype and responsiveness or unresponsiveness to GT, GAT, or (T,G)-AL. For example, offspring of nonresponders were unpredictably responders and vice versa. HLAidentical siblings were discordant for responder status. Based on the results of these studies, the authors concluded that a single dominant immune response gene did not control reactivity to the three synthetic antigens tested. Hsu et al. (1981) arrived at a slightly different conclusion in investigating the inhertance of immune reactivity to (H,G)-AL and (T,G)AL. These investigators showed concordant inheritance of HLA and responsiveness in families although there was no association between a specific HLA-A, Byor D allele in the general population and immune reactivity to either peptide. Based on the observation in two families that the mating between two nonresponders could produce responder offspring, the investigators proposed that gene complementation was involved in determining responder status. This hypothesis is consistent with the demonstration of a requirement for gene complementation in determining responder status to some synthetic polypeptides in mice. One very interesting family in this study (family 4000) demonstrated a recombination between HLA-B and HLA-D. The response pattern to both (T,G)-AL and (H,G)-AL indicated that Ir genes controlling these reactivities was telomeric and not centromeric to HLA-D (i.e., between HLA-D and B, but not between HLA-D and HLA-SB). This is of particular interest in view of the fact that HLA-DS (DC) may also be telomeric and not centromeric to HLA-D (see Fig. 1). While the studies of Hsu et al. provide convincing data to implicate HLA linked genes in controlling immune reactivity, they indicate that such control is polymorphic and not simply mediated by a single HLA-D gene. Moreover, not all inheritance patterns of responsiveness could be explained by a requirement for gene complementation. Three possibilities could explain a failure to show a significant association between a single HLA-D gene product in the general population and reactivity to synthetic polypeptides. First, it is possible that genes separate from, but closely linked to HLA-D actually represent the human Ir genes for reactivity to (T,G)-ALand (H,G)-AL. Likely candidates could be HLA-DS (DC) genes. According to our analysis, responsiveness to (T,G)-AL or (H,G)-AL in the study by Hsu et al. did

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THOMAS A. GONWA ET AL.

not correlate with the expression of a defined DS or DC molecule (i.e., DC-1, MT-3). A second possibility is that the response to (H,G)-ALand (T,G)-AL is encoded for by HLA genes which are not closely linked to HLA-DR or HLA-DS (DC) (e.g., HLA-SB). This possibility cannot be excluded. A third possibility is that the response to these two synthetic polypeptides is dependent on epitopes shared by distinct families of HLA-DR (or HLA-DS) molecules. In mice, it can be demonstrated that the immune response to a single synthetic antigen is controlled by genes present in several distinct H-2 haplotypes. For example, Ir genes associated with the H-2 haplotypes a, b, d, f, r, u, and v each determine responsiveness to GAT (Dorf, 1981).Although HLA-DR molecules are serologically distinct as defined by certain typing reagents, they may share epitopes which are important in determining immune reactivity to a specific antigen. Stated in another way, HLA-DR1, 4,and 7, for example, might share a common epitope necessary for presenting (T,G)-AL to reactive T cells. This possibility is consistent with the observation that the frequency of immune reactivity in the general population to any of the synthetic antigens tested (e.g., approximately 50%) is greater than the frequency of an individual HLA-DR determinant. It is also consistent with the observation that some serologically defined supratypic determinants actually reside within HLA-DR molecules defined as distinct by conventional typing reagents. Although HLA-DR molecules have been used as an example here, a similar consideration applies to HLA-DS molecules. Structurally distinct HLA-DS molecules can be demonstrated to share common, serologically defined epitopes (Shackelford et aZ., 1981). Several groups of investigators have studied the association between HLA phenotypes and the immune response to either environmental antigens or antigens used for routine immunizations. Marsh and colleagues (1981, 1982a,b) investigated the association between IgE and IgG antibodies with specificity for a highly purified component of ragweed pollen (Ra5) and HLA-D or HLA-DR types in 447 Caucasians who were naturally exposed to ragweed. Seventy-nine to 85% of individuals with IgG antibodies to Ra5 were HLA-D2+ while 93 to lo@% of individuals with IgE antibodies to Ra5 were HLA-D2+. The frequency of HLA-D2 in individuals Iacking anti-Ra5 antibodies was approximately 20%. It was noted that the association between antibodies to Ra5 and HLA-D2 was stronger than that seen with the serologically defined HLA-DR2. Specific MB or MT types were not associated with antibody positive, HLA-DR2-, D2-, or antibody positive HLA-DR2+, D2- phenotypes. Whether this finding indicates that

HUMAN

Ir

GENES

a7

genes closely linked to, but distinct from, those coding for HLA-DR actually constitute Ir genes for antibody responses to Ra5 is not known. It is interesting to note that Marsh and his group were not able to demonstrate a convincing linkage between IgE antibodies until they utilized a very pure Ra5 preparation (>99.% pure). This underscores the necessity for using simple, defined molecules with a single or limited number of antigenic determinants when analyzing the influence of Ir genes. Sasazuki and collaborators (l978,1980a,b) examined the influence of Ir genes on determining reactivity in the Japanese population to three antigens, tetanus toxoid, schistosomal worm antigen, and streptococcal cell wall antigens. Each of these complex antigens contains several different antigenic determinants and thus the immune response to the whole molecule would not be controlled by a single Ir gene. In order to circumvent this and examine the T proliferative response to only a single immunodominant determinant, the authors examined reactivity occurring in response to in vitro stimulation with low concentrations of each antigen (0.2-1 kg). For both tetanus toxoid and schistosomal worm antigen, a low response in the general Japanese population was associated with HLA-Dwl2. Seventy and 58% of low responders to schistosomal worm antigen and tetanus respectively were HLADw212. The respective frequency of HLA-Dw12 in high responders to each antigen was 13 and 11%. Low responsiveness appeared to be controlled by a dominant gene in that all of the low responders were heterozygous. HLA-Dw 12 individuals were not unresponsive to all antigens. They demonstrated normal reactivity to diphtheria toxin and C . albicans. Although the same group of investigators could demonstrate, in families, HLA-linked control of unresponsiveness to immunodominant determinants in streptococcal cell walls, they were not able to show an association between a specific HLA-D gene product in the general population and responsiveness to the same antigen. Sasazuki et at. interpreted these studies as indicating the presence of a dominant immunosuppressor gene which, during natural exposure to three antigens tested, caused preferential activation of suppressor T cells. This conclusion was supported by three findings (Hays et al., 1982). First, T cells from low responders to streptococcal cell wall did not respond to the antigen even when mixed with antigen presenting cells from haplotype-identical high responders. Second, purified T cells from low responders inhibited streptococcal cell wall reactivity when added to haplotype-identical, high responder cells. Finally, removal of suppressor effector cells from low responders by panning

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THOMAS A. GONWA ET AL.

with the monoclonal antibody Leu 2a resulted in the appearance of streptococcal wall reactivity among the remaining cells. (The Leu 2a determinant is displayed by the T cell population containing suppressor effector cells.) Whether or not similar T dependent suppressive influences are also involved in determining low reactivity to tetanus toxoid and schistosomal worm antigens associated with HLA-Dw 12 linked genes is not known. Another system in which HLA-D linked genes appear to influence the expression of immune reactivity by dictating the relative activity of helper vs suppressive influences is the Ir gene control of collagen reactivity (Solinger et al., 1982; Solinger and Stobo, 1982). These studies were initiated to determine the frequency of T dependent reactivity to collagen in patients with rheumatoid arthritis. The results of these studies indicated that while collagen induced production of the lymphokine, leukocyte inhibition factor (LIF), could be detected in 90% of patients with rheumatoid arthritis, it could also b e detected in approximately 30% of normal controls without any evidence of synovitis. Collagen induced production of LIF was demonstrated to require interactions between T cells and M4, was not specific for any single type of collagen, and could be elicited by the collagen-like synthetic polypeptide (Gly-Pro), which manifests no tertiary helical structure. Since rheumatoid arthritis is significantly associated with the HLA-DR phenotype, HLA-DR4, the association between collagen responsiveness and HLA-DR4 was investigated in both patients and normals (Table 111). All HLA-DR4+ individuals, either patients or COLLAGEN

TABLE I11 RESPONSIVENESSAND HLA-DR4 POSITIVITP Responders

HLA-DR phenotype

Rheumatoid arthritis

Nonresponders Rheumatoid arthritis

Others

~~

HLA-DR4+ HLA-DR4Totals

11 5

12 3

0 2

31

Others

Totals

0

23 38

~

28

30 ~

This is a 2 x 2 analysis of the relationship between collagen reactivity and the presence of the HLA-DR4 phenotype in patients with rheumatoid arthritis as well as in individuals with other forms of arthropathies and normals (these latter two groups are referred to as “others”). Only individuals in whom it was possible to assay for both collagen-induced LIF production and HLA-DR phenotypes are included. The xa for the relationship between collagen responsiveness and HLA-DR4 is 33.7, with a p value of less than 0.0001.

HUMAN

Ir

GENES

89

normals, were collagen responders. Of the 31 collagen responders, 23 were HLA-DR4+. This association is highly significant, p < 0,001,and suggests that genes linked to those coding for HLA-DR4 determine the expression of reactivity to collagen-like determinants inherent in the linear polypeptide Gly-Pro. One interpretation of these studies is that the HLA-DR4 molecule, or another Ia molecule encoded by closely linked genes, is the only human Ia molecule capable of restricting immune reactivity to collagen. HLA-DR4- individuals would be nonresponders by virtue of the fact that their accessory cells lacked the appropriate Ia determinant. Alternatively, HLA-DR4- individuals might contain T cells potentially reactive to collagen which are inhibited by specific suppressive influences. In this situation, HLA-DR4- individuals would be nonresponders by virtue of a predominance of suppressor, and not an absence of reactive, T cells. In order to distinguish between these two possibilities, advantage was taken of the fact that in some systems suppressive influences are radiosensitive while LIF production is radioresistant. Peripheral blood mononuclear cells (PBMC) from 20 HLA-DR4- collagen nonresponders were irradiated with 1000 rads and then assayed for LIF production in response to challenge with collagen. In each case the irradiated PBMC manifested collagen reactivity (Solinger and Stobo, 1982). Two points concerning the appearance of this reactivity among irradiated PBMC should be emphasized. First, it was specific for collagen in that there was no increase in the mean response of the irradiated PBMC to another antigen, C . albicans. In addition, irradiation did not result in the appearance of reactivity to purified protein derivative among the PBMC of two individuals who were unresponsive to this antigen. Second, the cellular requirements for and specificity of reactivity among the irradiated PBMC were identical to those noted among nonirradiated HLA-DR4+ individuals. Collagen-induced LIF production required both T cells and M$J and could be elicited by the collagen-like polypeptide (Gly-Pro),. If the appearance of reactivity to collagen among irradiated PBMC represents the elimination of suppressive influences, then it should be possible to inhibit reactivity among the irradiated cells by the addition of unresponsive, autologous nonirradiated PBMC. Indeed, this proved to be the case (Solinger and Stobo, 1982). Moreover, suppression requires the presence of T cells. Two other findings indicate that the absence of apparent collagen reactivity among HLA-DR4- individuals is due to the presence of suppressive influences. First, fractionation of T cells from' HLA-DR4-

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THOMAS A. CONWA ET AL.

collagen nonresponders on a 5-step discontinuous bovine serum albumin density gradient results in collagen reactivity among the high density population (Solinger and Stobo, 1982). Addition of low density T cells to this high density population suppressed this collagen reactivity. Second, treatment of PBMC from collagen nonresponders with the monoclonal antibody, OKT8, and complement (a procedure which depletes suppressor effector cells) resulted in the appearance of collagen reactivity among the remaining cells (Solinger and Stobo, 1982). In this system, therefore, the HLA-D linked expression of collagen reactivity represents the influence of these genes on determining the relative activity of collagen specific suppressive influences. Collagen reactive suppressor cells could be detected in association with each HLA-DR phenotype tested except HLA-DR4 (i.e., HLA-DR 1 , 2 , 3 , 5 , 7 , and 8). All HLA-DR4+ collagen responders except for one individual were heterozygous at the HLA-D locus. Their MHC, therefore, contains one of the alleles expressed in HLA-DR4- nonresponders. Irradiation (1000 R) of PBMC from HLA-DR4+ individuals did not result in any increase in reactivity to collagen suggesting that collagen reactivity in these individuals reflects an absolute absence of suppressive influences and not simply a reactive predominance of reactive over suppressor cells. These findings suggest the following two models to link HLA-DR4 positivity with collagen reactivity. First, generation of collagen specific suppressive influences might require complementation between two genes in the trans position. One of these genes would be linked to those coding for each HLA-DR type except HLA-DR4. Therefore, only one of the two required genes would be present in HLA-DR4 heterozygotes. Second, HLA-DR4 linked genes could code for events which prevent either the generation or activation of collagen reactive suppressive cells. This would be similar to the recently described contrasuppression circuit in mice in which it can be demonstrated that Ir genes can modulate immune reactivity by determining activity among cells capable of “suppressing” the activity of suppressor T cells (Gershon et al., 1981). Each of these possibilities is consistent with the observation that collagen reactivity and thus the failure to express collagen reactive suppressor cells is inherited as a dominant trait. In these studies, an association between HLA-D gene products (i-e., HLA-DR4) and expression of collagen reactivity was demonstrated. However, there were several HLA-DR4- collagen responders. Therefore, it is possible that the true collagen Ir genes occupy a locus which is distinct from, but closely linked to HLA-DR. The finding that rheumatoid arthritis is more closely associated with the HLA-DS mol-

HUMAN

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GENES

91

ecule, MT-3, than with HLA-DR4 supports this (Collier and Stobo, unpublished observations). Over the past decade it has been possible to demonstrate a striking association between specific HLA-D gene products and clinical disorders in which a given immune response contributes to the expression of disease (Schwartz and Shreffler, 1980). The association between HLA-DR2, Goodpasture’s syndrome, and the antibody response to basement membrane antigens in lung and kidney is but one example (Rees et al., 1978). In these associations, it has been hypothesized that Ir genes linked to the HLA-D locus determine responder status for the immune reactivity involved in the expression of the disease. However, for most associations, this has not been proven. For example, it has not been demonstrated that HLA-DR2 linked genes confer responder status for immune reactivity to basement membrane antigens. Only in the case of rheumatoid arthritis have HLA-D linked genes been implicated in an immune reactivity (i.e., T cell reactivity against collagen) which may contribute to the clinical manifestations of the disease. Several different autoimmune diseases each characterized by immune reactivity to a different auto-antigen are associated with the HLA-DR phenotype, HLA-DR3. These include systemic lupus erythematosus, Sjogren’s syndrome, dermatitis herpetiformis, myasthenia gravis, Graves disease, juvenile onset diabetes, Addison’s disease, and chronic active hepatitis (Schwartz and Shreffler, 1980). In this association, it has been hypothesized that genes linked to those coding for HLA-DR3 cause a generalized increase in immune reactivity which then provides the basis for a specific autoantibody response. This concept receives support from the demonstration that normal HLA-DR3 individuals exhibit immune hyperresponsiveness as manifest by an increase in spontaneous immunoglobulin production among peripheral blood mononuclear cells (Lawley et al., 1981; Ambinder et al., 1982). Two hypotheses have been provided to explain this immunologic hyperresponsiveness. The first is that the HLA-DR3 phenotype is associated with a generalized decrease in suppressive influences which normally serve to dampen immune reactivity. Ambinder et al. (1982) demonstrated diminished suppressor activity in normal HLA-DR3+ when compared to normal HLA-DR3- individuals. Four of 11 HLA-DR3+ individuals lacked any detectable suppressive influences. The assay system measured the ability of Con A activated T cells to inhibit immunoglobulin secretion by autologous peripheral blood mononuclear cells. A second hypothesis invoked to explain enhanced immune reactivity in HLA-DR3+ subjects is that there is decreased degradation of

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THOMAS A. GONWA ET AL.

antigen thus resulting in a persistent antigenic challenge. Legrand and colleagues (1982) studied the catabolism of sheep red blood cells by the peripheral blood macrophages of 100 normal individuals and demonstrated that 50% of individuals with diminished antigen degradation were HLA-DR3+. I n contrast, only )3% of individuals with rapid antigen degradation were HLA-DR3+. IV. Conclusions

The structural studies clearly indicate the existence of human l a molecules which are similar to those delineated in mice. However, it appears that there may be more human (4-6) than murine (2-3) Ia molecuIes. The functional studies indicate that HLA-DR and HLA-DS molecules can function as restriction elements which determine reactivity to simple antigens as well as immunodominant determinants present in complex antigens. The mechanisms by which human l a molecules control the expression of immune reactivity have only begun to b e explored. The available studies implicate these molecules in determining the relative activity of helper vs suppressive influences in controlling specific immune reactivity as well as generalized, i.e., polyclonal antibody formation. Future studies will generate genetic and molecular maps characterizing the whole range of human Ir genes and Ia molecules and indicate how they function to modulate immune reactivity in humans.

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Corte, G., Damiani, G., Calabi, F., Fabbi, M., and Burgelesi, A. (1981b).Proc. Natl. Acad. Sci. U.S.A. 78, 534. Cullen, S. E., Freed, J. H., and Nathenson, S. G. (1976). Transplant. Reu. 30,237. Das, H. K., Lawrence, S. K., and Weissman, S. M. (1983).Cell, in press. Dorf, M. E., ed. (1981). “The role of the Major Histocompatibility Complex in Immunobiology.” Garland, New York. Duquesnoy, R. J., Marrari, M., and Annen, K. (1979).Trunsplant. Proc. 11, 1757. Duquesnoy, R. J., Zeevi, A,, Marrari, M., and Halim, K. (1982). Clin. Zmmunol. Zmmunopathol. 23,254. Engleman, E. G., Charron, D. J., Benike, C. J., and Stewart, G. H. (1980).J.E x p . Med. 152,99s. Finn, 0. J., and Levy, R. (1982). Proc. Natl. Acad. Sci. U.S.A. 79, 2658. Geier, S. (1978).Cell. Zmmunol. 35, 392. Gershon, R., Eardley, D., Durum, S., Green, D., Shen, F., Hamauchi, K., Cantor, H., and Murphy, D. (1981).J.E x p . Med. 153, 1533. Gonwa, T. A., Picker, L., Raff, H. V., Goyefi, S. M., Silver, J., and Stobo, J. D. (1983). J . Immunol. 130, 706. Goyert, S. M., and Silver, J. (1981). Nature (London) 294,266. Goyert, S. M., Shively, J. E., and Silver, J. (1982).J.E x p . Med. 156, 550. Hartzman, R. J., Pappas, F., Romano, P. J., Johnson, A. H., Ward, F. E., and Amos, D. B. (1978). Transplant. Proc. 10, 809. Hays, E. F., Jones, P., Fatham, C. G., and Engleman, E. G. (1982). Clin. Zmmunol. Immunoputhol. 25, 283. Hsu, S . H., Chan, M. M., and Bias, W. B. (1981). Proc. Natl. Acad. Sci. U.S.A. 78,440. Hurley, C., Shaw, S., Nadler, L., Schlossmann, S., and Capra, J. D. (1982).J.E x p . Med. 56, 1557. Hushberg, H., Kaakinen, A., and Thorsby, E. (1976).Nature (London) 263,63. Johnson, J. P., Meo, T., Reithmuller, G., Schendel, D. J., and Wank, R. (1982).J. E x p . Med. 156, 104. Jones, P. P., Murphy, D. B., and McDevitt, H. 0. (1978a).J. E x p . Med. 148, 925. Jones, P. P., Murphy, D. B., Hewgill, D., and McDevitt, H. 0. (1978b).Zmmunochemistry 16, 51. Karr, R. W., Kannapell, C. C., Stein, J. A,, Fuller, T. C., Duquesnoy, R. J., Rodey, G. E., Mann, D. L., Gebel, H. M., and Schwartz, B. D. (1982).J. E x p . Med. 156, 652. Katz, D., Bentwich, Z., Eshhar, N., Lowy, I., and Mozes, E. (1981).Proc. Natl. Acad. Sci. U.S.A. 78, 4505. Kaufman, J. F., and Strominger, J. L. (1979). Proc. Natl. Acud. Sci. U.S.A. 76, 6304. Kaufman, J. F., and Strominger, J. L. (1982). Nature (London)297,694. Kaufman, J. F., Anderson, R. L., and Strominger, J. L. (1980).J.E x p . Med. 152, 37s. Kavathas, P., DeMars, R., Bach, F. H., and Shaw, S. (1981). Nature (London) 293,747. Klareskog, L., Tragardh, L., Rask, L., and Peterson, P. A. (1979).Biochemistry 18, 1481. Klein, J. (1979). Science 203, 516. Klein, J., Juretic, A,, Barevnis, C. D., and Nagy, Z. A. (1981).Nuture (London)291,455. Koch, N., and Hammerling, G. J. (1982a).J.Immunol. 128, 1155. Koch, N., and Hammerling, G. J. (1982b). Nature (Lortdon) 299,644. Korman, A. J., Knudsen, P. J., Kaufman, J. F., and Strominger, J. L. (1982).Proc. Natl. Acad. Sci. U.S.A. 79, 1844. Kratzin, H., Yang, C. Y., Gotz, H., Parely, E., Kolbel, S., Egert, G., Thinnes, F. P., Wemet, P., Altevogt, P., and Hilshmann, N. (1981).Hoppe-Seyler’s Z. Physiol. 362, 1665.

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Kvist, S., Wiman, K., Claesson, L., Peterson, P. A., and Dobberstein, B. (1982). Cell 29, 61. Lampson, L. A,, and Levy, R. (1980).J . Immunol. 125, 293. Larhammar, D., Gustafsson, K., Claesson, L., Bill, P., Wiman, E., Peterson, P. A., and Rask, L. (1982a).Cell 30, 153. Larhammar, D., Schenning, L., Gustafsson, K., Wiman, K., Claesson, L., Rusk, L., and Peterson, P. A. (1982b). Proc. Natl. Acad. Sci. U S A . 79, 3687. Lawley, T. J., Hall, R. P., Fauci, A. S., Katz,S. I., Hamburger, M. I., and Frank, M. M. (1981).N . Engl. J. Med. 304, 185. Lee, J. S., Trowsdale, J., and Bodmer, W. F. (1982).Proc. Natl. Acad. Sci. U.S.A.79,545. Legrand, L., Rivat-Perran, L., Huttin, C., and Dausset, J. (1982). Hum. Immunol. 4, 1. Letarte, M., and Falk, J. (1982).J. Immunol. 128, 217. Long, E. O., Gross, N., Wake, C. T., Mach, J. P., Carrel, S., Accola, R., and Mach, B. (1982a).EMBO J . 1,649. Long, E. O., Wake, C. T., Strubin, M., Gross, N., Accola, R. S., Carrel, S., and Mach, B. (1982b). Proc. Natl. Acad. Sci. U.S.A. 79, 7465. Longo, D. L., Matis, L. A,, and Schwartz, R. H. (1981).CRC Crit. Reu. Zmmunol. 2,83. McMillan, M., Frelinger, J. A., Jones, P. P., Murphy, D. B., McDevitt, H. O., and Hood, L. (198l).J. E x p . Med. 153, 936. McNicholas, J., Steinmetz, M., Hunkapiller, T., Jones, P., and Hood, L. (1982). Science 2.18, 1229. Mann, D. L., Kaufman, J., Orr, H., Robb, R., and Strominger, J. (1979).Transplant.Proc. 11, 668. Marsh, D. G., Meyers, D. A., and Bias, W. B. (1981). N . Engl. J. Med. 305, 1551. Marsh, D. G., Meyers, D. A., Freidboff, L. R., Ehrlich-Kautzky, E., Roebber, M., Norman, P. S., Hsu, S. H., and Bias, W. A. (1982a).J. Exp. Med. 155, 1452. Marsh, D. G., Hsu, S. H., Roebber, M., Ehrlich-Kautzky, E., Freidhoff, L. R., Meyers, D. A., Pollard, M. K., and Bias, W. B. (1982b).J. Exp. Med. 155, 1439. Moosic, J. P., Nielson, A., Hammerling, G. H., and McKean, D. J. (1980).J. Immunol. 125, 1463. Murphy, D. B., Jones, P. P., Loken, M. R., and McDevitt, H. 0. (1980).Proc. Natl. Acad. sci. U.S.A. 79,5504. Nadler, L. M., Stashenko, P., Hardy, R., Tomaselli, K. J., Yunis, E. J., Schlossman, S. F., and Pesando, J. M. (1981).Nature (London) 290, 591. Park, M. S., Terasaki, P. I., Bemoco, D., and Iwaki, Y. (1978).Transplant. Proc. 10,823. Park, M. S., Terasaki, P. I., Nakata, S., and Aoki, D. (1980). In “Histocompatibility Testing, 1980” (P. I. Terasaki, ed.), p. 854. Univ. of California Press, Los Angeles, California. Quaranta, V., Walker, L. E., Pellegrino, M. A., and Ferrone, S. (1980).J. Zmmunol. 125, 1421. Quaranta, V., Pellegrino, M. A., and Ferrone, S. (1981).]. Immunol. 126, 548. Rees, A., Peters, D., Compton, A., and Batchelor, J. R. (1978).Lancet 1, 966. Reinsmoen, N. L., Noreen, H. J., Friend, P. S., Giblett, E. R., Greenberg, L. J., and Kersey, J. H. (1978). Transplant. Proc. 10, 793. Sasazuki, T., Kohno, Y., Iwamoto, I., and Tanimura, M. (1978). Nature (London) 272, 5651. Sasazuki, T., Ohta, N., Kaneoka, R., and Kojima, S. (1980a).J. E x p . Med. 152,314s. Sasazuki, T., Kaneoka, H., Nishimura, Y.,Kaneoka, R., Hayama, M., and Ohkuni, H. (1980b).J. E x p . Med. 152,297s.

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ADVANCES IN IMMUNOLOGY, VOL. 04

Interferons with Special Emphasis on the Immune System1 ROBERT M. FRIEDMAN AND STEFANIE N . VOGEL Departments of Pothology and Microbiology, Uniformed Services University of the Healfh Sciences, Betherda, Morylond

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Interferon Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Actions of Interferons .................................. IV. Interferons and Defense against Viral Infections . . ......... V. Interferons and Other Mechanisms Related to Immunity and Inflammation .................................... VI. Antitumor Effects of Interferons in Animal Systems ...................... VII. Clinical Studies with Human Interferons ................................ References . . . . . . . . . . . . . ........................... ........

97 99 10 1 128

129 130 132 133

I. Introduction

Studies on interferons have become a special area of research. While in previous years a reasonably short review could give a complete summary of research on interferons, this is no longer possible; therefore, we shall attempt to highlight the areas of research on interferons that we feel are of special interest to immunologists. Interferons are induced, animal proteins. A variety of stimulating substances can act as interferon inducers and interferons inhibit a wide range of viruses by inducing an intracellular antiviral state; however, many interferons are species-specific in their antiviral activity (Isaacs and Lindenmann, 1957). Interferons were first described in 1957, but there is as yet no complete explanation of their induction, biological role, or biological activities. One reason for this is the impressive potency of interferons; the specific antiviral activity of human interferon, for instance, is more than lo9 international units per mg of protein (Rubinstein et al., 1978). With a molecular weight of approximately 20,000 this means that biological activity resides in 0.4 pg or lo7 molecules or 2 x M . This suggests that a few thousand molecules of interferon may induce an antiviral state; therefore, interferons are among the most active biological substances. As a consequence of this, The opinions or assertions contained herein are the private views of the authors and should not be construed as official or necessarily reflecting the views of the Uniformed Services University of the Health Sciences or Department of Defense. There is no objection to its presentation and/or publication.

97 Copyright 0 1983 b y Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-022434-8

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interferon preparations with great antiviral activity contain very little interferon. Interferon assays were, until very recently, biological. They are based on the ability of a preparation to inhibit the production of a virus or a viral product (Buckler, 1977). Although sensitive, they are time consuming and relatively imprecise. Because of the inherent inaccuracies of biological assays, a two- or threefold inhibition of a viral function is considered barely significant to define a unit; therefore, a level of uncertainty is present which is usually intolerable in a biochemical or biophysical system. Recent progress in the purification of interferons and the availability of monoclonal antibodies to interferons will soon lead to their immunochemical assay. One other problem has been that there are many species of interferon (Burke, 1977; Youngner, 1977). There are three general types of human interferon, designated alpha, beta, and gamma. When stimulated with virus, leukocytes in cultures produce predominantly the species called alpha interferon. There are at least 14 distinct genes for human alpha interferon (Nagata et al., 1980). Most alpha interferons contain little or no carbohydrate. Human fibroblast cultures, when stimulated with viruses or a chemical inducer of interferon such as the double-stranded RNA polymer, polyriboinosinic acid :polyribocytidylic acid [poly(I : C)], produce an interferon that is immunologically distinct from alpha interferons, and designated as beta (fibroblast) interferon. It is important to note, however, that leukocytes can produce beta interferon under some conditions, and that fibroblasts can be stimulated to produce some alpha .interferon. Beta interferon, a glycoprotein, is more hydrophobic than alpha interferon, so that beta interferon adheres to hydrophobic ligands such as hydrocarbons, that do not interact with alpha interferons. Beta interferon, but not alpha interferons, bind to lectins such as concanavalin A. One other striking difference between alpha and beta interferons is in their species specificity of antiviral activity. While human beta interferon is usually species-specific and for the most part induces antiviral activity only in human cells, human alpha interferons induce activity in human as well as some animal cell cultures. The third type of interferon, gamma (immune, type 11, or T interferon), like beta interferon, is a glycoprotein that differs from alpha or beta interferons in several fundamental respects (Youngner, 1977). It is antigenically distinct from them, and is more labile to acid. Most alpha and beta interferons are quite stable at pH 2, while the antiviral activity of gamma interferon is significantly reduced by this treatment. Gamma interferon is produced by lymphocytes in response to mito-

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gens or exposure to an antigen to which the cell had already been sensitized. Gamma interferon is, therefore, a lymphokine which appears to play a role in the regulation of immune response, and possibly in the antitumor effects of interferon treatment. Macrophages must be present for the production of gamma interferon by normal T lymphocytes. Also, a T to T cell interaction involving interleukin 2 may be required (Farrar et al., 1981). B lymphocytes produce alpha, rather than gamma interferon, whereas lymphoblastoid cell lines produce mixtures of alpha and beta interferons. Genes of all three types of human interferon have been cloned in microorganisms. So far, single genes have been definitely described only for gamma (Grey et al., 1982) and beta (Taniguchi et al., 1980) interferons, although there are some reports that additional types of beta interferon may be present (Sehgal and Sagar, 1980). There are at least 14 human alpha interferon genes, some of which may be alleles, and others nonexpressed pseudogenes. The various alpha species differ from each other by at most 15 to 30% in amino acid sequence. In contrast, the amino acid sequences of alpha interferons differ by about 85% with that of beta interferon (Nagata et aZ., 1980). It is not understood why there are so many types of alpha interferon, or indeed why there are three major gene types for interferons. II. Interferon Production

Alpha or beta interferons can be induced in animals or in cell cultures by living or killed RNA or DNA viruses (Burke, 1977); in addition, a number of natural and artificial nonviral substances are effective inducers of interferon production. The best studied of these are natural double-stranded RNA forms from fungi or synthetically produced molecules such as poly(1: C) (Vilcek and Kohase, 1977). It is possible that double-stranded RNA molecules are such excellent interferon inducers because they are similar in structure to a natural product that is the actual signal for turning on interferon production by cells. Many, but not all, viruses contain double-stranded RNA as a structural element or produce it during the course of their replication processes. There is a diverse group of additional substances which also induce interferon production in cell cultures or in animals (Merigan, 1973; Grossberg, 1977). These include bacteria that grow intracellularly such as Brucella abortus, Listeria monocytogenes, or Hemophilis influenzae, rickettisiae, mycoplasmae, protozoae, clamydiae; microbial products such as lipopolysaccharides; organic polymers, such as pyran copolymers or polyvinyl sulfate; and a variety of low-molecular-

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weight substances such as cycloheximide, kanamycin, tilorone, and toluidine blue. Some of the above induce interferon production only in animals; others, in vivo and in tissue cultures. This group is so divergent in structure, however, that the inducers may act by causing the production and release of a common intracellular substance such as a cellular double-stranded RNA. There are in animal cells naturally occurring double-stranded RNA forms that are themselves good interferon inducers (Stern and Friedman, 1970). Alternatively, the diverse substances which induce interferons and double-stranded RNA forms may interact with similar receptors on the cell surface. Many types of cells make interferons. Both T, B, and non-T, non-B lymphocytes produce alpha interferons; macrophages and most fibroblast cultures can also be induced to make both alpha and beta interferons. T cells produce predominantly gamma interferon. The inability of some cell lines to produce interferons has not been explained, but one interesting system involves mouse teratocarcinoma cell cultures: when undifferentiated, these cells do not produce interferons; however, after differentiation, production of interferons can be induced (Burke et al., 1978). The control of interferon production is not well understood, but it is likely that it involves a repressor mechanism. Since the cellular content of interferon mRNA can be assayed by several methods, the mRNA forms for alpha and beta interferons have been shown to be poly(A)-rich, distinct 8-12 S molecular forms (Cavalieri and Pestka, 1977). The best evidence for a repressor mechanism for interferon production is the phenomenon of superinduction in human fibroblast cultures (Vilcek and Kohase, 1977). Superinduction is the greater than normal production of beta interferon in fibroblast cultures that are induced to form interferon in the presence of inhibitors of protein synthesis (cycloheximide or puromycin), or of RNA synthesis (actinomycin D), or processing (dichloro-l-D-ribofuranosylbenzimidazole, DRB). Increased production of interferon is observed when the inhibitors of protein synthesis are removed or, if actinomycin D is added, after exposure to the inducer. Superinduction is at least in part due to a prolongation of the half-life of interferon mRNA. This could be related to an inhibition of the production of a repressor of interferon mRNA function; the repressor may normally be made so that interferon synthesis is usually not constitutive. There does appear, however, to be at least one cell line in which interferon synthesis is semiconstitutive; in this case the proposed repressor of interferon production may be defective (Jarvis and Colby, 1978). Gamma interferon production, like that of other lymphokines, is

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generally induced by substances that stimulate a mitogenic response in lymphocytes; these include antigens to which the donor of the lymphocytes had been exposed. The antigens can be viral or nonviral in origin and may be bacterial products such as tetanus and diphtheria toxoids or PPD. Plant-derived mitogens, e.g., phytohemagglutinin A (PHA), pokeweed mitogen (PWM), or concanavalin A (Con A), as well as other mitogenic stimulants, such as staphylococcal enterotoxin A (SEA) or antilymphocyte serum, are also effective in stimulating in vitro gamma interferon production; however, a recent report has demonstrated production of gamma interferon in the absence of cell proliferation (Landolfo et al., 1981). Unfortunately, the purification of gamma interferon has been difficult and has only recently been achieved (Yip et al., 1982). As noted above, lymphocytes produce gamma interferon. T cells produce gamma interferon within 3 days of exposure to mitogens. Interferon production in this system is dependent on the presence of macrophages in the culture so that pure cultures of normal lymphocytes cannot be stimulated to produce gamma interferon; however, lymphocytes depleted of macrophages can still be stimulated by viruses to produce alpha interferons. In addition, certain T cell lines have been shown to be capable of producing gamma interferon in the absence of macrophages. It is not known what factor the macrophages must supply to lymphocytes in order for them to produce gamma interferon, but the type of the interferon is determined by the species of lymphocyte which produce the interferon, not by macrophages in the culture (Youngner, 1977). Ill. Actions of Interferons

1. The Antiviral State

The mechanism of interferon action, for the sake of discussion, may b e divided into two phases (Friedman, 1977).The first relates to how interferon treatment induces an antiviral state or other activities in cells; the second relates to how these induced states are expressed on various cellular activities such as virus growth, cell replication, or the immune response. In order to bring about its effects on cells, interferons must first interact with the plasma membrane (Grollman et al., 1978). The significant reaction is an initial binding of interferons that is not an energy requiring process. The bound interferon can be released from the cell surface without inhibiting the later development of an antiviral state. Interferons seem to bind to a specific cell surface receptor; evidence

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for the specificity of the binding site is the finding that interferon action can be blocked in human cells by an antibody to a product of chromosome 21 (Revel et aE., 1976) or competitively by thyroid stimulating hormone, chorionic gonadotropin, or cholera toxin. Since the last three appear to have a similar or identical binding site, it follows that interferon must bind to the same or very similar site as substances in this general group (Grollman et al., 1978). Current work with purified, radioactive interferons should help to elucidate the nature of the binding reaction and the binding site(s) (Auget, 1980). The location of the putative interferon binding site appears to be on the outer surface of the plasma membrane. This was determined by stimulating human fibroblasts to produce interferon in the presence of antibody to beta interferon. Antiviral activity failed to develop in cells producing interferon; therefore, interferon had to be externalized to induce an intracellular antiviral state (Vengris et al., 1975). The chemical nature of the interferon receptor appears to be a complex of both ganglioside and glycoprotein components (Besancon and Ankel, 1977; Chang et al., 1978). How these interact to bind interferon on the cell surface and transmit information to an intracellular site is not clear, since interferons bind to either gangliosides or glycoprotein components. The glycoprotein component may represent an activation or amplification site for the induction of intracellular antiviral activity (Grollman et al., 1978). Once interferon has interacted with its receptors on the cell surface it is not clear what steps follow immediately. It is uncertain whether interferon is taken up by the cell, although interferons bound to Sepharose beads are active in inducing an antiviral state; however, it is uncertain in such studies how tightly the interferons are bound to the carrier. For the most part the activity of interferons can be accounted for by reactions initiated at the cell surface, but further studies with purified interferons will be necessary to answer definitively the question of whether interferon uptake is required for its biological activities. The great specific activity of interferons suggests that one or fewer molecules per cell can induce an antiviral state.

2 . The Inhibition of Virus Replication The development of antiviral activity following treatment with interferon requires cellular protein and RNA synthesis and in human cells chromosome 21 (Tan et al., 1977). After human interferons are bound to a receptor, biochemical reactions occur. This results in the production of specific mRNA forms which are, in turn, translated to

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give rise to proteins related to the antiviral state. In cultures of interferon-treated, virus-infected cells, viral messenger RNA was not efficiently translated. In some cases where virion-associated transcriptases were present, interferon treatment did not inhibit viral mRNA synthesis. In cell-free protein synthesizing systems derived from mouse cells treated with low concentrations of interferon, there was no inhibition of viral mRNA translation, unless the cells had also been infected with a virus. This suggested that interferon treatment induced a potential antiviral state which was not fully developed until the cells were virus infected (Friedman, 1977). Furthermore, addition of minute quantities of double-stranded RNA to cell-free extracts from interferon-treated cells resulted in the inhibition of virus-directed protein synthesis (Kerr et d.,1974). This might be related to the requirement for viral infection of interferon-treated cells in order to demonstrate an inhibition of translation of viral mRNA, because in many viral infections double-stranded RNA species are produced. Treatment of cell extracts from interferon-treated cells with doublestranded RNA resulted in an increase in the activity of three substances (Fig. 1) that might be related to antiviral activity (Farrell et al., 1978). These are a protein kinase, a series of adenosine polymers having more than two adenosines with a 2'5' linkage, the most important of which in most animal cells is pppA2'p5'A2'p5'AoH (2'5'A for short), and a synthetase capable of producing 2'5'A. The protein kinase, an enzyme activated by double-stranded RNA in interferon-treated cells, phosphorylates the small subunit of the protein synthesis initiation factor eIF-2. This is consistent with several observations strongly suggesting that initiation of viral protein synthesis is inhibited after interferon treatment. The 2'5'A and yet another enzyme, an endoribonuclease, that is usually constitutive, are closely related in the following manner: 2'5' adenylate synthetase, that is induced following interferon treatment, is activated by double-stranded RNA. It uses ATP as a substrate to form 2'5'A polymers that in turn may inhibit virus protein synthesis b y activating the endoribonuclease. The latter may be the active element in inhibition of virus protein synthesis, because it can hydrolyze viral mRNA. Thus, there are at least two ways in which interferon treatment inhibits viral protein synthesis. It is uncertain which if either of these is the more significant in a given virus infection of interferon-treated cells; it is also possible that both contribute to an antiviral state. In fact, rather than being unique antiviral mechanisms, the processes employed in the interferon system may well be adaptations of the normal systems that control cell growth and differentiation. It was not entirely

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ROBERT M. FRIEDMAN AND STEFANIE N. VOGEL ENDONUCLEASE (Inacllwe) ENDONUCLEASE

SYNTHETASE

MET-1RNAf GTP elF-2 40s RIBOSOME COMPLEX

(PHOSPHORYUTED)

405 RIBOSOME

PHOSPHORYIATED (37K)

elF-2 (37K) (PROTEIN SYNTHESIS INITIATION FACTOR)

FIG.1. Double-stranded ribonucleic acid (dsRNA)-related steps in the mechanism of interferon action. In the presence of dsRNA and adenosine triphosphate (ATP), an active protein kinase with a molecular weight of about 67,000 (67K)is induced. The phosphate added to the kinase may be removed by a phosphatase that is inhibited in the presence of dsRNA. The function of the active protein kinase appears to be to add a phosphate group to a subunit with a molecular weight of 37,000 (37K)of protein synthesis initiation factor eIF-2. Ordinarily, eIF-2 acts together with a ribosomal subunit (40 S), initiator transfer RNA (Met-tRNA,), and guanosine triphosphate (GTP) to initiate protein synthesis. In the presence of the phosphosylated 37K subunit of eIF-2, the initiation of protein synthesis is inhibited. In the case of the 2‘,5’-A synthetase, the addition of dsRNA activates the enzyme which forms oligoadenylate polymers (2’,5‘A,,) from ATP. Several “degradases” may inactivate 2‘5‘A,, but, if it is not destroyed, the 2 ’ 5 ’ k interacts with an endonuclease that is present in most cells. The active endonuclease degrades messenger RNA (mRNA). This in turn inhibits protein synthesis by stopping the elongation of proteins. Thus, both of these pathways may converge to inhibit viral protein synthesis.

unexpected, therefore, to find that interferons also have effects on the immune system, and on the growth of uninfected cells (Gordon and Minks, 1981). There are, however, several reports of interferon inhibition of steps in the virus replication cycle other than at the level of virus protein synthesis. This has been especially noted in tumor virus replication. Interferon treatment results in a marked decrease in the production of some oncogenic viruses and in the efficiency of cell transformation by virus, but, while interferons were originally thought to inhibit tumor viruses through the same process as that involved in the inhibition of other viruses, the mechanism of interferon action for at least some of these viruses appears to be more complex. Treatment of cells with

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interferon at various times after infection with SV40 virus yielded different results (Friedman, 1977; Revel, 1979). If interferon was added to cells prior to SV40 infection, virus production and viral T-antigen synthesis were inhibited, effects possibly resulting from blocking the transcription of SV40 DNA molecules by the cell RNA polymerase (Metz et al., 1976). Infection of these cells with SV40 DNA instead of intact virions, however, overcame the antiviral effect of interferon, which suggested that interferon was also inhibiting SV40 uncoating (Yamamoto et uZ., 1975). The latter results, however, are difficult to interpret since the large amount of infectious D N A used in these studies may be analogous to infecting with multiplicities of SV40 high enough to overcome the interferon block (Revel, 1979). Addition of interferon to cells during the early phase of SV40 infection (before viral DNA synthesis), however, failed to inhibit production of SV40 early RNA; but, addition during the late phase of the virus lytic cycle resulted in inhibition of viral protein synthesis at the level of translation with no inhibition of viral mRNA synthesis or of host cell protein synthesis (Revel, 1979). Transcription and translation of the SV40 T-antigen were not sensitive to interferon treatment in SV40transformed cells, although T-antigen production was sensitive during the late phase of the SV40 lytic cycle. Therefore, a single viral gene may be sensitive to interferon in certain phases of virus growth and resistant in others. Similar to SV40-transformed cells, treatment of cells acutely or chronically infected with C-type leukemia or B-type mouse mammary tumor virus (MMTV) viruses resulted in no inhibition of viral RNA or protein synthesis. RNA tumor viruses are, however, sensitive to an interferon mechanism that appears to act at a late stage of virus maturation. In some systems, interferon treatment resulted in marked inhibition of virus release, while in others, virus particle production appeared normal; however, the released virus was deficient in infectivity (Friedman, 1977). The data suggest that interferon may be inhibiting these viruses by altering the membrane through which these viruses are exported out of the cell, or b y altering cellular or viral protein(s) necessary for proper maturation of the virus particle. Studies with cells infected with an adeno-SV40 hybrid virus, that contains a combination of the interferon-sensitive (SV40 virus) and an interferon-insensitive (adenovirus) genome, have yielded interesting findings. I n simultaneous infection of cells with both complete viruses the sensitivity of adenovirus or of SV40 T-antigen production was characteristic of infection with either virus separately. I n contrast, in cells infected with an SV40-adenovirus hybrid, production of both

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T-antigens was as resistant as adenovirus T-antigen production in infection with adenovirus alone (Oxman et aZ., 1967). The SV40 genome is covalently linked to the adenovirus genome in the hybrid or to cellular DNA in SV40-transformed cells. The mRNA produced by the. integrated SV40 genome contained host sequences, and the mRNA of the hybrid had adenovirus and SV40 sequences. The resistance to interferon treatment of SV40 T-antigen production directed by an integrated viral genome may indicate that the primary sequence in the mRNA that specifies the viral protein does not determine sensitivity to interferon, but that other sites on the genome such as those concerned with initiation or control of genetic expression may be the loci of interferon action. This may also explain the lack of interferon-induced inhibition of murine RNA tumor virus protein production in chronically infected cells where the proviral DNA is integrated into host DNA. In experimental animal systems with virally induced tumors, such as hamsters inoculated with pol yoma virus or chickens infected with Rous sarcoma virus, interferon may inhibit development of tumors by inhibiting virus multiplication or an early virus-dependent step involved in cell transformation. It is unlikely, however, that the antiviral activities of interferons are responsible for inhibition of tumors that are apparently not virus-induced, or tumors in which virus replication is not involved in the progression of development. Interferons may inhibit replication of the tumor cell itself or may have effects on the host’s capacity for tumor rejection. For example, L1210 leukemia cells, that were resistant to the cell growth inhibition activity of interferons in uitro, could be inhibited in viuo. This suggested that the antitumor effect of interferons in these mice was not a result of direct inhibition of tumor cell multiplication and might be the result of interferon action on the immune system (Gresser et aZ., 1972). On the other hand, human interferon is effective in inhibiting the growth of human tumor transplants in nude mice. This suggested a direct antitumor effect of interferons (Taylor-Papadimitriou, 1980).

3. Znhibition of CeZZ Proliferation Since purified interferons inhibit both virus replication and cell multiplication (Knight, 1976; Gresser et aZ., 1979), it is now accepted that interferons inhibit the growth of a wide range of cell types. The sensitivity of cells to the growth inhibitory effects of interferons ranges from very sensitive to resistant and the same cell types can show varying sensitivities under varying assay conditions: for instance, growth of colonies in agar is more sensitive than growth on a solid support and

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sparsely seeded cultures appear to be more sensitive than the same cells seeded at high densities (Taylor-Papadimitriou, 1980). Tumor cells may be more sensitive to the growth inhibitory effects of interferons than are normal cells. The multiplication of HeLa cells was inhibited to a greater extent than that of human fibroblasts (Friedman, 1977); similarly, the inhibition of the multiplication of human osteosarcoma cells was greater than that of nontumor cells (Strander and Einhorn, 1977). In contrast, the multiplication of oncornavirus carrier cells (Billiau, 1975), or of X-ray transformed cells (Brouty-Boye et al., 1979) derived from C3H fibroblasts was less inhibited than that of nontransformed cells. Moreover, in the comparison of normal human mammary epithelial cells to breast cancer cells, or of 3T3 cells to SV40-transformed 3T3 cells, the normal cells were at least as sensitive to the growth inhibitory effects as the analogous transformed cells (Balkwill et al., 1978). There are also studies that suggest similar effects in uiuo. Interferons inhibited the multiplication of tumor cells and normal cells in animals (Gresser and Bourali, 1970b), of allogeneic lymphocytes and syngeneic bone marrow cells, when these were transferred into irradiated mice (Cerottini et al., 1973), and of regenerating liver cells in partially hepatectomized mice (Frayssinet et al., 1973). There are many possible sites at which interferons could inhibit the complex process of cell multiplication. Different approaches are being used to examine aspects of control of cell growth and what effects interferons have on these processes. These include (1)examination of interferon’s effects on the cell cycle; (2) study of cellular functions that may be involved in control of cell growth or cellular parameters that are altered in malignant cells; and (3) determination of whether the molecular mechanisms thought to be implicated in interferon’s antiviral activities play any role in its antiproliferative activities.

4 . Znterferons and the Cell Cycle Interferons do not arrest cells that are dividing asynchronously but synchronizes them into one phase of the cell cycle. Interferon treatment reduced the rate of entry into S phase and increased the duration of the G, and S + G2 phases (Balkwill and Taylor-Papadimitriou, 1978), so that the increased length of cell cycle time observed in treated cultures (Collyn d‘Hooghe et al., 1977) is probably due to the extension of these phases. Quiescent cells that can be stimulated to divide synchronously by mitogens provide an excellent system to study how interferon affect the events in G, crucial to the initiation of DNA synthesis. The events that occur after stimulation of cells with mitogens, but precede DNA synthesis, can be divided into “early” and “late.”

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Early events occur within minutes of mitogen stimulation, they are not dependent on cellular protein synthesis, and they include changes in intracellular CAMP levels and increased uptake of ions, nucleotides, and sugars. Late events, that occur hours after mitogen stimulation, are protein synthesis dependent; they include secondary increases in sugar and ion uptake and increases in the activities of certain enzymes. One of these enzymes, ornithine decarboxylase (ODC), catalyzes the first rate-limiting step in the synthesis of polyamines, that are involved in the regulation of various cellular reactions, including transcription and translation. Increases in ODC activity are associated with the proliferative response of cells in culture, in tumors, and also with tumor promotion (Janne et al., 1978). Interferon treatment of quiescent Swiss 3T3 cells at the time of mitogen stimulation had no effect on the early increase in uptake of ions, nucleosides, or sugars; however, it had a differential effect on protein synthesis-dependent events: induction of ODC activity was inhibited, while the second phase of stimulation of 2-deoxyglucose uptake was not affected. These results were observed with addition of serum, of a combination of growth factors, or of a tumor promoter serving as a mitogen (Sreevalsan et al., 1979, 1980). Similar findings were recently reported on the inhibitory effect of interferon on the induction of S-adenosyl-L-methionine decarboxylase, another enzyme involved in polyamine biosynthesis (Lee and Sreevalsan, 1981). There seems then to be a common interferon-sensitive' step involved in the stimulation of DNA synthesis by serum, tumor promoters, or growth factors. Further evidence for an interferon-sensitive step in DNA synthesis comes from the study of two clones of Swiss 3T3 cells with differential sensitivities to both the antiviral and antiproliferative activities of interferons. One clone was more sensitive to interferon in terms of inhibition of cell division, DNA synthesis, and induction of ODC activity, when interferon was added at the time of serum stimulation. Under the same conditions, NIH 3T3 cells sensitive to the antiviral effect of interferon against murine leukemia virus exhibited no inhibition of cell division, DNA synthesis, or ODC induction (Czarniecki et al., 1981). There is, however, evidence that interferoninduced inhibition of DNA synthesis is not dependent on the inhibition of ODC activation also caused by interferon. Concomitant inhibition of DNA synthesis and of activation of the enzyme was observed only when polypeptide hormones were used as stimulants; for instance, cholera toxin-stimulated DNA synthesis was inhibited, while toxin-stimulated ODC activation was not (Lee et al., 1980). These results indicated that a poor correlation exists between the

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activation of ODC and DNA synthesis in quiescent 3T3 cells, that are stimulated to proliferate.

5. Eflects of Interferons on Other Cellular Parameters Information concerning the many varied effects of interferons on cell structure and function is increasing rapidly (for reviews see Gresser, 1977; Stewart, 1979; Taylor-Papadimitriou, 1980). Interferon treatment of cells resulted in significant alterations of the cell surface including increased expression of certain cell surface antigens or receptors (see below), increased net negative charge on the cell surface (Knight and Korant, 1977), decreased thymidine uptake (Brouty-Boye and Tovey, 1977), and alteration in the density of the plasma membrane (Chang et al., 1978). Interferon treated cells also exhibited changes in the binding of cholera toxin and thyrotropin (Kohn et al., 1976). Alterations in the cell membrane resulting from interferon treatment may play a role in the inhibition of murine leukemia viruses discussed earlier. Additionally, SV40 transformed cells that normally produce and release plasminogen activator (PA) seem to accumulate PA at the plasma membrane after interferon treatment (Schroeder et ul., 1978), so that interferon might alter the cell surface in a manner that prevents C-type virus shedding. There is, however, an increase in secretion of plasminogen activator after exposure of human macrophages to leukocyte interferon (Hovi et al., 1981). Since cell to cell contact plays a role in cell growth regulation, alterations induced in the plasma membrane could well cause alterations in cell DNA synthesis and growth. A direct negative effect on cell growth would, of course, be an ideal mechanism of action for an antitumor agent. In many respects interferons would seem to be growth control factors; but, they are an unusual class of biological substances, since almost all growth factors that have been studied stimulate cell replication. Indeed, many of the “toxic” effects observed in interferon therapy, such as leukopenia, thrombocytopenia, and hair loss, may be extensions of its growth inhibitory properties. Changes in the cytoskeIeton, composed of microtubules, microfilaments (actin and myosin), and the cytoplasmic matrix, have been observed in conjunction with transformation. Such changes may be a cause of or result in transformation. The cytoskeleton may be altered by interferon treatment so that microfilament and fibronectin organization are changed and there is increased rigidity of the plasma membrane lipid bilayer (Pfeffer et al., 1979, 1980a,b). Drugs such as colchicine, that disrupt the cytoskeleton, inhibit the development of the

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antiviral action of interferon, while compounds such as sodium butyrate, which has been reported to promote cytoskeletal organization, appear to enhance interferon action (Taylor-Papadimitriou, 1980). The antiviral activities of interferons were shown to be potentiated by dibutyryl-CAMP (Friedman and Pastan, 1969). Interferon-treated cells contained increased levels of cAMP (Weber and Stewart, 1975; Meldolesi et al., 1977). Since it appeared that increased levels of cAMP might be involved with inhibiting cell growth rates of several systems (Rozengurt, 1979), it was thought that membrane adenylate cyclase activities might play a role in the antiproliferative response to interferons; however, a longer exposure to interferon was necessary for alteration of cAMP levels than for detection of cell growth inhibition (Tovey et al., 1979). Additionally, growth of Schwann cells, human keratinocytes, and human mammary epithelial cells is stimulated by both cholera toxin and cAMP analogs (Taylor-Papadimitriou, 1980); therefore, the relationship between interferons and cAMP appears to be more complex than originally suggested.

6. Antiviral and Antiproliferative Activities of Interferons One important question is whether the proteins induced by interferon treatment are involved in interferon’s cell growth inhibitory activity. In one report, mouse embryonal carcinoma stem cells that were insensitive to the antiviral and antiproliferative effects of interferon did not demonstrate kinase induction after interferon treatment. After differentiation, kinase activity was induced and growth of these cells was inhibited (Wood and Hovanessian, 1979); however, many changes occur within a cell upon differentiation, and it is difficult to assign responsibility for acquired sensitivity to any one of these changes. The role of the synthetase in these antiproliferative effects has been studied by examining the inhibitory effects of 2’51 directly on cell protein synthesis (Williams and Kerr, 1978; Hovenessian et al., 1979). The dephosphorylated trimer, that can apparently pass through the cell membrane, has also been shown to inhibit DNA synthesis in lymphocytes stimulated by lectins (Kimchi et al., 1979). NIH 3T3 cells were sensitive to the antiviral effects of interferon against murine leukemia virus; however interferon treatment resulted in no antiviral activity against a lytic virus such as encephalomyocarditis virus (EMC), and no inhibition of cell division, or DNA synthesis. Both synthetase and kinase activities were induced by interferon but the endonuclease ordinarily activated by 2’5’ oligoadenosine appeared to be absent. The results suggested that kinase activity is not sufficient for cell growth inhibition, and also indicated a possible role of the 2’5’A activated

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pathway in the antiproliferative activities of interferons (Epstein et al.,

1981).

7 . Effects of Interferons on the Immune System. Effects of Interferons on the Humoral Immune System The immunoregulatory functions of interferons were recognized only within the past decade. The effects of interferons on the production of antibodies were among the first examined. Interferons can have both suppressive or enhancing effects on the production of antibodies, depending upon the dose and time of administration of interferon relative to antigenic challenge. Early in vivo studies indicated that when low doses of interferon were administered simultaneously with sheep erythrocytes (SRBC), an augmented antibody response was seen; however, if high doses were administered with the antigen, the antibody response was depressed (Braun and Levy, 1972). Subsequently, it was observed that when interferon was administered to an animal prior to antigenic challenge, both the primary antibody response (Chester et al., 1973; Merigan et al., 1975) as well as the induction of an anamnestic response (Brodeur and Merigan, 1975) were suppressed. The antigens used in these various studies were shown to fall in both T-dependent (i.e., SRBC) and T-independent (i.e., lipopolysaccharide) classes. The results of these in vivo studies were confirmed and extended in vitro. Gisler et al. (1974) and Johnson et al. (1974, 1975) demonstrated interferon-mediated suppression of the antibody response to SRBC in vitro when the interferon was added simultaneously with the antigen, but found an augmentation of the antibody response if added to the cultures 2-4 days after the antigenic stimulus. Pretreatment of purified B cells with interferon and the subsequent co-culture of these cells with T cells, macrophages, and antigen, resulted in significant inhibition of antibody production (Gisler et al., 1974), suggesting a direct suppressive effect on B cells. Interferon was found to reduce dramatically the number of clones that proliferate in response to antigen (Booth et al., 1976a,b).The in vivo findings were further confirmed in vitro using the T-independent antigen, lipopolysaccharide (LPS), as the antigenic stimulus (Johnson et al., 1975), although the T-dependent anti-SRBC response was found to be more readily induced by interferon (Johnson and Baron, 1976; Johnson, 1977). The finding that the T-dependent, B cell antibody response was more sensitive to suppression by interferon than the T-independent response to LPS suggests differences in the mechanisms by which each antigen initiates proliferation. Recent findings by Attallah et al. (1980), that

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interferon fails to suppress pokeweed mitogen-driven, human B cells to produce plaque-forming cells or to secrete antibody, suggest that polyclonal activiation may overcome the antiproliferative effects of interferon on B cells. Gamma (Type I1 or immune) interferon was subsequently tested for its effects on the antibody response. It is important to remember that until very recently, all gamma interferon preparations contained a mixture of a number of lymphokines. Several different groups have demonstrated that gamma interferon also suppresses the in vitro and in vivo immune response to SRBC (Virelizier et al., 1977; Sonnenfeld et al., 1977; Lucero et al., 1980). Two of these reports found that on the basis of antiviral activity, gamma interferon was significantly (20-250 times) more suppressive than preparations of alpha or beta interferons (Virelizier et al., 1977; Sonnenfeld et al., 1977). As observed for alpha and beta interferon preparations, gamma containing preparations also enhanced the production of anti-SRBC forming cells if administered in vivo or in vitro 48 hours after antigen stimulation (Sonnenfeld et al., 1978). The in vitro response to LPS was also inhibited by gamma interferon-containing preparations 24 hours prior to antigen; however, the gamma preparations failed to augment the PFC response when added to LPS-stimulated cultures at 48 hours. These findings (1) strongly support the role of gamma interferon as an immunoregulatory lymphokine, and (2) are consistent with the hypothesis that interferons suppress the antibody response b y exerting antiproliferative effects on those B cells which would normally be proliferative in response to antigen, but augment the immune response to antigen later by inhibiting the proliferation of suppressor T lymphocytes. The suppressive effects of interferons on the production of plaqueforming cells are mimicked by the addition to cultures of oxidized glutathione, and the effects of both are reversed by the addition of sulfhydryl reducing agents, such as 2-mercaptoethanol (2-ME). p-Hydroxymercuribenzene sulfonic acid binds sulfhydryl groups only at the cell surface and its suppressive effects on the antibody response are not inhibited by 2-ME (Johnson, 1980). Johnson (1980) proposed that interferons suppress by binding to sulfhydryl groups on the cell surface, or by functioning as a thiol-oxidizing agent. Since both interferon- and oxidized glutathione-treated cells or cell lysates possess a ribosome-associated protein kinase that may inhibit protein synthesis (Farrell et al., 1978), inhibition of protein synthesis may underlie the inhibition of B cell proliferation, or provide a second mechanism leading to the depression of antibody synthesis. The ability of interferons to modulate the antibody response of

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human cells has only recently been addressed (Parker et al., 1981). When human peripheral blood lymphocytes were stimulated with a preparation of leukocyte interferon, a rather different pattern from that seen in murine cultures was observed. In contrast to murine cultures, where interferon pretreatment suppressed the production of antibody, interferon pretreatment of human cells stimulated the plaque-forming cell response (PFC) to a T-dependent antigen; however, simultaneous addition of interferon and antigen to the cultures led to a suppression of the PFC response. The kinetics of antibody production or the ratio of T to B cells in the human vs murine cultures might underlie the apparent differences following interferon treatment; however, until these studies are verified using highly purified interferon preparations (the preparation used in this study was <1% pure), one cannot overlook the possible effects of contaminating factors. Interferons also exert an immunoregulatory effect on the IgE system. Treatment of murine spleen cells from sensitized animals with interferon resulted in a decreased ability to transfer cutaneous anaphylaxis (Ngan et al., 1976). Interferon was also found to increase the release of histamine from basophils after exposure to ragweed antigen or to antiIgE antibodies (Ida et al., 1977). Interferon enhancement of IgEmediated histamine release from human basophils was found to require a 6-9 hour induction period and new RNA synthesis (Hernandez-Asensio e t al., 1979). Hooks et al. (1980) demonstrated that the addition of infectious or UV-inactivated viruses to human leukocyte cultures facilitated histamine release, when the cultures were subsequently exposed to anti-IgE. These authors demonstrated a temporal relationship between the augmentation of histamine release and the induction of interferon in the cultures. They further demonstrated the efficacy of gamma interferon containing preparations in the augmentation of histamine release. It is in this context that interferon has been ascribed a potential role in the development of asthma during upper respiratory tract infections (Hooks et al., 1980; Sonnenfeld, 1980). Additionally, interferons may serve to induce other cofactors associated with the reagenic response, such as the IgE binding factors (Yodoi et al., 1981). Perhaps one of the most exciting observations relating to the role of interferons in the regulation of humoral immunity resides in the recent findings of Hooks et al. (1979, 1980) and Preble et al. (1981, 1982). These investigators demonstrated that the levels of interferon in sera of patients with autoimmune disorders (systemic lupus erythematosus, rheumatoid arthritis, scleroderma, Sjogren’s syndrome) were significantly elevated. During periods when the disease was clinically inac-

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tive, serum interferon levels were generally reduced. Based on the sensitivity of interferon from patients with autoimmune disease to pH 2 treatment, this interferon was characterized as type gamma. A recent report by Preble et al. (1982) indicates that, although the interferon of patients with autoimmune disease is pH 2-labile7both monospecific and monoclonal antibodies to alpha interferon were efficacious in neutralizing the interferon of SLE patients. This suggests that the interferon detected in the sera of autoimmune patients is an unusual form of alpha interferon. It is of interest that Heremans et al. (1978) found that interferon treatment of NZB/NZW mice accelerated the progression of autoimmune manifestations. Finally, the finding that strains of mice that spontaneously develop autoimmune disorders also have circulating titers of alpha interferon supports the notion that interferon may play an important role in the pathophysiology of some autoimmune diseases (Friedman et al., unpublished observations).

8 . Effects of Znterferons on Cellular Zmmune Responses. Znterferon Modulat~onof Surface Markers of Cells of the Immune System It is likely that many of the observed effects of interferon on the cellular immune system stem from interferon-induced changes in membrane-associated antigens and receptors on lymphoid cell types. These changes may be analogous to the previously discussed interferon-induced changes in the cell surface. Interferon has been demonstrated to augment in vitro the expression of histocompatibility antigens on murine thymocytes (Lindahl et al., 1974; Lonai and Steinman, 1977; Sonnenfeld and Merigan, 1979), and splenic lymphocytes from young mice (Lindahl et al., 1974; Lonai and Steinman, 1977) at concentrations between 500 and 4000 U/ml of alpha and beta interferon. Substantially higher concentrations (30,000 U/ml) of these interferons were required to induce Ia and Lyt 1,2, and 3 antigens on thymocytes (Sonnenfeld and Merigan, 1979). No increase in theta antigen was observed upon treatment with alpha and beta interferon preparations (Lindahl et al., 1974; Lonai and Steinman, 1977; Sonnenfeld and Merigan, 1979). Gamma interferon-containing preparations were found to induce histocompatibility antigens at significantly lower concentrations of interferon (3-30 U/ml). Similarly, the induction of Ia and Lyt antigens, as well as theta, was inducible with 500-1000 Ulml (Sonnenfeld and Merigan, 1979). These findings are consistent with the observation that gamma interferon was more efficacious in suppressing the antibody response than alpha and beta preparations. Interferon was also found to enhance the binding of branched synthetic polypeptide antigens to Ly2+ T cells (Lonai and Steinman, 1977). Re-

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cently, Hokland et al. (1981) confirmed the interferon-mediated expression of &-microglobulin and histocompatibility antigens (HLA-A, B, and C) on human peripheral blood lymphocytes previously reported for other systems (Heron et al., 1978; Fellous et al., 1979; Attallah and Strong, 1979). They showed that pure interferons elicited the same effect, thus demonstrating that interferon is the substance responsible for the expression of these antigens in partially purified preparations. In contrast, interferon was found not to augment Ia antigen expression (Attallah and Strong, 1979). Stimulation of human lymphocyte preparations with beta interferon (2-20 U/ml) was shown to result in a dose-dependent increase in IgG Fc receptor (FcR) binding capacity, as assessed by rosette formation with IgG-coated ox erythrocytes; however, the ability of these cells to bind IgM-coated erythrocytes via the IgM Fc receptor was decreased upon exposure to interferon (Itoh et al., 1980). Similar results were obtained by Fridman et al. (1980). These investigators found that partially purified or electrophoretically pure mouse interferon induced an increase in IgG Fc receptors in the murine T2D4 cell line (derived from a T cell hybrid line). They also found that human leukocyte interferon enhanced the expression of this receptor on human Burkitt cells, but did not affect its expression on mouse cells. These findings were subsequently confirmed in vivo (Aguet et al., 1981). When partially or highly purified virus-induced interferons were administered to C3H mice, an increase in IgG Fc receptors was detectable in splenic lymphocytes, mesenteric lymph node cells, and thymocytes from cortisone-treated mice. Interferon-enhanced IgG Fc receptor (FcR) binding by murine macrophages has been demonstrated by Vogel et al. (1982) by measuring binding of Wr-labeled, opsonized (rabbit IgG) sheep erythrocytes to macrophages cultures. As was observed for the induction of thymocyte membrane antigens, gamma interferon preparations were found to be considerably more efficacious (by approximately 33-fold) in the induction of FcR. Interferon was also found to augment macrophage binding capacity of Wr-labeled sheep erythrocytes opsonized with either monoclonal IgG2, or IgGZbopsonins, suggesting an increase in expression of both types of Fc receptors. In a recent abstract, Steeg et al. (1981) reported that enhancement of Ia antigens on murine peritoneal macrophages was interferon-mediated. Interferon has also been used to enhance the binding of concanavalin A to L1210 leukemia cells (Huet et al., 1974). Finally, interferon treatment of cultured human melanoma cells resulted in enhanced expression of HLA-A, HLA-B, and p,-microglobulin antigens but did not augment the expression of melanoma associated antigens or Ia-like antigens (Imai et al., 1981).

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These findings support the role of interferon as a major regulator of surface antigen and receptor expression.

9. Interferon-Induced Modulation of Macrophage Functions

.

One of the earliest recognized actions of interferons relate to their ability to enhance phagocytic activity. The enhancement of phagocytosis by interferon is due to an increase in both the number of particles ingested per macrophage and the number of macrophages engaged in phagocytosis. Inducers of alpha and beta interferon administered in uiuo or in vitro or preparations known to contain heat labile, pH 2 stable antiviral activity stimulated macrophage uptake of colloidal carbon, latex beads, Escherichia coli, and opsonized erythrocytes (Huang et al., 1971; Donahoe and Huang, 1973,1976; Imanishi et al., 1975; Huang, 1977; Hamburg et al., 1978, 1980a,b; Manejias et al., 1978; Degre and Rollag, 1980; Vogel et al., 1982). Direct comparison of the various systems utilized to examine interferon-induced enhancement of phagocytosis in vitro is especially difficult, since experimental conditions such as the source of macrophages (strain of animal, elicited or nonelicited), culture conditions, and quality of interferon preparations differ greatly from study to study. The augmentation of ingestion of substances such as latex beads or colloidal carbon particles would indicate that interferons increase the rate and/or the capacity of phagocytic cells to ingest small particles; however, in the case of Fc-mediated phagocytosis, it is likely that several mechanisms contribute to the increase in particle uptake. Interferon-induced enhancement of Fc-mediated phagocytosis requires new RNA and protein synthesis (Hamburg et al., 1980b). Wang et al. (1981) demonstrated that opsonized erythrocytes, bound by thioglycollateelicited macrophages at 4"C, were ingested 2-3 times more rapidly in interferon-treated cultures (1000-5000 U/ml beta interferon) in the 2 minute interval after raising the temperature to 37°C. In contrast to the findings of others, these investigators observed a concomitant suppression of pinocytosis. Using resident peritoneal macrophages, Hamburg et al. (1980b) suggested that the interferon-induced increase in Fcmediated phagocytosis was also related to an increase in efficiency of uptake, rather than increased Fc receptor expression, since rabiolabeled monoclonal anti-IgGZbreceptor antibody failed to bind preferentially to IFN-treated cultures. These authors recognized the possibility that the anti-Fc receptor antibody used in this study may not have recognized a determinant expressed by Fc receptors, either newly synthesized or exposed after interferon treatment; however, using macrophages derived from the C3H/HeJ mouse strain, that lose

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their ability to bind and phagocytose opsonized sheep erythrocytes with time in culture, Vogel et al. (1982) demonstrated that interferon treatment (beta or gamma interferon) of these cells resulted in a parallel enhancement of Fc receptor-mediated binding and phagocytosis of opsonized sheep erythrocytes. The findings of Guyre et al. (1981) which demonstrated by Scatchard analysis increased numbers of IgG, Fc receptors in human monocyte cultures or human macrophage cell lines treated with gamma interferon-rich supernatants, support the notion that IFN-augmented Fc receptor expression contributes to augmented Fc receptor functions such as phagocytosis. It is interesting to note that in both human lymphocytes and human polymorphonuclear leukocytes, interferon-induced enhancement of Fc receptor binding capacity is accompanied by an enhancement of antibody-dependent cellular cytotoxicity (ADCC) (Itoh et al., 1980; Hokland and Berg, 1981). Interferon has also been reported to augment another macrophage function associated with an increased state of differentiation, spreading in culture. Macrophages derived from mice administered interferon inducers in vivo (Rabinovitch et al., 1977) or macrophages treated in oitro with interferon or interferon inducers (Schultz e t al., 1977) appear well spread in vitro, with marked granulation of the cytoplasm in comparison to the rounded, refractile appearance of untreated macrophages. The increase in spreading was accompanied by an increase in the cytoplasmic concentration of the enzyme lactate dehydrogenase (Schultz, 1980). Such morphologic changes may be related to previously discussed alterations in the cytoskeleton induced by interferon treatment (Pfeffer et al., 1979). The role of interferon as a macrophage activating agent is not limited to early manifestations of macrophage differentiation, such as increased spreading or phagocytic activity. Rather, the interferon-treated macrophage has been demonstrated to be capable of nonspecific tumor cell killing (Schultz et al., 1977, 1978; Schultz and Chirigos, 1978; Taramelli et al., 1981; Boraschi and Tagliabue, 1981). The impetus for recognition of an interferon-induced, host-mediated defense stemmed from the findings that interferons exerted antitumor action in a number of animal models (reviewed in Chirigos, 1977, and Schultz, 1980). The tumoricidal effect was not due to a direct action of interferon on the tumor cells, but rather, was host-mediated (Gresser et al., 1972). Using the interferon inducers poly(1: C) and dextran sulfate, Schultz et al. (1977) demonstrated that treatment of murine macrophages in vitro led to the killing of interferon insensitive MBL-2 leukemia cells. Cell to cell contact between the macrophage and tumor cell appeared to be

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essential for tumor cell killing. Other polyanionic compounds were also found to activate the macrophage to a tumoricidal state (Schultz et al., 1977). These authors also found that affinity purified, murine beta interferon (2 x lo7 U/mg specific activity) rendered the macrophages cytotoxic. The maximum inhibition of tumor cell growth was observed at concentrations between 103 and 104 U/ml, with significant killing detectable at 100 U/ml. Since these findings were reported, there has been a great deal of speculation as to the relationship between interferons (and specifically, the lymphokine, gamma interferon) and macrophage activating factor (MAF),the lymphokine(s) previously demonstrated by a number of investigators to activate macrophages to a tumoricidal state (Hibbs et aZ., 1977; Russell et al., 1977; Ruco and Meltzer, 1978; Weinberg et al., 1978). Most of these studies proposed that MAF provides an essential differentiative signal to the macrophage, but only in the presence of endotoxin will complete activation occur. Several lines of evidence would suggest that these two entities differ chemically and activate macrophages by different mechanisms. First, Leonard et al. (1978) has described MAF as heat labile at 56°C for 30 minutes. In contrast, gamma interferon, which is also produced under the conditions these authors use to produce MAF, is heat stable (Youngner and Salvin, 1973). Taramelli et al. (1981) examined M A F and interferon-mediated activation in C3H/HeJ and A/J macrophages. These cells possess defects in macrophage activation (reviewed by Vogel et al., 1981; Vogel and Rosenstreich, 1981) and fail to become tumoricidal in response to MAF; however, both interferon and poly(1: C) were capable of activating these macrophages to tumor cytotoxicity, but this required a longer exposure to these agents than normal C3H/HeN macrophages (10- 12 vs 2 hours). Boraschi and Tagliabue (1981) also found differences between MAF preparations and partially purified beta interferon preparations in their ability to provide sequential activation signals to macrophages from normal C3H/HeN mice and C3H/HeJ mice. These authors found that M A F could provide both “priming” and “triggering” signals to the normal macrophages and only the “priming” signal to the C3H/HeJ macrophage. In contrast, interferon could “prime” the normal macrophages, but not C3H/HeJ macrophages, for tumor cytotoxicity. Furthermore, interferon was unable to provide the “second signal” to MAF-primed C3H/HeN macrophages. In addition, these authors reported no correlation between antiviral activity and “MAF” activity in their lymphokine preparations. The elucidation of the functional and biochemical differences between M A F and interferons may ulti-

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mately provide a rational approach for differential antitumor therapy using these different cytokines.

10. E8ect.s of Interferons on Cell-Mediated Immunity A role for interferons in the regulation of cell-mediated immunity has been recognized for some time (reviewed by DeMaeyer and DeMaeyer-Guignard, 1977); however, the specific mechanisms by which interferons exert their varied effects are far from being elucidated. One of the earliest recognized and most striking effects of interferon on cell-mediated immune responses in vivo was the demonstration that administration of interferon inducers or interferon-rich culture supernatants significantly delayed the rejection of skin grafts across major or minor histocompatibility differences in mice (Mobraaten et al., 1973; Hirsch et al., 1974). Consistent with these findings, it was subsequently demonstrated that administration of interferon inducers or interferon-rich preparations to mice previously sensitized with picryl chloride or sheep erythrocytes resulted in inhibition of delayed type hypersensitivity (ear or foodpad swelling) upon challenge with the sensitizing antigen (DeMaeyer et al., 1975; DeMaeyer-Guignard et al., 1975). Interferon not only inhibited the delayed type hypersensitivity reaction upon challenge, but also depressed sensitization when administered prior to the sensitizing injection (DeMaeyer-Guignard et al., 1975; Gresser et al., 1979). Tilorone (an interferon inducer in mice) administration inhibited the cellmediated response to a number of intracellular parasites as well as to sheep erythrocytes (Collins, 1980). As there are many examples of depressed cell-mediated immunity following virus infection (Howard et al., 1969; Notkins et al., 1970; Mortensen et al., 1973),these findings strongly implicated interferons as suppressive agents. The immunoregulatory effects of interferons on delayed-type hypersensitivity have been further examined by DeMaeyer and DeMaeyer-Guignard (1980). The timing of administration of interferon was found to be critical with regard to the modulation of delayed hypersensitivity to SRBC. As was observed for the modulation of antibody synthesis (see above), administration of interferon prior to sensitization with antigen led to a depression of the cell-mediated response to SRBC; however, when administered after the sensitizing antigen, an augmentation could be demonstrated. The dosage of antigen was also critical, so that interferon-induced enhancement of the cell-mediated response occurred only when suboptimal or nonsensitizing doses of antigen were used. Lastly, interferon modulation of delayed hypersensitivity was

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affected by the host genotype. For instance, BALB/c mice were more sensitive to the immunosuppressive effects of interferons than C57BL/6 mice, wherease the immunoenhancing effect was more marked in the C57BL/6 strain. Thus, the inherent sensitivity of the immune system to interferons may profoundly control the resultant effect of an interferon on delayed type hypersensitivity responses. Interferons have also been shown to modulate a number of in vitro responses felt to reflect in uiuo cell-mediated immunity. LindahlMagnusson et al. (1972) demonstrated that interferon depressed the proliferative responses of lymphocytes to the T cell mitogen, phytohemagglutinin (PHA), or to an allogeneic stimulus. Subsequently, it was demonstrated that the T cell response to concanavalin A (Con A) was also depressed by in vitro exposure to interferon (Rozee et al., 1973) and that in vivo administration of interferon led to the depression of proliferation in response to PHA or Con A in uitro (Brodeur and Merigan, 1975). Evidence provided by Kadish et al. (1980) strongly suggests that interferon mediates the suppression exhibited by Con A-induced human suppressor cells, including the response of normal lymphocytes to Con A. Taken collectively, these findings suggest that interferons can exert on T lymphocytes a strong antiproliferative effect analogous to that observed for B lymphocytes (seeabove). It is interesting to note that Dinh et a2. (1980)demonstrated that exogenous interferon enhanced or depressed the production of lymphokines by Con A-stimulated human leukocytes, depending on the concentration of interferon added to the cultures. Since the manifestations of delayed type hypersensitivity are closely associated with the production of lymphokines (reviewed by Oppenheim, 1981), this might provide an underlying mechanism for the observed effect of interferon on delayed type hypersensitivity responses in uiuo and in uitro. One of the more puzzling aspects of the findings presented thus far is an apparent paradox: interferons induce in murine T lymphocytes increased expression of H-2 antigens (Lindahl et al., 1974; Lonai and Steinman, 1977; Sonnenfeld and Merigan, 1980), yet interferon treatment results in prolonged allograft survival across H-2 barriers (Hirsch et al., 1974) and depressed proliferation in one-way mixed leukocyte reactions (MLR) in vitro (Lindahl-Magnusson et al,, 1972). In fact, the immunoregulatory role of interferon in the MLR and the concurrent generation of cytotoxic T lymphocytes (CTLs) is an area of active and intensive investigation. During the course of a mixed leukocyte reaction i n oitro, interferon activity is generated in the resulting culture supernatants (Giffordet al., 1971; Virelizier et al., 1977). These find-

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ings have since been confirmed by many others; gamma interferon appears to be the predominant species produced (Virelizier et al., 1977).Landolfo et al. (1981)demonstrated that differences at the K, D, or I-S-G regions of the H-2 complex stimulated the production of gamma interferon in mixed leukocyte cultures (MLC); however, differences at the I-S-G regions were more stimulatory. When mino! histocompatibility (Le., M l s locus) existed in the MLC, gamma interferon was also produced, even in the absence of proliferation. In addition, various preparations of interferon have been shown to augment the generation of CTLs (Lindahl et al., 1972; Heron et al., 1976; Simon et

al., 1979). These findings led investigators to hypothesize that interferons provide an essential signal in the generation of cytolytic effector cells; however, the precise role of interferons in the generation of CTLs and the requirement for additional cytokine signals is not yet understood. Farrar et al. (1981) found that partially purified, gamma interferonfree, interleukin 2-rich (IL 2) preparations could induce an MLR in cultures that had been lymphocyte-enriched by Sephadex G- 10 and nylon wool column purifications. These cultures were called “macrophage depleted,” although a number of investigators have found that they may contain small numbers of potentially contributory residual macrophages (reviewed by Weinblatt et al., 1981).The B cell enriched stimulator population was not irradiated. In the “macrophage depleted” MLC, the IL 2-rich preparation was required to induce proliferation and the generation of CTLs. Both the proliferative response and the generation of CTLs were blocked by the presence of a rabbit heteroantiserum previously demonstrated to have specificity for gamma interferon (Osbourne et a,?., 1980). These findings strongly suggested that IL 2 provides a signal for the generation of CTLs and that the development of this effector function is dependent upon the production of gamma interferon; however, several other recent reports have suggested that lymphocyte and macrophage factors other than IL 2 and interferon contribute to the in vitro allogenic response (Hsu et al., 1981; Finke et al., 1981; Reddehase et al., 1982).In the studies of Hsu et al. (1981) and Finke et al. (1981), the allogeneic stimulator cells were heated at 45°C for 1 hour in lieu of UV irradiation. Although the viability and alloantigens of the stimulator cells remained intact, this treatment was thought to block the release of factors required for the MLR. As in the “macrophage depleted” system of Farrar et al. (1981), CTLs were generated by adding to the cultures cytokines derived in vitro from mice previously infected with L. monocytogenes. Preparations depleted of interferons (alpha, beta, or gamma) were still effica-

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cious in generating CTLs (Hsu e t al., 1981). Finke et al. (1981) subsequently demonstrated that both T cell and macrophage products distinct from IL 2 and interleukin 1(IL 1, a macrophage-derived inducer of IL 2; Farrar et al., 1980) could participate in the induction of CTLs. Purified IL 2 was not active. These findings do not preclude a role for gamma interferon in the generation of CTLs, but suggest that cytokines in addition to IL 2 participate in the induction of this response. The findings of Reddehase et al. (1982) further support this hypothesis and suggest that the generation of CTLs is dependent upon a synergy between IL 2 and another soluble factor produced late in culture by the irradiated, I-region incompatible stimulator cells. If, in fact, gamma interferon is a key factor in the induction CTLs, the mechanism of its action will require careful analysis. One possibility is that once the gamma interferon is produced in the MLR, it can augment the sensitivity of T cells to IL 2 by increasing the number of IL 2 receptors. This, in turn, could lead to the proliferation of the cytotoxic T cell subset, a possibility which is perfectly consistent with earlier findings that interferon modulates T cell surface antigens (see above) and the recent identification of an IL 2 “receptor” (Hilfiker and Farrar, 1981; Robb et al., 1981). This idea is supported by the finding that poly(1: C) treatment of spleen cells or Thy 1-depleted spleen cells renders them capable of absorbing interleukin 2 (Kuribayashi et al., 1981) and that gamma interferon pretreatment of “macrophage depleted” T cells renders them sensitive to IL 2-induced proliferation (Farrar et al., 1981). The intriguing possibilities presented by these findings merit future examination to dissect fully mechanisms by which interferons modulate T cell function.

11. Eflector of Interferons on Nonspecific Cytolysis, with Special Attention to Natural Killer Cell Function Natural killer (NK) cells comprise a heterogeneous subpopulation of lymphoid cells that possess spontaneous cytolytic activity both in vivo and i n vitro against a variety of cellular targets. No previous sensitization with the target cell is required for their lytic activity. They exist in man, mouse, and a wide variety of other species (reviewed in Herberman et al., 1980) and are felt to form the cellular basis for nonspecific host resistance to tumors as well as to intracellular infectious agents. NK cells were originally recognized by their characteristic ability to lyse a broad range of tumor cells in vitro; however, other studies have since extended the lytic reactivity of NK cells to include virusinfected, as well as certain normal cells, such as hematopoietic bone marrow cells. In this regard, it has been suggested that NK cells may

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play a role not only in surveillance and resistance against neoplasia and viral disease, but also in the control of normal development of hematopoietic cells (Herberman, 1980). The general characteristics of NK cells and their relationship to other lymphoid cell types have been extensively presented in several recent reviews (Kiessling and Wigzell, 1979; Herberman et al., 1980; Herberman and Ortaldo, 1981) and in recent reports by Minato et al. (1981) and de Landazuri et al. (1981). The participation of interferons as modulators of NK cell activity has been recognized for approximately 5 years during which time a large number of reports have demonstrated for murine and human systems that the in vivo and in v itro administration of interferons or interferon inducers resulted in a 2- to 10-fold augmentation of NK cell activity (Gidlund et al., 1978; Santoli et al., 1978; Trinchieri et al., 1978; Trinchieri and Santoli, 1978; Oehler et al., 1978; Zarling et al., 1979; Djeu et al., 1979, 1980, 1981a; Senik et al., 1979; Huddlestone et aZ., 1979; Quinnan and Manischewitz, 1979; Einhorn, 1980; Herberman et al., 1980, 1981). Subsequently, it was demonstrated that OQ the basis of antiviral activity, gamma interferon was comparable to alpha and beta containing preparations in its ability to augment NK cell activity (Senik et al., 1980). This is in contrast to numerous reports in other immunoregulatory systems in which gamma interferon has been found to be a more potent biological response modifier (see above). Moreover, it has been found that gamma interferon requires a longer time period to induce an increase in NK activity than do alpha or beta interferon preparations (Wigzell, 1981). The effects of interferons or interferon inducers are more evident in the older mice, in whom spontaneous NK cell activity is diminished. Also, the ability of interferons to affect NK cell activity varies from mouse strain to mouse strain. Strains which possess high NK cell activity are more susceptible to the enhancing effects of interferons. These findings imply that the host’s genetically inherited sensitivity to interferons may play an important role in the interferon-inducible lytic efficiency of NK cells (Herberman et al., 1980). The mechanism(s) by which interferons augment NK cell activity is an area of active investigation. Herberman and colleagues (personal communication) have recently shown that NK activity is increased in large granular lymphocytes transfected with 2’5’-oligoadenylate derivatives. Unlike the interferon-induced augmentation of antibodydependent cellular cytotoxicity (ADCC) described for a number of different cell types (Itoh et aZ., 1980; Herberman et al., 1980; Hokland and Berg, 1981), the enhancement of NK cell activity does not depend on the presence of antibody directed toward the target tumor cells,

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although ADCC is augmented in this population as well (Herberman et al., 1980). The increase in ADCC observed may be the result of an interferon-induced enhancement of Fc receptor capacity analogous to that observed in macrophages (see above). The effects of exogenous interferon on NK cell function in vitro are rather dramatic. Exposure of NK cells to interferon for 1 minute at either 0 or 37°C led to a significant enhancement of cytotoxicity 4 hours later (Herberman et al., 1980). The metabolic requirements for interferon-induced modulation of NK cell function have also only recently been addressed. Djeu et al. (1981a) found that although the binding of interferon to NK cells occurred very rapidly and was independent of temperature and new protein synthesis, the acquisition of augmented NK cell activity required new mRNA and protein synthesis in the first few hours after exposure to interferon. Inhibitors of DNA synthesis (e.g., mitomycin C) did not alter the effect of interferon on N K cell activity, which indicated that cell proliferation was not required for increased NK cell activity. The new mRNA and protein synthesis may, therefore, serve either to differentiate pre-NK cells to fully mature NK effectors or to increase the lytic potential of existing NK cells. Both of these possibilities are consistent with published reports. Ortaldo et al. (1981) found that human NK cells treated with interferon exhibited increased lytic activity in both the presence and absence of augmented effector to target binding, depending upon the target cell utilized. Thus, one mechanism may involve an interferoninduced increase on NK cells of specific receptors for the target tumor cells. Saksela et al. (1980) demonstrated by immunofluorescence that a subpopulation of human NK cells produced interferon when exposed to target tumor cells. This finding provided the first experimental clue for a mechanism involving an autostimulatory pathway for NKmediated lytic activity. Moreover, these authors also found a second subset of NK cells that responded well to exogenous interferon to develop their full cytotoxic potential. Based on this latter finding it was proposed that interferon exerted its effects on pre-NK cells to differentiate them to the fully cytolytic form. The findings of Minato et al. (1980) indicated that spleen cells from nude mice could be induced to produce interferon and exhibit NK cell activity in response to persistantly viral-infected cells. Both the interferon producing cell and the NK effector expressed the Qa5+, Ly5+ phenotype. When these investigators first treated the spleen cells with anti-Ly5 antiserum plus complement, they removed both the NK activity and the ability to produce interferon. If at this point interferon was added back to the cultures, they retained both NK activity and the Ly5+ antigen. Treat-

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ment with anti-Qa5 antiserum plus complement eliminated NK cell activity and this could not be restored by exogenous interferon. Chun et al. (1976) had previously demonstrated that interferon induces NK cells that bear Qa5+ antigens. Based on their findings, Minato et al. (1980) suggested that a population of Ly5+ cells responds to persistently virus-infected tumor cells by producing interferon, that, in turn, stimulates Ly5- precursors to become Ly5+ NK effector cells. Djeu et al. (1981b) have since found that the production of interferon by NK cells is not limited to contact with tumor cells. Rather, they found that a wide variety of stimulants including tumor cells, poly(1: C), mitogens, and certain viruses and bacteria led to an augmentation of both NK activity and interferon production by NK cells purified by discontinuous Percoll gradient centrifugation. The interferon produced by NK cells in response to tumor cell stimulation was found to be labile to both heat (56°C for 1 hour) and pH 3.0 treatment. Further studies have been carried out by Minato et al. (1981) in which four distinct subpopulations of murine cells exhibiting NK activity have been identified based on their cell surface markers, target cell specificities, and sensitivity to interferon or interleukin 2. The NK, cells (Thyl-, Lyt2-, Qa5+) lysed measles-infected target cells but not P815 mastocytoma cells. These cells comprise the bulk of NK activities described in the literature and interferon was shown to augment the activity of this subpopulation. NK, cells (Thyl+,Lyt2-, Qa5+) have the same target range as NK, but fail to respond to interferon. TK cells (Thyl+, Ly2+, Qa5-, Ly5+) are T killer cells which derive spontaneously from euthymic but not nude mouse spleen cultures and possess target specificity opposite that of NK, and NK, cells. Interferon fails to augment the activity of these cells, although they do respond to IL 2. Lastly, NK,, cells (Ly5+)derived from bone marrow cultures kill virus-infected cells but not the mastocytoma cells and are not augmented by interferon. These findings strongly support the hypothesis that NK cells are comprised of a heterogeneous group of phenotypically and functionally distinct subpopulations, not all of which are responsive to augmentation by interferon exposure. The findings of Henney et al. (1981) support this idea. In their system, interleukin 2 was found to augment NK cell activity, and the effect of IL 2 was additive to the enhancement seen with interferon. Thus, IL 2 may be acting on a discrete subpopulation of NK cells not affected by interferon treatment. The effect of interferon on NK target cells presents a curious paradox. Several investigators have demonstrated that interferon treatment of the target cells decreases their sensitivity to NK cell

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cytolysis in a dose-dependent fashion. Under optimal conditions, this inhibition of cytotoxicity can be >99% (Trinchieri and Santoli, 1978; Hansson et al., 1980; Moore et al., 1980; Bergeret et al., 1980; Trinchieri et al., 1981). These findings might well account for some of the conflicting data in the literature. Since interferon treatment results in the augmentation of NK activity, but also renders target cells more resistant to NK-mediated cytolysis, the experimental design becomes of critical importance. Whether the NK cells are first incubated with interferon and then exposed to the target or simultaneously cocultured with targets and interferon may result in very different outcomes. A number of studies have been carried out that examine the mechanism underlying interferon-induced resistance to NK killing. Based on cold target inhibition studies, Hansson et al. (1980) proposed that interferon-treated target thymocytes express less of the NK target structure. Similarly, Trinchieri et a2. (1981) found that unlabeled normal fibroblasts could compete for NK-mediated cytotoxicity on Wr-labeled target fibroblasts, whereas interferon-treated fibroblasts failed to compete as targets. The effect of a pulse of interferon on the target cell was found to be transient and reversible and appeared to be specific for NK activity since interferon treatment of the targets failed to alter their sensitivity to killing by ADCC, PHA-stimulated lymphocytes, or cytotoxic T cells. Interferon treatment of the fibroblast targets did not block the binding of NK cells to individual target cells; however, a significant proportion of the bound NK cells lysed only the untreated fibroblasts. Interestingly, NK cytotoxic activity for W r labeled, untreated fibroblasts was inactivated by preincubation of the NK cells with untreated, but not interferon-treated, fibroblasts. Thus, target cells, that for one reason or another fail to be acted upon by interferons, possess the capacity for significantly reducing NK activity. When the phenomenon of interferon-mediated protection of target cells was originally recognized, many proposed that differential interferon sensitivity of normal vs neoplastic cells might provide a discriminatory mechanism for NK-mediated cytolysis. Strong experimental support for this hypothesis has not yet been provided and differential interferon sensitivity of normal vs tumor targets has not been consistently demonstrated; however, the finding of Trinchieri et al. (1981), that only target cells which have not been exposed to interferon can interact with NK cells to reduce significantly subsequent killing by the same NK population, may provide the host with an alternate mechanism for discriminating desirable targets from normal cells. An additional level of control may be provided by a balance between the efficiency of interferon to augment NK cell activity vs the ability to

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induce target resistance to NK cell killing. An important clinical study which emphasizes the urgency of our greater understanding of the mechanisms involved in interferon-mediated NK cell activity has been reported recently by Mathe (1981). In one patient with chronic lymphocytic leukemia (CLL), NK cell activity (measured in uitro) increased throughout the course of his first cycle of 10 injections of leukocyte interferon. In a second CLL patient who had received three cycles of interferon treatment, NK activity was first augmented and then subsequently depressed. This finding has been experimentally reproduced in mice. Mice injected one time with interferon exhibited marked augmentation of splenic and peritoneal NK activity. Six injections led to an equally marked depression of NK activity. Many possible mechanisms could account for depressed NK activity following persistent administration of interferon. The NK precursor pool may have been exhausted or perhaps interferon ultimately results in NK cell anergy or complete target resistance. Alternatively, interferon may ultimately result in the generation of a suppressor of NK activity. Elucidation of the control mechanisms operative in the interferonmediated modulation of both NK activity as well as target resistance to NK killing should provide a powerful tool in future therapeutic intervention.

12. Interferons and the Pathogenesis of Lymphocytic Choriomeningitis V i m s (LCMV) Disease Infection with LCMV, an arenavirus, gives rise to two disease patterns in mice. The first is a rapidly fatal form of meningitis in adult mice; the second is related to the establishment of a chronic state of infection in which high titers of virus are present in the mouse blood and tissues. The mice with chronic infections are innoculated at birth with virus; after about 6 months, such mice develop a chronic renal disease, glomerulonephritis. Immune complexes consisting of complement, LCMV, and antibody to LCMV have been reported in the glomerular basement membrane of these mice; therefore, it was likely that such complexes were related to the pathogenesis of the renal disease associated with chronic LCMV infection (Hotchin, 1962; Oldstone and Dixon, 1969, 1970, 1971). Studies involving the administration of antibody to mouse interferon at the time of infection of newborn mice with LCMV have suggested a role for interferon in LCMV-induced renal disease (Gresser et al., 1978b). Mice that had received antibody to interferon had less disease than did controls, in spite of the fact that there were high titers of LCMV in the blood and tissues of mice receiving the anti-interferon

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antibody (Riviere et al., 1977). This result suggested that interferon production induced by LCMV infection was related to the pathogenesis of the renal disease (Merigan et al., 1977). When newborn mice infected with LCMV were checked for interferon production, they were found to have high titers of circulating interferon (Riviere et al., 1977). Newborn mice treated with interferon, in order to determine whether interferon itself could cause the renal disease, developed glomerulonephritis at about the same time as did the mice injected with LCMV at birth (Gresser et al., 1976a).This suggested that the renal disease may result from production of high titers of interferon in newborn mice (Gresser et al., 1981). Additional work has indicated that in adults the pathogenesis of LCMV strains is related to their ability to induce the production of interferon; those strains of virus which are better interferon inducers are more virulent (Riviere et al., 1980). This finding may be due to localization of virus in the central nervous system (CNS) in the case of strains producing interferon. Even though titers of virus are relatively low in this situation, the immune response to virus takes place entirely in CNS, and this response results in the death of the animal. In LCMV stains, poor in the ability to induce interferon, virus spreads throughout the body. This results in a more wide spread immune response and less severe disease. It has also been noted that treatment of monkeys infected with arenaviruses with an interferon inducer results in exacerbation of disease rather than its mitigation (Jacobson et al., 1981). IV. Interferons and Defense against Viral Infections

The immune response plays a critical role in prevention of many viral infections. This is the reason that there are so many vaccines directed against the spread of viral diseases. The immune response also seems important in the recovery of animals from some primary virus infections; however, the ability of patients with severe immunodeficiencies and of animals with blocked cellular and humoral immune responses to recover from many virus infections indicated that factors other than the immune response are involved in recovery from such infections (Baron, 1973). Evidence that interferons have such a function is as follows: administration of exogenous interferons can inhibit the development of virus diseases; interferons are produced early and in large quantities in viral diseases; also, studies employing antibody to mouse interferon suggested that interferons do indeed have an important function in recovery from some primary virus infections, because, when infected animals were treated with antibody to mouse

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interferon, they developed diseases with uncharacteristically short latent periods, unusual severity, and high mortality rates (Gresser et d., 1976a,b,c). The severity of all viral infections was not increased b y treatment with antibody to interferon. For instance, anti-interferon antibody did not activate chronic herpes virus infections in mice, a result which suggested that immune responses are critical in the control of this infection; however, the modulation of the immune response b y interferon indicates that it may even play an important role in infections in which the immune response is involved. V. Interferons and Other Mechanisms Related to Immunity and Inflammation

While the antiviral activity of interferons is indeed important in recovery from many primary infections with viruses, there are other mechanisms that play roles of varying importance in recovery from different viral diseases. Cell-mediated immunity is thought to act as an antiviral mechanism in that viruses, that alter the plasma membrane by insertion of viral proteins, bring about changes that make these cells targets for natural killer (NK) or killer (K) cell lysis. This mechanism is therefore likely to be important in infections with herpesviruses and other viruses that alter cell surfaces, so that they can bud from it. This would include such agents as influenza virus, paramyxoviruses, most arthropod-borne viruses, and RNA tumor viruses (Sonnenfeld, 1980). The febrile response is also important in recovery from some virus infections. Many wild-type viruses do not grow as well at 40°C as they do at body temperature, 37°C. Indeed, some attenuated virus strains are temperature-sensitive mutants. Elevation of body temperature or local increase in temperature, such as is seen in an area of inflammation, may serve as a potent antiviral mechanism. Other aspects of the inflammatory response may act as significant antiviral factors. For instance, during the inflammatory reaction, acid products are generated and the p H is often lowered locally. White blood cells and tissue monocytes are brought into the area of infection during the inflammatory reaction. The granulocytes and monocytes provide phagocytic activity important in engulfing viruses and thus removing them from the site of infection. If the phagocytic cell is capable of killing the virus, such activity would be of importance in recovery. The inflammatory response and the febrile reaction may act to hasten the antibody response and to increase the level of antibody produced. This could be important in infections in which antibody is thought to play a role in recovery (Baron, 1973). As some of the non-antiviral activities of interferons have come to

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light, it is apparent that interferons may alter many of the mechanisms that were previously thought to be independent. Interferons stimulate the phagocytic activity of macrophages; this may eventuate in the destruction of infecting viruses. Antibody production itself can be stimulated by interferon treatment, when low concentrations of interferon are employed, or when interferon appears after viral antigen, which is certainly the sequence that occurs during infections with viruses. Interferons directly or indirectly also stimulate the inflammatory and the febrile responses (Ida et al., 1977; Yaron et al., 1977; Desomer et al., 1977; Kauppinen et al., 1977). Finally, some aspects of the cellmediated immune response, that are stimulated by interferons, may play an important role in recovery from viral infections; this includes the stimulation of cell lysis by K and NK cells, activities that can be directed against infected cells in which the plasma membrane has been altered b y viral infection. The action of interferons that prevents murine RNA tumor viruses from budding from the cell surface may have the same effect; that is, a cell surface associated viral antigen is more likely to “interest” immune surveillance cells. VI. Antitumor Effects of Interferons in Animal Systems

Very soon after the discovery of interferons as factors capable of protecting cells from virus challenge, it was reported that crude interferon preparations could also exert an inhibitory effect on the multiplication of mouse L-cells (Paucker et al., 1962). Since the interferon preparations used were extremely impure, the effect of interferon on cell multiplication was thought to be due to contaminants in the preparations. More recent studies with purified preparations of both mouse and human interferons have provided evidence they are antiviral substances, and exert effects on a wide range of activities in both normal and malignant cells. Several hypotheses have been proposed to explain the antitumor effects of interferons. These include inhibition of tumor virus replication or cell transformation by virus; inhibition of tumor development through primary effects on the immune system of the host; and direct inhibition of the growth of the tumor cell itself. Both interferon and interferon inducers have been used to study inhibitory effects on virus, chemical, and radiation induced tumors as well as on transplantable and spontaneous tumors (Gresser, 1977; Stewart, 1979). The findings that interferons could inhibit the multiplication of oncogenic viruses as well as nononcogenic viruses led to the discovery that interferon preparations could also inhibit cell transformation and tumor production caused by these oncogenic viruses. In

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animals inoculated with polyoma, Rous sarcoma, or Shope fibroma viruses, tumors were inhibited and animal survival increased if treatment was prior to virus inoculation. In mice inoculated with Friend or Rauscher leukemia viruses, however, it was necessary to continue treatment after infection in order to inhibit the various manifestations of these leukemias (Gresser, 1977). Since virus multiplication takes place throughout the course of these diseases, continued repression of virus multiplication by interferon may have been necessary for inhibition of evolution of the disease. I n order to examine the possibility that interferons might b e inhibiting multiplication of the tumor cells themselves, or might be enhancing tumor cell rejection in the host, Gresser and colleagues (Gresser and Bourali, 1969, 1970a; Gresser et al., 1969) studied the growth of inoculated tumors in several strains of mice. These tumors developed from transplanted tumor cells, not from transformation of host cells by virus. Less success was usually attained in the treatment of solid transplantable tumors than in the treatment of ascites tumors. Interferon therapy was most effective when the tumor inoculum was low, but once the tumor was well established, regression did not occur (Gresser, 1977); however, treatment with interferon inhibited the development of both the subcutaneous nodules of the Lewis lung carcinoma at the site of transplantation and the development of pulmonary metastases from the transplant (Gresser and Bourali, 1972). Another approach to the study of direct effects of interferons on tumor growth employed immunosuppressed hosts. Gresser and Bourali-Maury ( 1973) found that treating mice with anti-lymphocyte serum or X-irradiation did not alter the inhibitory effect of interferons on transplantable tumors. The development of tumors from transplanted HeLa cells and xenografts of human breast cancers in nude mice was markedly suppressed by human interferon (TaylorPapadimitriou, 1980). While such studies suggested the possibility that the inhibitory activity of interferons against at least some tumors may result from a direct antigrowth action on the tumor cells themselves, bypassing the immune response of the host, studies with interferon resistant L121O cells in mice, suggested that immune responses may play a role in interferon-induced resistance to tumor transplantation (Gresser et al., 1972). So far, there have not been many reports on the antitumor effects of gamma interferon, due to the lack of availability of sufficient quantities of gamma interferon preparations. I n the treatment of certain tumors, however, activity of gamma interferon was more potent per antiviral unit than of alpha or beta preparations (Salvin et al., 1975; Crane et al.,

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1978;Glasgow et al., 1978).Because of the possible presence of many lymphokines in these gamma interferon preparations, conclusions cannot yet be made concerning its antitumor activity. Interferons have also been used in conjunction with other antitumor agents to determine whether the effects are additive, synergistic, or antagonistic. In one study, interferon treatment inhibited murine leukemia only after the number of tumor cells was first reduced by treatment with 1,3-bis(2-chloroethy1)-l-nitrosourea (Chirigos and Pearson, 1973). Direct injections of interferon followed by poly(1: C) after several hours led to a synergistic inhibitory effect on autochthonous Moloney murine sarcoma virus-induced tumors (Stewart, 1979),while interferon and cyclophosphamide had additive effects in increasing survival from lymphomas in AKR mice (Gresser et al., 1978a).It would also be of interest to test combinations of the different alpha interferons cloned thus far. VII. Clinical Studies with Human Interferons

Most reported trials in humans have employed alpha interferons, because of the availability of quantities of these interferons, but many additional studies with recombinant alpha and beta interferons are now in progress. Exogenous alpha interferon decreased virus growth and severity of disease in a rhinovirus-induced respiratory infection, in vaccinia virus skin infections, and in ocular herpesvirus (Tyrrell, 1977; Guerra et al., 1977).Prompt treatment with 5 X 1W units per kg per day after the onset of Herpes xoster in patients with lymphomas decreased viral spread; it also alleviated the most distressing symptom of this disease, pain. Interferon-treated patients recovered more quickly and had less post-herpetic neuralgia than did untreated controls (Jordan et al., 1974). Interferon treatment was reported effective in selected patients with certain forms of chronic hepatitis B virus infection. Clinical studies employing interferon treatment in patients with long-standing, static disease demonstrated a decrease in circulating hepatitis B virus surface antigen, Dane particles, and infectious virus. The liver biopsies of such patients showed less chronic inflammation and some healing after treatment (Greenberg et al., 1976). Because of the effects of interferon on animal tumors, on tumor virus replication, and on cell multiplication, there have been attempts to treat human cancers with alpha interferon preparations. In one such study, Dr. H. Strander from Stockholm has for several years treated osteogenic sarcoma patients with human alpha interferon. The results so far may indicate that the recurrence and mortality rates after surgi-

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cal removal of primary tumors is decreased in the patients receiving long-term therapy (Strander, 1977). While this is an uncontrolled test, the results are suggestive of an inhibitory effect on the spread of the tumor. They have in turn sparked enthusiasm that has led to further trials. In the case of nodular poorly differentiated lymphomas (Merigan et al., 1978), three patients appeared to undergo some clinical improvement and tumor regression while on treatment; however, this is a capricious disease, in which therapy is difficult to evaluate. The results to date of interferon studies on the growth of the other tumors are marginal, negative, or, if positive, on too few patients to be considered significant; however, it may well be that interferons will find a place in the therapy of tumors but, at present, it is difficult for us to imagine that they alone will be a panacea for human cancer. Due to the current availability of relatively large supplies of human interferons for studies in man, it is likely that definitive results, which support or contest a role for interferons in the therapy of viral diseases or tumors, will soon be forthcoming.

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ADVANCES IN IMMUNOLOGY, VOL. 34

Acute Phase Proteins with Special Reference to C-Reactive Protein a n d Related Proteins (Pentaxins) a n d Serum Amyloid A Protein M. B. PEPYS A N D MARILYN L. BALTZ lmmunofogicol Medicine Unit, Deporiment of Medicine, Royof PostgroduoteMedical Schoof, Hommersmifh Hospitol, London, Englond

111.

V.

VI.

VII.

141 eins . . . . . . . . . . . . . . . 145 145 A. General Considerations 147 C. Interleukin 1.. . . .. . . . . . . ... . .. . . . . . . . . , . . . . . . . . . . . . . . , . . . . . .. .. . ... 148 D. Prostaglandins . . . . . . . ............ 1fj0 C-Reactive Protein, Serum and Related Proteins (Pentaxins): Definition 151 and Nomenclature . . . . . . . . . . . . . . . . . . . . 156 A. Structure and Ligand Specificity.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . 156 160 162 168 D. Functions ....................... E. Measurem ................................ . . . 175 183 Serum Amyloi ....................... 183 A. Structure . . . . .. .. . . .. . . . . . . . . . . .. 184 C. Serum Levels . .. . . . . . . . . . . . . . . . . . 187 D. SAP and Amyloidosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . 188 E. SAP-Related Material in Normal Human Tissues . . . . . . . . . . . . . . . . . . . . . 189 190 Serum Amyloid A Protein . . . . . . . . . . . . . . . . . . . . . . . 190 A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191 ....................... B. ApoSAA . . . . . . . . . . . . . . . . . . . . . . . . . 192 C. Polymorphism . . . . . . . . . . . . . . . . . . . 192 . . . . . . . . . . . . . . . . . . . . . . . 193 E. Functions.. . . . . . . . . . . 195 F. Measurement in Clinical Practice . . . . . . . . . . . . . . . . . . . . . . . . . . 198 Summary .......... . .. . ................................... 199 References . . . . . . . . . . . . . . . . . . . . . . . . . 211 .

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I . The Acute Phase Response

The acute phase response is the name given to a characteristic pattern of alteration in concentration of a number of plasma proteins (Table I) which occurs following a wide variety of different forms of 14 1 Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-022434-8

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TABLE I PLASMA PROTE?IN CONCENTRATIONSIN THE ACUTE PHASE RESPONSE Increased Proteinase inhibitors Coagulation proteins

Complement proteins

Transport proteins

a,-hntitrypsin al-Antichymotrypsin Fibrinogen Prothrombin Factor VIII Plasminogen Cls C2, B c 3 , c 4 , c5 c9 C56 ClINH Haptoglobin Hemopexin Ceruloplasmin Ferritin

Lipoproteins Miscellaneous

C-reactive protein Serum amyloid A protein Ceruloplasmin a,-Acid glycoprotein Fibronectin Gc globulin

Decreased Inter a-antitrypsin

Properdin (P)

High-density lipoprotein Low-density lipoprotein Albumin Prealbumin

infection, inflammation, or tissue damage (Crockson et al., 1966; Werner, 1969; Aronsen et al., 1972; Koj, 1974; Ganrot, 1974; Kindmark, 1976; Gordon, 1976; Kushner, 1982). The term “acute phase” was introduced by Avery and his colleagues to refer to serum obtained from patients who were acutely ill with infectious disease and which contained C-reactive protein (CRP) (Abernethy and Avery, 1941; MacLeod and Avery, 1941). This protein had been discovered by Tillett and Francis in 1930 (Tillet and Francis, 1930)as a precipitin for the then recently identified somatic C-polysaccharide of pneumococci and with the techniques which were then available it was demonstrable only in acute phase sera. CRP was therefore also known as “acute phase protein,” but as assays for specific, individual plasma proteins were developed, it became clear that the concentrations of a number of these are also raised in acute phase sera and they are now collectively designated as acute phase proteins. The increased levels of acute phase proteins are an important com-

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ponent of the overall systemic response to local or generalized injury. This response includes a variety of cellular, neurological, biochemical, endocrine, and other metabolic changes such as leukocytosis, fever, decreased serum zinc and iron concentration, increased serum copper concentration, increased protein catabolism and gluconeogenesis, negative nitrogen balance despite increased total protein synthesis, and increased synthesis of glucagon, insulin, ACTH, cortisol, catecholamines, growth hormone, TSH, T4, aldosterone, and vasopressin (reviewed by Kushner, 1982). Recent work suggests that many of these changes may be brought about by a variety of as yet incompletely characterized regulatory mediators which are transiently produced early after injury (Sipe and Rosenstreich, 1981; Kushner, 1982; Bornstein, 1982; Kampschmidt et al., 1982). In a sense all these acute phase processes, including the early mediator production, the physiological and biochemical alterations, and the increased levels of acute phase proteins may be considered to constitute the acute phase response. However, historically this term has been applied principally to the acute phase plasma protein changes and it is that sense in which it will be used here. The stimuli which induce the acute phase response are diverse, but all include as a common denominator the production of cellular or tissue injury or death (Table 11). Certain very toxic substances, such as abrin (M. L. Baltz, A. J. S. Davies, K. Gomer, and M. B. Pepys, unpublished observations), or materials with great biological potency, such as Gram-negative bacterial endotoxin, may be exceptional in that low doses can provoke major acute phase responses without causing other clinical evidence of toxicity. This may result from their potent capacity to stimulate and activate macrophages. Recent work implicates macrophage products as important signals in the initiation of acute phase protein synthesis (see Section 11). Any substance capable of appropriately triggering macrophages may therefore cause an acute phase response even if it has no other action or toxicity. It has also become clear from both clinical and experimental studies that while the acute phase TABLE I1 STIMULIWHICH INDUCETHE ACUTE PHASERESPONSE Chemical or physical trauma Chemical, toxic, or allergic inflammation Bacterial, viral, fungal, or parasitic infection Ischemic necrosis Malignant neoplasia

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response is nonspecifically triggered by most forms of tissue damage, and generally reflects quantitatively the extent of such damage, there are some circumstances in which the acute phase response overall or the response of particular proteins may be lacking or inappropriately modest (see Sections IV,E and V1,F). Such observations are of some value in differential diagnosis and clinical management as well as being likely to facilitate elucidation of the control mechanisms of the acute phase response. Acute phase responses of plasma proteins and the various physiological, metabolic, and endocrine changes following tissue injury, infection, or inflammation occur in all homoiothermic animals and therefore presumably have beneficial overall functions concerned with restricting injury and promoting resolution and repair. The roles of coagulation proteins, complement proteins, transport proteins, and proteinase inhibitors in these processes seem fairly obvious. The functions of some of the miscellaneous proteins are less clear, although suggestive information is available in some cases. Ceruloplasmin possesses significant oxidase activity and, in addition to its role in copper transport, it probably also contributes to mobilization of iron from tissue storage sites, regulation of circulating levels of biologically active amines and protection of lipids from autooxidation (Goldstein et al., 1982). Recently ceruloplasmin has been shown to catalyze conversion of superoxide anion to hydrogen peroxide and oxygen in a manner similar to the intracellular enzyme, superoxide dismutase (Goldstein et al., 1979a). Superoxide anion is a highly reactive free radical which is produced into extracellular fluids by “activated” polymorphonuclear leukocytes and seems to be an important mediator of inflammation and tissue injury (McCord, 1974; Salin and McCord, 1975; Johnston and Lehymeyer, 1976). Increased levels of ceruloplasmin during the acute phase response may thus make a significant contribution to scavenging superoxide anion in the circulation (Goldstein et al., 1979b, 1982). Fibronectin (reviewed by Mosher, 1980; Ruoslahti et at., 1981), by virtue of its capacity to bind collagen and glycosaminoglycans and its ability to interact at cell surfaces, seems to be involved in the nonspecific phagocytic function of the reticuloendothelial system. Fragments of fibronectin can, under different circumstances, both enhance and depress uptake by mononuclear phagocytes (Czop et d., 1981; Ehrlich et al., 1981). Plasma levels of fibronectin tend to fall immediately after injury and in patients with overwhelming infection and septic shock they remain low. However, in some situations of inflammation and tissue damage an acute phase rise occurs and may

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then, perhaps, contribute to processes of resolution and repair. As well as enhancement of phagocytosis of circulating debris by intact fibronectin (Bevilacqua et al., 1981) proteolytic cleavage fragments apparently have chemoattractant properties for fibroblasts (Postlethwaite et al., 1981). The functions of CRP and of serum amyloid A protein (SAA) are not known but are likely usually to be beneficial because of the stable evolutionary conservation of these proteins (see Sections I11 and VI). Proteins with amino acid sequences homologous with human SAA are present in birds (Gorevic et al., 1977) as well as a number of subprimate mammals, and all vertebrates which have been studied possess plasma proteins belonging to the CRP family and this is true even of an invertebrate, Limulus polyphemus, the horse shoe crab (Robey and Liu, 1981). CRP and SAA are of particular interest for several reasons: the rate and extent of their increase in concentration in the acute phase response, which exceed those of any other proteins, the value of serum CRP measurements in clinical management of a variety of different disorders and the role of SAA in pathogenesis of reactive systemic, AA-type, amyloidosis. Apart from this introductory section, and the discussion below of the induction and control of the acute phase response, the remainder of the present article will mainly be devoted to a detailed consideration of CRP and related proteins and of SAA. Extensive reviews of the other specific proteins and groups of proteins listed in Table I are readily available in the literature (Davie and Hanahan, 1977; Evered and Whelan, 1980; Koj, 1974; Lachmann and Peters, 1982; Miller and Lewis, 1981; Putnam, 1975; Schmidt, 1975) and an up-to-date account of many aspects of the acute phase response is provided by Volume 389 of the Annals of the New York Academy of Sciences which comprises the proceedings of a 1981 symposium on the subject. II. Induction and Control of Synthesis of Acute Phase Proteins

A. GENERAL CONSIDERATIONS The raised circulating levels of acute phase proteins which occur during the acute phase response are mainly due to increased de nouo synthesis, although in some cases there may be a contribution from release of preformed stocks of protein (Koj, 1974). Even when there is major utilization of amino acids, as in regeneration of damaged or excised tissue, synthesis of acute phase proteins proceeds unabated suggesting the importance and probable survival value of the acute phase

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response (Koj, 1974). It has been shown with several proteins, including fibrinogen, haptoglobin, rabbit CRP, and mouse SAP, that the fractional catabolic rate is independent of the plasma level. In some cases, such as consumptive coagulopathy or in vivo complement consumption, increased utilization of particular proteins may mask their increased production in the acute phase response. The turnover of certain plasma proteins may increase during acute phase responses but, because synthetic and catabolic rates remain in balance, plasma levels do not change and these proteins are therefore not identified as acute phase reactants. Such considerations may underlie the different behavior with regard to plasma levels of otherwise very similar proteins such as SAP and CRP in different species (see Section 111). From a functional point of view, increased turnover in one species may be able to provide for the same in vivo role of the protein as an increased absolute circulating concentration in another species. Most of the acute phase proteins are synthesized by hepatocytes, in some cases, such as CRP, exclusively so, while others, such as complement components, may also be synthesized by mononuclear phagocytic cells at least in vitro. (Colten, 1976; Colten and Einstein, 1976; Whaley, 1980).While there is convincing evidence that most of the plasma complement components are made only by the liver, local production by macrophages in inflammatory lesions may have considerable biological importance (Colten, 1976; Lachmann and Peters, 1982). al-Acid glycoprotein is produced by lymphocytes, monocytes, and polymorphs as well as by hepatocytes (Gahmberg and Andersson, 1978). It exists as an externally located integral membrane protein of the leukocytes and is then shed into the external medium. Fibronectin is synthesized by endothelial cells, fibroblasts, various other tissue cells, macrophages, and monocytes. The source of the plasma fibronectin is not known, it may be vascular endothelial cells and is probably not hepatocytes. The murine serum glycoprotein gp70 is of particular interest in that it is a polymorphic protein closely related to a retroviral envelope protein. It forms a part of the antigen-antibody complexes, levels and tissue deposition of which correlate closely with pathogenesis of the lesions in the NZB/W and MRWl murine models of spontaneous autoimmune, lupus-like disease (Izui et al., 1979). gp70 is expressed independently of virions on the surface of lymphoid cells but these are not the source of the serum gp70 (Obata et al., 1978). It has recently been demonstrated that gp70 behaves like a typical acute phase protein in several but not all mouse strains and is synthesized and secreted by hepatocytes (Hara et al., 1982). In these respects its

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properties correspond to those of a normal host constituent rather than a product of the viral genome. Different patterns of acute phase response of different proteins may be seen following different stimuli, or in different individuals or different species following the same stimulus (Crockson et al., 1966; Werner, 1969; Aronsen et al., 1972; Koj, 1974; Ganrot, 1974; Kindmark, 1976; Kushner, 1982; Baltz et al., 1980b, 1982a,b, and see Sections IV,E and V1,F). Some of this complexity may reflect consumption of certain proteins, such as clotting factors, complement proteins, or proteinase inhibitors. However, where it reflects synthetic rates, complexity of the response may depend on the different cells of origin of the various proteins, and the nature of the specific molecular signals to which they respond. The precise nature of these signals is as yet incompletely understood, though there has been significant progress recently in elucidating the events involved in biosynthesis in the liver.

B. BIOSYNTHETICMECHANISMS I N THE LIVER In experimental studies of acute phase proteins of hepatic origin it seems that increased production results from an increased number of hepatocytes being recruited to active synthesis. These cells are initially located in a periportal distribution but with increasingly intense inflammatory stimulation most hepatocytes can become involved (Kushner and Feldmann, 1978; Benson and Kleiner, 1980; Baltz et al., 1980a; Courtoy et al., 1981). There is no clear evidence for specialization of individual hepatocytes and all of them seem able to make several acute phase proteins simultaneously (Courtoy et al., 1981). The molecular basis for the increased hepatocyte synthesis of acute phase proteins appears to be an increased abundance of the appropriate species of mRNA. This has been demonstrated for mouse SAA with a 500- to 1000-fold increase in abundance of the messenger, (Morrow et al., 1981), for rat al-acid glycoprotein with a 90-fold increase (Ricca et al., 1982) and for baboon a,-antitrypsin (Chandraet al., 1981). At the peak of the acute phase response the mRNA for rat a,-acid glycoprotein constitutes 2.7% of the total liver messenger (Ricca et al., 1982), while mouse SAA protein synthesis comprises up to 2.5% of total hepatic protein synthesis (Morrow et al., 1981). Concomitantly there is a decrease, of about 5-fold in the rat and 3-fold in the mouse, of abundance of mRNA for albumin (Ricca et al., 1982; Morrow et nl., 1981). This probably explains the fall in levels of plasma albumin and possibly also some other proteins (Table I) which accompanies the acute phase response.

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The intracellular processes leading to increased mRNA for acute phase proteins and decreased mRNA for albumin in hepatocytes are not yet known. The very large increment in abundance of mRNA for mouse SAA is associated with increased transcription, the underlying mechanism of which is not known. It does not seem to be related to the level of cytosine methylation in the DNA (Stearman et al., 1982).There are no differences between the 3’-poly(A) sequences of mRNA from acute phase and normal rat liver, suggesting that addition of these sequences is not responsible for stabilization of mRNA which could contribute to its persistence and high levels (Demczuk and Chandler, 1982).Kinetic studies of the increase in poly(A)+RNA and of the onset and rate of acute phase protein synthesis suggest that an early contribution to plasma levels may derive from increased efficiency of translation of existing mRNA before new and greater amounts of mRNA appear (Demczuk et al., 1982). Further clarification of these mechanisms will follow from the availability of cloned probes for particular proteins and those €or mouse SAA (Morrow et al., 1981; Stearman et al., 1982), rat a,-acid glycoprotein (Ricca et al., 1982), and primate al-antitrypsin (Chandra et al., 1981; Kurachi et aZ., 1981)have already been prepared. Recombinant DNA technology should help to shed valuable light on the structure, polymorphism, and possibly the function of these proteins as well as other aspects of their biosynthesis, intracellular processing, and secretion. It will also be of interest to compare the control mechanisms of the acute phase response with those of other phenomena of rapidly inducible gene expression, such as production of heat shock proteins in Drosophila, (Ashburner and Bonner, 1979) and de no00 expression of MHC-coded proteins on lymphocytes exposed to interferon (Burrone and Milstein, 1982). C. INTERLEUKIN 1 The onset of acute phase protein synthesis in the liver following the stimulus of an acute inflammatory or necrotic lesion at a distant local site implies the existence of humoral andlor neural mediation. The peripheral, periportal distribution of the actively synthesizing cell supports this view, since both circulating and neural connections enter the liver lobules in this area. Substantial evidence has accumulated recently that a peptide product of mononuclear phagocytic cells, now designated as interlevkin 1 (IL-1), is involved in triggering hepatocytes to acute phase protein synthesis (Sipe et al., 1979; Selinger et al., 1980a,b; Sztein et al., 1981; McAdam and Dinarello, 1980; McAdam et al., 1982; Kampschmidt et al., 1982; Bornstein, 1982).IL-1 seems to be a family of peptides of around 15,000 molecu-

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lar weight which have slightly different isoelectric points and which can aggregate into larger molecular forms while retaining biological activity (Oppenheim and Gery, 1982). IL-1 is actively synthesized by monocytes and macrophages when they are stimulated by microorganisms, by microbial products such as lipopolysaccharides (endotoxin) or muramyl dipeptides, by phagocytosis even of inert particles, and by activated T lymphocytes or their lymphokine products, such as macrophage-activating factor or colony-stimulating factor (reviewed by Oppenheim and Gery, 1982). IL-1 production depends on de novo synthesis of mRNA and protein and preformed stocks of IL-1 are not present. Various biological properties which are now ascribed to IL-1 were previously studied separately and ascribed to materials which were named accordingly. Thus endogenous pyrogen (EP) or leukocytic pyrogen (LP), which is responsible for fever in all higher animals, probably regardless of the exogenous pyrogen administered, was first obtained from leukocyte preparations which were predominantly polymorphs (Bornstein, 1982). More recent work suggests that monocytes/macrophages are much richer sources of EP, if indeed polymorphs make it at all (Bornstein, 1982). Preparations obtained in the same way as EP provoke rapid acute phase responses when injected i n vivo and the activity responsible was called leukocyte endogenous mediator (LEM) (Wannemaker et d., 1975; Merriman et al., 1975; Kampschmidt et al., 1978). Products of activated macrophages capable of promoting the activation of lymphocytes by antigens or mitogens were designated as lymphocyte activating factor (LAF) and subsequently as IL-1 (reviewed by Oppenheim and Gery, 1982). It now seems clear that during isolation of these substances the different biological activities of pyrogenicity, lymphocyte activation, and acute phase protein stimulation copurify (McAdam and Dinarello, 1980; McAdam et al., 1982). It remains to be determined whether all the biological properties reside in each peptide or whether they belong to slightly different materials which have not yet been separated. The absolute amounts of these potent mediators are very small indeed so that such questions may prove difficult to answer. The mechanism of induction of IL-1 production and its effects on hepatocytes are not known. Intravenous injection of IL-1 preparations may not evoke acute phase responses of the same intensity as those seen following an acute local inflammatory stimulus (Bornstein, 1982; M. L. Baltz, K. P. W. J. McAdam, C . Dinarello, and M. B. Pepys, unpublished observations) and there may be a greater response of one acute phase protein than another, for example, mouse SAA compared

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to mouse SAP (Sipe et al., 1982). IL-1 may therefore be only one part of the triggering mechanism or may normally act indirectly via other mediator systems. Alternatively IL-1 may only act with high efficiency at short range and may need to be released in the appropriate microenvironment, for example by the Kupffer cells, to have maximum effect. Intracerebral inoculation of LP has been shown to be more effective in stimulating acute phase protein synthesis than comparable intravenous injections (Turchik and Bornstein, 1980), but it is not known whether this is because a direct neural or a neurohumoral message to the liver is involved, nor whether, if the central nervous system effect is genuine, it conveys a permissive or a necessary signal. Little is known of the way in which macrophages are triggered to produce the acute phase proteins which they make themselves but clearly the local secretion of these proteins within inflammatory lesions, for example the rheumatoid synovium (Ruddy and Colten, 1974), may be of considerable biological importance (Colten, 1976; Whaley, 1980). It is possible that the same stimuli are involved which also evoke IL-1 production, thereby promoting lymphocyte responses, causing fever and inducing the acute phase response by the liver. Products of activated T cells can enhance synthesis of complement components by macrophages (Littman and Ruddy, 1977, 1978) but some stimuli may induce IL-1 production without doing so. In mice the acute phase response of C3 to T cell-dependent immunologically mediated tissue injury is abolished by T cell depletion. This is the case, for example, in infective processes, such as HymenoZepsis nana infestation (Baltz et aZ., 1982d) and schistosomiasis. In the latter direct tissue damage still occurs in T cell-deprived hosts and hepatic acute phase proteins are produced while C3 levels remain normal (Pepys et aZ., 1980b; Baltz et al., 1982d). Such observations suggest that in some situations the acute phase component of the plasma level of some complement proteins may be derived from macrophages activated via immunological mechanisms. The baseline synthesis b y the liver of these same proteins and the increased production by the liver of other acute phase proteins may be regulated separately.

D. PROSTAGLANDINS There is some evidence that prostaglandins may be involved in induction of the acute phase response. Rabbits bearing a prostaglandin (PGE2)-producingtumor developed acute phase protein levels which correlated closely with the plasma levels of PGE2 and administration of indomethacin, a prostaglandin synthetase inhibitor, reduced PGEz and acute phase protein levels in parallel (Voelkel et al., 1978). Simi-

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larly administration of prostaglandins PGE1, E2, F,,, or A, to rabbits all induced acute phase rises in haptoglobin concentrations (Shim, 1976). Infusion of PGE, in man has recently been shown to cause major elevations in the concentrations of CRP, SAA, and other acute phase proteins (Whicher et al., 1980, 1982). Injection of PGE, into mice tolerized to endotoxin failed to induce an acute phase response, while injection of IL-1 had its usual effect (Whicher et al., 1982). This suggests that in the course of inducing the acute phase response both prostaglandins and endotoxin may act on macrophages, causing them to release IL-1, rather than directly on the liver cells. This is supported by the observation that prostaglandins had no effect on biosynthesis of CRP by isolated rabbit hepatocytes in vitro (Schultz et al., 1982). It is thus possible that prostaglandins or other products of arachidonic acid metabolism generated at local sites of inflammation or tissue damage may be involved indirectly in mediation of the acute phase response. Prostaglandins themselves are however not likely to be essential, since administration of indomethacin in doses sufficient to block prostaglandin synthesis did not block the CRP response to intramuscular turpentine in rabbits (Schultz et al., 1982). In repeating the experiments in which prostaglandin infusions have caused acute phase responses in vivo it will be very important to exclude even trace contamination with endotoxin or other IL-l-inducing materials. Ill. C-Reactive Protein, Serum Amyloid P Component (SAP), and Related Proteins (Pentaxins): Definition and Nomenclature

The existence in man of a protein distinct from but related to CRP was first demonstrated by the discovery that a putatively newly described protein, which at that time was called Clt, shared amino acid sequence homology with CRP and had a similar appearance in the electron microscope (Osmand et al., 1977). C l t had been isolated during the preparation of subcomponents of the first component of complement, C 1, b y calcium-dependent affiity chromatography on IgGSepharose and was thought to be a fourth subcomponent of C1 (Assimeh and Painter, 1975). However, the isolation of the so-called C l t in this way was a result of its calcium-dependent binding to the agarose of which Sepharose beads are composed (Pepys et al., 1977a,b) and this protein has nothing to do with the structure or function of C1 (Painter, 1977; Ziccardi and Cooper, 1977; Cooper and Ziccardi, 1979). In fact, C l t was identical with a known normal serum protein (Pinteric et at., 1976; Pepys et al., 1977b), which had been independently isolated in different laboratories. It was named 9.5s

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al-glycoprotein on the basis of its biophysical properties (Haupt et al., 1972), and serum amyloid P component (SAP) (Bladen et al., 1966; Cathcart et al., 1965) because of the pentagonal appearance of its molecules in the electron microscope and because it was first identified as a constituent of amyloid deposits. Human CRP and SAP resemble each other as follows: (1)both have the capacity for calcium-dependent binding to particular specific ligands (see below); (2) both are composed of noncovalently associated polypeptide subunits, of a single type in each molecule, which are arranged in an annular disc-like configuration with cyclic pentameric symmetry (Osmand et al., 1977) (Fig. 1);(3) they share substantial, approximately 60%) homology of amino acid sequence (Osmand et al., 1977; Oliveira et al., 1977, 1979; Anderson and Mole, 1982) (Fig. 2). C-reactive protein derives its name from its calcium-dependent reactivity with pneumococcal C-polysaccharide (CPS) (Abernethy and Avery, 1941) and the proteins which can be isolated from the sera of other animals on the basis of this same reactivity are also designated as CRPs. They closely resemble human CRP in molecular appearance, subunit composition, amino acid composition, and, where available, amino acid sequence (Bach et al., 1977; Pepys et al., 1978a, 1980a, 1982c; Oliveira et al., 1980; de Beer et al., 1982a; Baltz et al., 1982a). Human SAP undergoes calcium-dependent binding to agarose but not to CPS (Pepys et al., 1977a,b) and it can be quantitatively isolated from whole serum at over 80% purity by calcium-dependent affinity chromatography on agarose (de Beer and Pepys, 1982). Serum proteins in lower animals which share with human SAP the capacity to bind to agarose but not to CPS have been designated as SAPS (Pepys et al., 1978a).They are demonstrably members of the same family in terms of molecular appearance, subunit composition, and amino acid sequence (Pepys et al., 1978a, 1980a, 1982a; de Beer et al., 1982a; Baltz et al., 1982a).Furthermore in those species (ox and mouse) in which amyloid occurs, the selectively agarose-binding protein is demonstrable in the amyloid deposits, confirming the relationship to human SAP (Baltz et al., 1980a; E. Gruys, F. C. de Beer, and M. B. Pepys, unpublished observations). In the presence of calcium, human CRP binds weakly to agarose (Pepys et al., 1977a; Volanakis and Narkates, 1981), rabbit CRP binds more strongly (M. B. Pepys, unpublished observations), and rat CRP even more so (de Beer et al., 1982a). These interactions may cause problems during isolation procedures. However since, with the possible exception of the Syrian hamster female protein (see below), there is no reactivity of SAP with CPS it is generally possible to separate,

FIG.1. Electron micrographs of negatively stained preparations of human C-reactive protein (a) and human serum amyloid P component (b). Most molecules are seen face-on but arrows in (a) and insets in (b) (at higher magnification) show views of the disc-like molecules side-on. Magnification, x320,OOO. Micrography performed by Dr. E. A. Munn, Department of Biochemistry, ARC Institute of Animal Physiology, Babraham.

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SAP

90 Y I OR]

SAP CRP

FTKPQL

I

:b-m P

FIG.2. Amino acid sequences of human C-reactive protein (Oliveira et al., 1977, 1979) and human serum amyloid P component (Anderson and Mole, 1982). Deletion gaps have been introduced at positions 45,46, and 182 of SAP to maximize homology; the numbering system used is for SAP (Anderson and Mole, 1982)

purify, and define CRP and SAP molecules on the basis of their calcium-dependent ligand specificity. Despite the stable evolutionary conservation of overall molecular structure and binding specificity in the CRP-SAP family there are notable differences in some properties of the proteins in different, even closely related species. For example, in man CRP is not glycosylated (Gotschlich and Edelman, 1965) and is the classical and dramatic acute phase reactant with levels rising from a median normal of less than 1mg/liter (Shine et aZ., 1981) to as much as 300 mg/liter or more within 24-48 hours of an acute stimulus (Kushner et al., 1978; de Beer et al., 1982~). The circulating concentration of human SAP is relatively stable at about 30-40 mg/liter and changes little with acute stimuli, though it does tend to be higher, within the overall normal range of up to 90 mg/liter, in patients with chronic inflammatory diseases (Pepys et al., 197813, 1980a). In mice CRP is a trace protein present at normal

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levels in the microgram/liter range, rising to not more than 2 mg/liter during the acute phase response (Siboo and Kulisek, 1978; Pepys, 1979a), while SAP is a major acute phase reactant, levels of which can rise from normal of 5 to 100 mg/liter in different inbred strains to over 600 mg/liter (Pepys et al., 1979a; M. L. Baltz and M. B. Pepys, unpublished observations). In rats the SAP concentration corresponds closely to that seen in man, but CRP which, unlike its human and rabbit counterparts, is glycosylated, is normally present at about 300 mg/liter even in specific pathogen-free animals and can rise to around 1000 mg/liter (de Beer et al., 1982a). Such differences from man, particularly in the behavior of the rat protein, have led to difficulties both at the philosophical and semantic level (Gotschlich, 1982) and to some terminological confusion in the literature. Pontet et al. (1981) have recently isolated a rat serum protein and called it SAP, despite the fact that the protein has the known ligand specificity of human CRP and that they did not even look for a rat counterpart of human SAP. In fact their protein is immunochemically indistinguishable from the protein we have identified as rat CRP (M. B. Pepys and M. Pontet, unpublished observations). Nagpurkar and Mookerjea ( 1981) have independently isolated and characterized the same protein, confirmed as being identical with our rat CRP (M. B. Pepys and S. Mookerjea, unpublished observations), and named it “phosphorylcholine-binding protein” on the basis of its ligandbinding reactivity. They considered the possibility that it might be related to CRP but failed to show the calcium dependence of its binding properties through inappropriate experimental design and were put off by their failure to show immunochemical cross-reactivity with human CRP or to isolate a comparable protein from normal human serum. In fact immunological cross-reactivity between CRPs of different species is exceptional (Baltz et al., 1982a),perhaps because of their highly conserved structure, and the normal level of human CRP is about 300 times lower than that in the rat. Nagpurkar and Mookerjea did not examine their protein in the electron microscope, but when this is done it reveals the very close resemblance of rat CRP to human and other CRPs and confirms its membership of the family (see Note Added in Proof, A). Human CRP and SAP molecules both have a pentagonal appearance in the electron microscope, CRP being composed of a single pentameric disc and SAP of two such discs interacting face-to-face. On the basis of this common structural feature Osmand et al. (1977) proposed the name “pentraxins” for this protein family. A more correct derivation from the Greek is actually “pentaxin” (M. Papamichail, personal

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communication) and we suggest here that it may be useful to adopt this term more generally than has hitherto been the case. This would be convenient and help to avoid confusion in the increasing number of cases in which these proteins in different species do not share precisely tbe same properties as their human counterparts. It seems unlikely that the physiological raison d’etre of the proteins is either to react with pneumococcal somatic C-polysaccharide or to be deposited with amyloid fibrils, respectively, and perhaps a new nomenclature such as pentaxin 1, pentaxin 2 ( P l yP2, etc.) may be more suitable. IV. C-Reactive Protein

A. STRUCTUREAND LIGAND SPECIFICITY The subunit of human CRP consists of 187 amino acids with PCA at the N-terminal, proline at the C-terminal, and a disulfide bridge between the cysteine residues at positions 36 and 78 (Fig. 1)(Oliveira et al., 1977, 1979). Partial sequences are available for rabbit (Bach et at., 1977), plaice (Pleuronectes platessa L., a marine teleost) (Pepys et al., 1980a, 1982d), and Limulus polyphemus (the horseshoe crab) CRP (Liu et al., 1982). There are significant homologies between the different species, as high as 90% for rabbit and man but more tenuous for the invertebrate. The human CRP sequence shows no significant repeating sequences, indicating that it has not evolved by gene duplication, nor does it show any statistically significant homology with any other known proteins, except other pentaxins (Oliveira et al., 1977, 1979). Distant homologies have been noted with the C,2 domain of IgG and with C3a but they are insufficient to support a common evolutionary origin (Oliveira et al., 1977, 1979). However in view of the capacity of human CRP to bind and activate C l (see Section IV,C,3) it is of interest that a peptide dimer containing two copies of the 282-292 region, part of the Cy2 domain of human IgG, protein Eu, is as efficient as whole intact IgG, in inhibiting binding and activation of C1 by antibody-coated erythrocytes (Lukas et al., 1982). Sequence homologies between residues 90 and 105 in CRP and another putative Clq-binding site, residues 312-317, in the Cy2 have also been pointed out (Osmand and Short, 1981) (see Note Added in Proof, A). Circular dichroism studies of human CRP show that it contains a significant amount of a-helix (34%) and about 45% of p-structure (Young and Williams, 1978). This differs from immunoglobulins which do not contain a-helix. There are also significant differences in the

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circular dichroism spectrum of CRP in the presence and absence of calcium and, by analogy with other calcium-binding proteins, the results obtained suggested that the peptide oxygen of tyrosine residues may be involved in calcium-binding by CRP (Young and Williams, 1978). Human CRP molecules consist of 5 subunits (Gotschlich and Edelman, 1965; Osmand et al., 1977) as do the CRPs of rabbit (Bach et aZ., 1977) and rat (de Beer et al., 1982a), while plaice CRP contains 10 subunits (Pepys et al., 1980a, 1982~).Rat CRP differs from CRP and SAP of all other species studied so far in that one pair of subunits in each molecule is covalently linked by an interchain disulfide bridge (de Beer et al., 1982a). In all the other proteins the subunits are noncovalently associated, though there is direct or presumptive evidence of an intrachain disulfide bridge in all of them (Baltz et al., 1982a). The various vertebrate CRPs differ from each other with respect to glycosylation and plaice in particular is unique in that it contains a mixture of glycosylated and nonglycosylated subunits (Pepys et al., 1982~).Although no polymorphism has been detected in human CRP there appears to be some heterogeneity at certain residues in human SAP (Anderson and Mole, 1982) and isoelectric focusing of mouse and rat SAP and rat CRP, even from inbred strains, demonstrates a series of molecules in each with slightly different pZ (Baltz et aZ., 1982a). LimuZus CRP differs more significantly than do the vertebrate proteins among themselves. It appears to consist of 12 complex subunits, each containing glycosylated A and B moieties, which are noncovalently associated in a double-stacked hexamer (Liu et al., 1982), and it has sialic acidspecific lectin properties in addition to its binding capacity for phosphorylcholine (Roche and Monsigny, 1974). Within the intact molecule of human CRP each subunit binds one or two calcium ions (Gotschlich and Edelman, 1965) and having done so is then able to bind a variety of different ligands with varying degrees of affinity. The ligand which it binds best is phosphorylcholine (Volanakis and Kaplan, 1971) with a K, of 1-2 X M (Anderson et al., 1978) but it also binds other phosphate monoesters with a stoiochiometry of 1 mole per mole of CRP subunit (Gotschlich and Edelman, 1967, 1965; Gotschlich et al., 1982). Replacement of the phosphate monoester group by other acidic groups or by conversion to a phosphate diester markedly diminishes or abolishes the affinity of binding (Young and Williams, 1978; Gotschlich et al., 1982). The much higher affinityfor phosphorylcholine compared with other phosphate monoesters suggests that the binding site within CRP recognizes

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the positively charged trimethylammonium group as well as the phosphate. CPS and many other complex polysaccharides of microbial, fungal, or metazoan parasite origin with which CRP reacts contain phosphorylcholine (Tomasz, 1967; Tillet et aZ., 1930; Pepys and Longbottom, 1971; Baldo et at., 1977a,b; Pery and Luffau, 1979). However, human CRP also reacts with materials which lack both phosphate and choline, such as depyruvylated type 4 pneumococcal polysaccharide (Heidelberger et aE., 1972), and more weakly with some galactans including agarose (Pepys et al., 1977a; Volanakis and Narkates, 1981; Uhlenbruck et aZ., 1979). Rat CRP and rabbit CRP, as well as their reactivity with CPS and phosphorylcholine, bind much more strongly than human CRP to agarose, as does Syrian hamster FP (see Section 111). Rabbit CRP also shows significant differences from human CRP with respect to its ligand requirements in the phosphorylcholine-binding site in terms of the presence and spacing of phosphate and cationic groups (Oliveira et al., 1980). Since all these reactions are calcium dependent and inhibitable by phosphorylcholine, they presumably involve the same binding site in the CRP molecule. This may therefore have specificity for galactosyl or related residues in some particular conformation or for some other minor or trace constituent such as anionic groups, e.g., carboxylate residues in these biological polymers. Although apparently surprising, the behavior of CRP in this respect resembles that of anti-streptococcal antibodies of known anti-phosphorylcholine specificity which can still bind to the polysaccharide backbone of streptococcal carbohydrates from which all the phosphorylcholine has been removed (Bennett and Glaudemans, 1979). There has been some recent progress in biophysical and chemical oharacterization of the phosphorylcholine-binding site of CRP. On the basis of comparisons with the known sequences of phosphorylcholine-binding myeloma proteins, in which the residues involved in the binding site have been directly identified (Phe-Tyr-Met-Glu in the first hypervariable region), it has been proposed that the sequence Phe-Tyr-Thr-Glu in CRP may be part of the binding site (Young and Williams, 1978). There are additional, though less marked, similarities between the CRP sequence and the portion of the second hypervariable sequence known to be involved in binding by these antibodies (Young and Williams, 1978). Further evidence of structural similarities between CRP and anti-phosphorylcholine antibodies is provided by the fact that monoclonal antiidiotype antibodies directed against the anti-phosphorylchoIine myeloma protein HOPC8 will also react with CRP (Volanakis and Kearney, 1981).They do so, however,

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only in the presence of calcium ions which CRP requires to recognize phosphorylcholine but which, of course, the anti-phosphorylcholine antibodies do not. These observations suggest that either the calcium itself or the conformational change its uptake induces, as shown by circular dichroism studies, is essential for the functional configuration of the binding site. Comparisons of the inhibition of binding to phosphorylcholine by CRP and HOPC8, using various phosphate monoesters, choline derivatives, and dipeptides, have shown that the main reactivity of CRP is for the anionic group and that carboxylate can substitute for phosphate (Barnum et al., 1982). In contrast, HOPC8 recognizes predominantly the cationic choline moiety. Inhibition of CRP but not HOPC8 binding by certain di- and tripeptides which have to have both a free carboxyl and free a-amino group confirms the zwitterionic nature of the CRP binding site (Barnum et al., 1982). The carboxy-terminal residue also has to be a hydrophobic amino acid and it has to be in the appropriate (L not D) conformation, indicating the presence of a stereospecific secondary binding site in the binding pocket (Barnum et al., 1982). Electron spin resonance studies using spin-labeled derivates of phosphorylcholine as ligands for CRP have suggested that the binding pocket is rather shallow (Liu et al., 1982). In the absence of calcium ions human CRP binds polycations such as poly-L-lysine and poly-L-arginine polymers, lysine-rich and argininerich histones, myelin basic protein, and leukocyte cationic protein (Siege1et al., 1974, 1975; di Camelli et al., 1980; Potempaet al., 1981). At appropriate concentrations of CRP and ligand the complexes aggregate and precipitate. These interactions can be modulated by the simultaneous presence or subsequent addition of heparin, a polyanion. Similar properties have been observed with rabbit CRP though it seems to react selectively with the arginine polycations (Potempa et al., 1982). All these reactions are inhibited by calcium ions but are promoted by phosphorylcholine in the presence of calcium. It is not known whether there is a polycation-binding site in the CRP subunit distinct from the binding site for phosphorylcholine or whether the part of the latter site with specificity for the cationic moiety is involved in these reactions. These however are important questions since the ligand-binding properties of CRP are among the stably conserved features of the molecule and are likely to be central to its biological role. More definitive structure-function analysis of the CRP molecule will require its three-dimensional delineation by X-ray crystallography and such studies have been initiated (C. Buxton, A. Wonnacott, D. M. Blow, and M. B. Pepys, unpublished observations).

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B. SYNTHESIS AND TURNOVER CRP is synthesized by hepatocytes (Hurlimann et al., 1966; Kushner and Feldmann, 1978) and there is no evidence for its production by any other cell type. Rabbit CRP closely resembles human CRP in its structure and in its behavior as an acute phase protein and almost all experimental work on induction and control of synthesis and on turnover of CRP has been performed in rabbits. Following an acute stimulus increased CRP synthesis starts in the periportal area, as mentioned in Section I17B7and then spreads to involve all cells across the liver lobule. Perfused rabbit livers or primary hepatacyte cultures continue to synthesize CRP in vitro at levels which correspand closely to the serum levels of CRP at the time of removal of the liver (Macintyre et al., 1982). These synthetic rates do not increase with time but their peristence suggests that continued presence of a circulating mediator is not required. Furthermore the failure to switch off synthesis in vitro suggests that a separate negative control mechanism may operate in vivo to regulate the duration of acute phase protein production. However much more work is required to establish the validity of these concepts since when hepatocytes are cultured in vitro they may show different patterns of protein secretion than they do in vivo (Guillouzo et aZ., 1981). The in vivo half life of labeled CRP in the circulation of rabbits is about 4-6 hours and is the same in normal animals or during the course of acute phase responses induced by various stimuli (Chelladurai et al., 1982). The fractional catabolic rate is also the same in stimulated and unstimulated animals and the amount of CRP catabolized per day is thus proportional to the plasma concentration. Furthermore the plasma level is evidently determined by the synthetic rate and can vary between 0.16 mg/kg body weighvday in control animals and 2.9-13.5 mg/kg/day in stimulated animals (Chelldurai et al., 1982). The sites to which CRP is cleared and the mechanism of its catabolism are not known. Direct estimation of in vivo turnover of labeled CRP poses ethical problems in man and could only be undertaken with CRP purified from the test individual’s own acute phase serum. This has not yet been done. However studies of the CRP response to myocardial infarction show that the peak level of CRP which is attained correlates well with the severity of tissue damage (Kushner et al., 1978; de Beer et al., 1982c) and that this level depends on the duration of increased CRP production rather than upon the initial rate of rise (Kushner et al., 1978: Macintyre et al., 1982).The kinetics of the human CRP response

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are compatible with rates of synthesis and turnover comparable to those seen with rabbit CRP or mouse SAP (see Section V,C). Apart from experiments which clearly show that leukocyte endogenous mediator preparations can induce CRP production in rabbits (Merriman et al., 1975) there is also indirect evidence indicating that the reticuloendothelial system and macrophages play an important role. For example administration to rabbits of carageenan, a mixture of long chain sulfated galactans which is known to be toxic to macrophages and to cause reticuloendothelial blockade (Schwartz and Leskowitz, 1969; Thompson et al., 1979), inhibits the CRP response to turpentine given shortly afterward (Macintyre et al., 1982). Direct evidence regarding IL-1 and initiation of acute phase protein synthesis in man is not yet available but intravenous injection of tiny doses of a nonpyrogenic endotoxin, which provokes peripheral blood neutrophil leukocytosis, also evokes a small CRP response (M. B. Pepys, F. C . deBeer, and A. J. Pinching, unpublished observations). Endotoxin is known to be a potent stimulus for the production of IL-1. Since CRP is a molecule composed of identical subunits the processes underlying synthesis and intracellular assembly of the subunits are of considerable interest. They have been investigated so far only in the rabbit and neither within hepatocytes nor in the secreted CRP has any material corresponding to free subunits or precursor forms of either the subunits or the whole molecule been detected using antiCRP antibodies (Kushner and Somerville-Volanakis, 1976). It is of course possible that an uncleaved precursor polypeptide might lack the epitopes of the native, secreted CRP. Answers to these questions should be provided by studies of CRP synthesis using recombinant DNA techniques and these are currently in progress. The control of CRP synthesis in lower vertebrates has been subjected only to limited investigation (White et al., 1981, 1982). In the plaice the serum CRP level is about 55 mg/liter and is the same in males and females. In contrast the lumpsucker has CRP levels which are two- to eightfold higher in males than in females and which may exceed 1 g/liter (Fletcher et al., 1977). PIaice which were maintained at a raised temperature, 18.5 compared to 10-12"C, showed no rise in their serum CRP concentration but injection of endotoxin caused an increase of about 10%in the CRP at 24 hours. Subsequently the CRP level returned to normal. Carageenan also provoked a similar rise with a peak at 4 days but other inflammatory agents such as turpentine, etiocholanolone, and Freund's complete adjuvant all caused a slow, progressive decrease in the CRP concentration. High doses of colloidal carbon, administered to plaice with the aim of blockading the re-

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ticuloendothelial system, had no effect on the CRP response to subsequent injection of endotoxin.

C. BIOLOGICAL PROPERTIES

1 . Zntroduction A number of different properties of CRP have been reported in terms of interactions with other biological macromolecules and with various cell types. All these properties are related to or initiated by the ligand-binding capacity of CRP. Some of them are readily reproducible and clearly of physiological or pathophysiological importance in viuo, but others which have been claimed, particularly some of the effects upon cellular functions, have either been refuted or remain to be confirmed. Human CRP is a very effective precipitin and agglutinin, of soluble and particulate ligands, respectively, and such ligands are widely distributed both within the body and in a range of different microorganisms, parasites (see Section IV,A), and lower animals (Baldo et al., 1977b). Human CRP which has been aggregated or formed a complex with a ligand is a potent activator of the classical complement pathway and this seems likely to be important for its in vivo function, though interestingly man is the only species identified so far in which isologous CRP activates isologous complement.

2 . Interactions with Lipids Binding of CRP to various lipids, in the form of emulsions of cholesterol with phospholipids and in the form of more carefully characterized liposomes, can be demonstrated directly with radiolabeled pure CRP and indirectly by agglutination of lipid particles and by complement activation. CRP can apparently bind to these lipids either via the phosphorylcholine polar head group in the phospholipid moiety or via positively charged groups on the ligand particle. Somewhat different observations have been made by various groups. With liposomes formed from dimyristoyl phosphatidylcholine, cholesterol, galactosylceramide, and stearylamine the binding is calcium independent, not inhibited by phosphorylcholine and not affected by substitution of dimyristoyl phosphatidylethanolamine for dimyristoyl phosphatidylcholine (Mold et al., 1981b). The actual ligand group is apparently provided by the positively charged stearylamine (Tsujimoto et al., 1981). With liposomes formed from phosphatidylcholine alone or together with cholesterol, no binding of CRP takes place and in this case inclusion of some stearylamine had no effect (Volanakis and Wirtz, 1979). However, incorporation of lysophos-

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phatidylcholine into the liposomes provided for excellent binding of CRP and the binding constant (7.1 X lop5M ) was of the same order as that between CRP and free phosphorylcholine (1.6 x M ) (Anderson et al., 1978; Volanakis and Wirtz, 1979). Further increases in affinity of binding occurred when small amounts of galactosyl ceramide are incorporated in the liposomes. All the binding was inhibitable by free phosphorylcholine and was calcium dependent. These observations suggest that in the absence of lysolipids the phosphorylcholine polar head group of the phospholipids in the artificial lipid membranes is not accessible to CRP but that when the structure is altered by their presence, binding of CRP can occur and is then enhanced by an accessory or secondary interaction with galactosyl residues (Volanakis and Wirtz, 1979). Analogous observations have been made with natural cell membranes. CRP does not bind to the intact plasma membranes of living cells i n vivo or i n vitro but if erythrocytes, as a model system, are modified appropriately they then express ligands for CRP. Such modifications include incubation with lysophosphatidylcholine, treatment with exogenous phospholipase A,, which specifically cleaves phosphatidylcholine to generate lysophosphatidylcholine, or simply osmotic lysis (Narkates andvolanakis, 1982).This was true both for human and for rabbit red cells but not for sheep red cells, which are known not to contain any phosphatidylcholine. Such results are compatible with the i n vivo finding of deposition of CRP on or in damaged and necrotic cells following local inflammation or ischemia (Kushner and Kaplan, 1961; Kushner et al., 1963), and indicate that despite the apparent capacity of CRP to bind a wide variety of ligands which are ubiquitous in the body it will do so only if rather strict requirements regarding the stereochemical presentation of the ligand(s) are met. This clearly has important implications for the i n vivo behavior and function of CRP. Another aspect of the interaction between CRP and lipids which may be relevant i n vivo is the effect of aggregation of CRP molecules on their ligand-binding properties. Expression of reactivity by aggregated or complexed CRP which is not manifested by the native molecule has previously been noted with respect to C l q and classical pathway complement activation (see Section IV,C,4). More recently it has been shown that aggregated human CRP selectively binds lowdensity lipoprotein (LDL) and traces of very low-density lipoprotein (VLDL) when exposed to whole normal human serum in vitro (deBeer)et al., 1982f).This contrasts with the behavior ofnative CRP in the plasma or serum where it coexists with all the phospholipidcontaining lipoproteins but does not seem to bind to them (de Beer et

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al., 1982e). Rabbit and rat CRP also show the same behavior, binding LDL from the isologous serum but in our hands only when the CRP has been aggregated (M B. Pepys, F. C. de Beer, M. L. Baltz, I. F. Rowe, and A. K. Soutar, unpublished observations; see Note Added in Proof, B). Earlier reports have described the coisolation of lipoproteins with human CRP and the presence of human CRP-lipoprotein complexes (Wood, 1963; Sat0 and Hara, 1968) but we were unable to detect any such complexing in acute phase sera examined by gel filtration or analytical ultracentrifugation (de Beer et al., 1982e). Using the same techniques we also failed to detect complexes between CRP and lipoproteins in rat or acute phase rabbit sera (M. L. Baltz, F. C. de Beer, and M. B. Pepys, unpublished observations) though other groups have reported that rabbit CRP in serum is either entirely in a complex with LDL (Pontet et al., 1979), or partly in a complex with VLDL (Cabana et al., 1982). These discrepancies may be due to different serum lipoprotein levels in the various animals tested, since in both rats and rabbits LDL levels are normally very low, especially by comparison with man. Alternatively, or in addition, it is possible that some in vitro aggregation of CRP may have permitted interaction with lipoproteins to occur. Thus in the procedures originally described for isolation of human CRP the initial step was precipitation, that is aggregation, of the CRP (MacLeod and Avery, 1941; McCarty, 1947; Wood et al., 1955) and there is evidence that exposure of isolated rabbit CRP to free ionized calcium causes aggregation (Potempa et al., 1982; see Note Added in Proof, B). In any event the binding of plasma lipoproteins, particularly the selective binding of LDL by aggregated human CRP, is of considerable interest and may have important implications both for the function of CRP and for the behavior of LDL. If CRP which had become complexed in the circulation with ligands of autologous or of extrinsic origin was capable of binding LDL the clearance and metabolism of both the CRP and the LDL could be affected. If, on the other hand, the CRP was complexed with ligands on damaged cells in situ, for example, in or beneath the vascular endothelium, then the secondary binding of LDL could result in local deposition of LDL, This may be relevant to the pathogenesis of atherosclerosis. Some indirect evidence for the in vivo uptake of LDL by CRP complexed with an extrinsic rather than an autologous Iigand is provided by the recent observation that Mycobacterium Zeprae organisms within the dermal lesions of human leprosy are coated with both CRP and LDL (M. Ridley, F. C. de Beer, and M. B. Pepys, unpublished observations).

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3. Interactions with Other Ligands Polysaccharides and peptido-polysaccharides containing phosphorylcholine, and with which CRP reacts, are widely distributed among bacteria, fungi, and metazoan parasites. CRP can also bind to certain materials which lack phosphorylcholine, such as, depyruvylated type 4 pneumococcal polysaccharide, dextran sulfate, nucleic acids, histones, and other polycations (see Section IV,A). Among common bacteria of clinical interest CRP binds much better to pneumococci than to any others, and best of all to type 27 which contains phosphorylcholine in the capsule as well as in the cell wall (Mold et al., 1982). There is some binding to Escherichia coli but none to Staphylococcus aureus (Kindmark, 1972). Binding of CRP to type 27 pneumococci brings about the capsular swelling reaction (Lofstrom, 1944) in the same way as antipneumococcal capsular antibody. Binding of CRP to ligands in, or derived from other organisms can evoke various secondary effects, including precipitation, agglutination, complement activation (see Section IV,C,4), and perhaps also the actions of complexed CRP on cells which have been proposed (see Section IV,C,5). 4 . Complement Activation Human CRP, which has been aggregated by itself or has reacted with phosphorylcholine-containing or cation-containing ligands or polycation-polyanion complexes, activates the classical complement pathway (Kaplan and Volanakis, 1974; Volanakis and Kaplan, 1974; Siege1et al., 1975; Osmandet al., 1975; Claus et al., 1977a,b; Richards et al., 1977, 1979) via C l q and C1 activation, and does so as efficiently as IgG antibody. All the functional and biological effects of complement activation are generated by CRP, including opsonization by fixation of C4b and C3b and lysis, if the ligand is at a cell surface (Mortensen et al., 1976; Mold et al., 1982; Osmand et al., 1975). In addition insoluble complexes of CRP with CPS are solubilized by complement activation in the same way as antigen-antibody complexes (Volanakis, 1982a,b). The precise mechanism by which complexed CRP binds and activates C l is not known, though possible sequence homologies between parts of CRP and the Cy2 domain, which is thought to be important in C l q binding, have been noted (see Section IV,A). In experiments with liposomes it has been found that the presence of some galactosyl residues in addition to accessible phosphorylcholine groups significantly enhances CRP binding, C l q binding, and C4 activation (Volanakis and

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Narkates, 1981). Similarly, desialation of erythrocyte membranes, which exposes terminal galactosyl residues, promotes uptake of CRP when these membranes have also been treated with phospholipase A2 or subjected to osmotic shock, and increases the efficiency of C l q binding (Narkates and Volanakis, 1982; Volanakis, 1982a). It is not yet clear whether these results are due to assumption of a different conformation by CRP which has interacted with both phosphorylcholine and galactosyl groups or to some other mechanism. Some of the ligands to which CRP binds, including positively charged liposomes and whole pneumococci, activate complement by the alternative pathway. When CRP becomes bound to these materials it inhibits activation by this pathway and initiates instead activation by the classical pathway (Mold and Gerwurz, 1981; Mold et al., 1982). The mechanism of this effect is not known but it clearly seems to involve attachment of CRP to the activating surface, since it is not seen with alternative pathway activators, such as rabbit erythrocytes, zymosan, lipopolysaccharide, and E . coli, to which CRP does not bind. Human CRP complexed with CPS activates rat (de Beer et al., 1982a) and mouse (Mold et al., 1982) complement but has no effect on rabbit (M. B. Pepys, unpublished observations) or guinea pig complement unless human C l q is provided (Volanakis and Kaplan, 1974). Rabbit CRP does not activate rabbit complement (H. Gerwurz, personal communication) and rat CRP does not activate rat complement (de Beer et al., 1982a). Plaice and lumpsucker (Cyclopterus lumpus L., another marine teleost) CRP can both activate human complement (Baltz et al., 1982a) while other interspecies compatibilities have not been tested.

5. Znteractions with Cells a. Introduction. The capacity of CRP to bind to intact, living cells and affect cellular functions would be of considerable importance for the biological role of CRP and there have been many reports in recent years of such phenomena. They have served to arouse great interest in CRP but unfortunately some of the effects described have proved to be due to contaminants of the CRP preparations or not to be reproducible for other as yet undefined reasons. This whole area is therefore in a rather fluid state and there will probably need to be substantial independent confirmation from different laboratories of the various interactions currently reported before a clear and generally accepted picture emerges. b. Lymphocytes. Isolated pure CRP does not bind to viable lymphocytes in culture nor does it significantly affect their activation by

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antigens, mitogens, or mixed lymphocyte culture reactions (James et al., 1981a,b, 1982; M. L. Baltz, V. M. Rumjanek, and M. B. Pepys, unpublished observations). Reports that human CRP selectively binds to human T cells and modifies their functions, that it blocks E-rosetting, and that it generates suppressor T cells in cultures of mouse spleen cells have not been confirmed either by the laboratory which first made the observations or by others (Mortensen et al., 1975, 1977; Mortensen and Gerwurz, 1976; Croft et al., 1976; Mortensen, 1979). Recent work by James et al. (1981a,b) indicates that CRP which has been complexed with CPS or aggregated on its own is bound b y a subset of those human peripheral blood lymphocytes which have Fc ( y ) receptors and includes B cells, T cells, and so-called “null” cells. The CRP-binding cells comprise only some 3.0% of total lymphocytes, though complexed or aggregated CRP was taken up by a larger proportion of monocytes (James et al., 1982). Exposure to these forms of CRP had no significant specific effect on lymphocyte functions tested in vitro (James et al., 1982). Williams et al. (1978, 1980; Williams, 1982) have described the presence of CRP on the surface of 10-35% of peripheral lymphocytes of patients with rheumatic fever, poststreptococcal chorea, or acute streptococcal infections, although there was no correlation with plasma level of CRP at the time the cells were taken. In contrast patients with acute myocardial infarction (Williams, 1982) or various other inflammatory and neoplastic diseases (M. B. Pepys, unpublished observations), all of whom had high serum CRP levels, generally had 5% or less of peripheral lymphocytes bearing detectable CRP and this did not differ from values obtained in healthy controls. The significance of these observations is not clear though in view of the results of James et al., (1981a,b, 1982), it is possible that streptococcal infection may provide suitable ligands to generate the type of CRP complex which is able to be bound by mononuclear cells. The nature and significance of the CRP detected on the surface of the small number of peripheral lymphocytes in healthy subjects and in patients undergoing acute phase responses also remain to be determined (James e t al., 1982). c. Platelets. The suppressive effect of some preparations of CRP on platelet aggregation, activation, and release reactions (Fiedel and Gewurz, 1976a,b; Fiedel et al., 1977) is now known to have been due to a coisolating low-molecular-weight material which contaminated these preparations (Fiedel et al., 1982a). This material has yet to be biochemically characterized and identified. Pure, isolated, native CRP does not affect platelet function (Fiedel et al., 1982a,b) but it has recently been reported that aggregated CRP or CRP complexed, particu-

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larly with polycations rather than with CPS, is able to aggregate and activate platelets (Fiedel et al., 1982b,c). d . Phagocytic Cells. There has been some controversy about whether CRP itself can function as an opsonin (Williams and Quie, 1968; Kindmark, 1971; Mortensen and Duskiewicz, 1977). It clearly does bind to some microorganisms and to ligands of autologous origin and, in the case of human CRP, there is no doubt that complement activation can then generate both chemotactic factors and the opsonic fragments of fixed complement proteins. There is evidence from in vitro studies with monocytes that, following complement fixation by CRP binding to a particulate ligand, the presence of both the CRP and the fixed complement is required for internalization, and that the cell surface interaction with bound CRP takes place via the Fc(y) receptor (Mortensen et al., 1976; Mortensen and Duskiewicz, 1977). Comparable results have been obtained in vivo in mice where there was enhanced splenic clearance of CPS-coated isologous erythrocytes only when they were coated with both human CRP and mouse complement (Mold et al., 1982). The presence of CRP alone did not promote clearance in decomplemented animals, nor did complement coating of the particles have any effect if the CRP was eluted in vitro before in vivo testing (Mold et al., 1982). Given bacteria to which CRP can bind, that is particularly pneumococci, there is convincing evidence that CRP can both opsonize in vitro and provide complete protection in vivo against otherwise lethal infection (Mold et al., 1981a, 1982; Edwards et al., 1982; Yother et ul., 1982). These in vivo experiments have been performed in mice into which isolated human CRP has been passively transferred and the protection conferred is comparable to that of antipneumococcal antibody. In contrast isolated SAP has no protective effect (Yother et al., 1982). D. FUNCTIONS

1 . Introduction The stable conservation of the existence, structure, and ligandbinding specificity of CRP suggests that it has an important biological role which is presumably beneficial to the organism. No human case of CRP deficiency has yet been described. However the secondary effects of ligand binding by CRP are different in different species, for example, rat CRP does not precipitate or agglutinate its ligands despite binding to them and neither rat nor rabbit CRP activate their isologous complement (de Beer et al., 1982a, and see Section IV,C,4).

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If indeed the function of CRP is the same or similar in all species then these secondary effects cannot be essential for whatever this function may be. The discovery of some secondary effect of binding which was common to CRP of all species would clearly be of importance and it is therefore interesting that aggregated rat and rabbit CRP both bind LDL from their autologous serum in oitro, in the same way as does human CRP (de Beer et al., 1982f; I. F. Rowe, F. C. de Beer, M. L. Baltz, A. K. Soutar, and M. B. Pepys, unpublished observations). Apart from, the general proposition that CRP, through its interactions with complement (in man at least) and with various cell types, may be involved in “modulation” of inflammatory reactions (Gewurz et al., 1982), three more specific hypotheses have been advanced regarding its function. Only the first of these is at present supported by direct experimental evidence.

2 . CRP as a Defense Mechanism against Microbial Infection The specific and yet broad spectrum ligand-binding reactivity of CRP in all species enables it to recognize products of a wide range of different microorganisms (see Sections IV,A and IV,C,3). Simply binding to these products may reduce their toxicity or role in pathogenicity, while involvement of secondary processes such as complement activation and phagocytosis may also contribute to protection against infection. The efficacy of this function, at least for human CRP in mice infected with pneumococci, has been clearly demonstrated (Mold et al., 1981a, 1982; Yotheret al., 1982) and supports the concept that CRP may act in this way both in lower animals, which have not evolved specific antibody mechanisms, and in higher animals at an early stage of infection, before production of specific antibody gets underway.

3 . CRP in the Resolution and Repair of Damaged Tissue Following acute inflammatory or ischemic tissue damage in rabbits, CRP is deposited on necrotic cells (Kushner and Kaplan, 1961; Kushner et al., 1963), and we have recently made similar observations in rats (S. Holford and M. B. Pepys, unpublished observations). Volanakis has shown that human CRP does not bind to the intact membranes of healthy living cells, or to artificial membranes which mimic their composition and structure, but does bind to damaged or altered cell or artificial phospholipid membranes, and having done so then activates complement (see Sections IV,A and IV,C,2). He has therefore suggested that the role of CRP may be to recognize damaged cells and their products in situ, and by then activating complement to generate the chemotactic and opsonic activities required to promote

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phagocytosis, leading to resolution and repair of the lesion (Volanakis, 1982a). This is an attractive concept but is not yet supported by any direct in vivo evidence. The failure of CRP in species other than man, which have been investigated so far, to be complement fixing (see Section IV,C,4), and the relative paucity of reports of CRP deposited at local sites of inflammation or tissue damage in man (see Section IV,D,5), must be considered.

4 . CRP and the Clearance of Abnormal Materials

from the Circulation Increased production of CRP is stimulated as efficiently by autologous damage in the absence of infection as it is by infection or by nonviable microbial products and the ligand specificity of CRP is also directed toward many substances of autologous origin. During the course of tissue damage a variety of tissue and cellular constituents, which are not normally found there, may gain access to the plasma. Since CRP is a plasma protein it has been suggested that a major role of CRP may be to complex with such abnormal materials in the circulation and to detoxify them and/or facilitate their clearance (Pepys, 1981a). If an abnormal material of extrinsic origin, such as a microorganism or microbial product, is in the plasma it too may have its clearance enhanced by CRP provided that it bears appropriate ligand(s). This function of CRP has been demonstrated with pneumococci and with CPS-coated erythrocytes (Section IV7C,5,d).It has yet to be demonstrated for ligands of autologous origin and in fact using analytical ultracentrifugation, gel filtration chromatography, and radiometric assay for complexed CRP it has been shown that only trace amounts, if any, of the CRP in human acute phase serum exist in the form of complexes with high-molecular-weight ligands (de Beer et al., 1982e,f). The absence of circulating complexes of CRP does not of course exclude the clearance hypothesis since, if the latter is correct, it is to be expected that any such complexes would be rapidly cleared and not persist in the plasma. Various mechanisms are possible by which circulating complexes of CRP with its ligands may be cleared. Direct interaction of complexed or aggregated CRP with cell surface receptors on fixed or circulating phagocytic cells, or even on other cells, should be considered, though the existence of receptors for CRP has not yet been firmly established (see Section IV,C,5). In man, complement fixation by CRP provides an obvious candidate mechanism, while in man and the other species in which binding of LDL occurs, this interaction may be involved. The mere physical size of aggregates between CPR-ligand complexes and

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LDL particles should promote their uptake and clearance while cell surface LDL receptors could also play a part. Further work is required to determine whether and to what extent these phenomena occur in vivo. 5 . Znjlammation Mediated by CRP Intradermal or intracutaneous injection of CPS in patients with acute pneumococcal pneumonia, with other acute infections, or with rheumatic fever, elicits an immediate wheal and flare reaction which resolves over an hour or so and is then followed by a nonimmediate reaction (Francis and Abernethy, 1934; Finland and Dowling, 1935). This starts at 2-3 hours, is maximal at 6-10 hours, and gradually resolves over the next 24-72 hours. The lesion is edematous and erythematous and may have a central hemorrhagic area. In normal healthy subjects, or convalescent patients whose serum CRP levels have fallen toward normal, injection of CPS has no effects. Further studies of these reactions in man have not been reported in the literature since their first description in the 1930s. Despite their having been called “delayed” reactions by the original workers, the time course and clinical features correspond more closely to the antigen-antibody complex-mediated, type 111, or Arthus reaction than to classical delayed type, tuberculin, or type IV hypersensitivity. Experimental evidence also favors the Arthus-type mechanism. Isolated human CRP, aggregated chemically with bis-diazotized benzidine, activates human complement and generates chemotactic attractants for human neutrophils and eosinophils in vitro (Parish, 1977). Incubation of neutrophils with aggregated CRP and complement leads to phagocytosis and intracellular degradation of the aggregates and elicits release of lysomal enzymes into the medium. Aggregated IgG and complement have similar effects but, while aggregated IgG is still active in the absence of complement, the aggregated CRP is not (Parish, 1977). Similarly intracutaneous injections of aggregated human CRP into guinea pigs or rabbits had no inflammatory effect (Parish 1977) and it is known that human CRP does not activate complement in these species (see Section IV,C,4). However, injection of aggregated CRP which had previously been incubated with human complement and had fixed human Clq, so that it was capable of activating both guinea pig and rabbit complement, elicited typical lesions with neutrophil infiltration and fibrinoid necrosis (Parish, 1977). It therefore seems clear that, at least in man, CRP has the potential for causing or enhancing inflammation as a result of its ability, having become aggregated or complexed with one or more of its many possi-

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ble ligands, to activate the complement system. Some direct evidence of such a role for CRP in human pathology is already available (see below) and the possibility of this mechanism being operative should be considered in any inflammatory or tissue-damaging process in which circulating CRP levels are high. Immunohistochemical studies of the lesions of spontaneous cutaneous vasculitis have revealed the presence of CRP in up to 40% of cases, with a higher incidence in lesions consisting chiefly of neutrophils rather than mononuclear cells (Parish, 1976). C3 was usually also found in the same sites. While it is possible that the CRP in this disorder may be enhancing inflammation and contributing to the chronicity of the lesions, it may also be promoting resolution and itself being degraded by the neutrophils. In this respect it is of interest that rabbit CRP has been demonstrated, predominantly within neutrophils, in the spinal lesions of experimental allergic encephalomyelitis. No CRP was found in lesions infiltrated exclusively with mononuclear cells (du Clos et al., 1981). The presence of CRP apparently bound to nuclear structures within synoviocytes and histiocytes has been reported in synovial biopsies from patients with rheumatoid arthritis (Gitlin et al., 1977). The significance of this finding is not known. CRP was not detected in similar biopsies or autopsy specimens from osteoarthritic or normal joints. No CRP was present in renal biopsies from cases of anaphylactoid purpera sclerosing or proliferative glomerulonephritis (Gitlin et d.,1977), and we have also failed to detect CRP in renal biopsies of patients with systemic lupus erythematosus, other collagen vascular diseases and systemic vasculitides, membranous glomerulonephritis, mesangiocapillary glomerulonephritis, diabetes, or minimal change nephritis (R. F. Dyck, D. Turner, D. J. Evans, and M. B. Pepys, unpublished observations). Further work is evidently required to clarify the extent to which tissue deposition of CRP is a feature of various inflammatory or other tissue-damaging processes in man and, if it is present, the manner in which it contributes to local lesions (Figs. 3 and 4). A fascinating example of a human pathological process in which CRP may be essential is provided by the studies of the mechanisms of hemolysis caused by the venom of the brown recluse spider, Loxosceles reclusa (Hufford and Morgan, 1981). The venom is not directly lytic but sensitizes human erythrocytes for lysis by complementsufficient human serum. This complement-dependent hemolysis is itself dependent on the presence of the trace amounts of CRP which are normally present in serum of healthy adults. Cord serum contains extremely low levels of CRP and despite adequate complement does not

173

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FIG.3. Serum C-reactive protein (CRP) levels in patients with rheumatoid arthritis. Each point represents the concentration in a different individual (Mallya et al., 1982b).

lyse venom-treated red cells. Addition of as little as 200 ng/ml of isolated pure CRP enables cord serum to mediate lysis. Apart from intravascular hemolysis, which occurs only rarely, there is normally a characteristic, gangrenous skin lesion at the site of the spider bite. In experimental animals, it has been shown that polymorphs and complement are required for evolution of this lesion (Smith and Micks, 1970) and it is possible that CRP may be involved here also. The mechanism of the immediate wheal and flare reaction to injection of CPS in patients with raised CRP levels is not known, though it may result from generation of the anaphylatoxins, C3a and C5a, in the course of CRP-mediated complement activation. Alternatively it is possible that complexed CRP itself may be able to interact directly

174

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FIG. 4. Serum C-reactive protein (CRP) levels and erythrocyte sedimentation rates (ESR) in patients with systemic lupus erythematosus. Each point represents the median of several measurements in an individual patient while he or she was in a particular disease category (Pepys et al., 1982e).

with mast cells, basophils, or other cell types capable of producing or releasing rapidly acting inflammatory mediators. However, we have not been able so far to show that either CRP alone or CRP-CPS complexes cause histamine release from whole human peripheral blood or separated peripheral blood leukocytes either in the presence or absence of complement (S. Holford and M. B. Pepys, unpublished observations). The capacity of CRP from other mammals to mediate inflammatory processes has not yet been investigated but CRP in at least some species of teleost fish resembles human CRP in being able to cause immediate erythematous reactions (Fletcher and Baldo, 1974; Baldo and Fletcher, 1975a,b; Fletcher et al., 1980; Baltz et al., 1982a). In-

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tradermal injection of CPS, or CPS-like materials of fungal origin, in the plaice, which has normal serum levels of CRP of about 55 mg/liter, elicits immediate erythema and this can be prevented by pretreatment of the fish with cromoglycate or diethylcarbamazine but not by antihistamines or anti-serotonin drugs. The flounder (PEatichthysflesus L.) is a marine teleost closely related to the plaice but which does not have any demonstrable CRP in its serum and in which injection of CPS elicits no reaction. Passive transfer of whole plaice serum or of isolated plaice CRP confers on the flounder the capacity to react to CPS and i n vitro incubation of excised skin reaction sites from passively sensitized flounders yields prostaglandin E-like material (A. Anderson, A. White, and T. C. Fletcher, unpublished observations). E. MEASUREMENTI N CLINICALPRACTICE

I. lntroduction With the advent of precise immunochemical assays for CRP there has been renewed interest in the value of serum CRP measurements in clinical practice. The nonspecific nature of the acute phase response, coupled with the relatively crude semiquantitative assays which were previously available, caused clinical CRP tests to fall into disfavor. However it is now becoming increasingly clear that availability at the bedside or in the clinic of measurements of CRP, and possibly also other acute phase proteins such as SAA, can make a useful contribution to patient management.

2 . CRP Response in Disease Major elevations of the serum CRP concentration are seen in most severe infections, in individuals of any age, the actual degree of elevation usually corresponding reasonably well with the severity of the infection (Sabel and Hanson, 1974; Sabel and Wadsworth, 1979; Moodley, 1981). Bacterial infections are usually the most potent stimuli to CRP production and in patients with bacterial meningitis the CRP level is apparently always greater than it is in cases of viral or aseptic meningitis (Corral1et al., 1981; Peltola, 1982). This distinction is not true with infections elsewhere, for example, in the respiratory track (Salonen and Vaheri, 1981). Less information is available about parasitic and fungal diseases though any serious infection, even with opportunistic organisms in immunocomprised hosts, usually causes high CRP levels. In all the other conditions listed in Table I11 the CRP

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TABLE I11 C-REACTIVE PROTEIN RESPONSE I N DISEASE Conditions associated with major elevation of serum CRP Infections Allergic complications of infection Rheumatic fevera Erythema nodosum leprosumb Rheumatoid arthritisC(Fig. 3) Inflammatory disease Juvenile chronic arthritisd Ankylosing spondylitise Polymyalgia rheumaticaf Systemic vasculitis‘ Behcet’s syndromeh Reiter’s disease‘ Psoriatic arthritid Crohn’s diseasek Familial Mediterranean fever’ Malignant neoplasiam Lymphoma, Hodgkin’s Carcinoma, sarcoma Ischaemic necrosis” Myocardial infarction Surgery Trauma” Burns Fractures Anderson and McCarty, 1950; * Srivastava et al., 1975; McConkey et al., 1972, 1973, 1979; Amos et al., 1977; Walsh et al., 1979; Mallya et al., 1982b,c; Pepys, 1981b; Mellbye et al., 1978; Cowlinget al., 1980; R.K. Mallya and M. B. Pepys, unpublished observations; 0 C. R. K. Hind and M. B. Pepys, unpublished observations; Lehner and Adinolfi, 1980; R. F. Dyck and M. B. Pepys, unpublished observations; Laurent et al., 1981; Pepys et al., 1977c; Fagan et al., 1982; I Frensdorff et al., 1964; Wood et al., 1958; Graf and Rapport, 1958; Holm and Pompeius, 1961; McFarlane et al., 1967; Child et al., 1978, 1980; Cooper et al., 1978, 1979; Bastable et al., 1979; te Velde et al., 1979; Cooper and Stone, 1979; Trautner et al., 1980; Drahovsky et al., 1981; O’Quigley et al., 1981; Rashid et al., 1982; Richards et al., 1982; ” Kroop and Schackman, 1957; Levinger et al., 1957; Johansson et al., 1972; Kushner et al., 1978; Crockson et al., 1966;Aronsen et al., 1972; Fischer et al., 1976.

’

J

level reflects activity of the disease and in severe cases high levels are always seen. Furthermore, following the acute onset or exacerbation of disease the CRP level rises rapidly, reaching levels of over 200 mg/ liter in 48 hours in some cases and with rapid remission occurring spontaneously or after therapy, for example, with steroids or antibiotics, it can fall at approximately the same rate. In contrast there are some disorders, listed in Table IV, in which, despite active tissuedamaging inflammatory processes, the CRP level rises only modestly if at all.

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TABLE IV C-REACTIVEPROTEIN RESPONSEIN DISEASE Conditions associated with minor elevation of serum CRP Systemic lupus erythematosusO (Fig. 4) Scleroderma* DermatomyositisC Sjogren’s syndromed Ulcerative colitise Leukemid a Hill, 1951; Honig et al., 1977; Pepys et al., 1978b, 1982d; Perreira da Silva et al., 1980; Bravo and Alarcon-Segovia, 1981; *Whither et d., 1980; “Haas et al., 1982; Montsopoulos et al., 1983; Pepys et al., 1977c; Fagan et al., 1982; Mackie et al., 1979; Rose et al., 1981; Starke et al., 1983.

’

3. Clinical Applications of CRP Measurement On the basis of these observations a case can be made for measurement of serum CRP in diagnosis and management of a wide range of different clinical conditions. The general situations in which it is useful are shown in Table V (for specific references see Tables I11 and IV). Elevation of the serum CRP concentration is a sensitive and unequivocal sign of an organic tissue-damaging disease process and CRP measurement therefore provides a useful screening test, for example, in outpatient consultation. This case is strengthened by the fact that a precise, reproducible objective serum test is involved and rapid, simple assay systems are becoming available. In those disorders, activity of which is associated with major elevation of serum CRP, CRP levels are often the best and most sensitive objective index of disease activity. This is useful for monitoring the response to established and experimental forms of therapy, particularly

TABLE V CLINICALAPPLICATIONS OF SERUMCRP MEASUREMENT Screening for organic disease Monitoring of extent and activity of disease Infection Inflammation Malignancy Necrosis Detection of intercurrent infection

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in diseases such as Crohn's disease (Fig. 5) or some forms of systemic vasculitis in which the clinical manifestations are less accessible to direct assessment than they are, for example, in rheumatoid arthritis. It is also useful in management of microbial infections which are not

severe moderate mild Predn isolone (183)

t

i

;eb *

July 1979 (days) 1979 (months) 1980 Hospital admission FIG.5. Twenty-six-year-old man with pan-colonic Crohn's disease. Single episode of bloody diarrhea in 1973. Admitted with severe exacerbation 6 July 1979, temperature 38"C, pulse llO/minute, 16 stooldday, hematocrit 41.5%, leukocytes 13.8 x 10Vliter. Rectal mucosa severely inflamed with histiocytic granulomata on biopsy. Rapid improvement occurred on treatment with oral and rectal prednisolone, ampicillin, and metronidazole with complete clinical and histological remission on day 11 (17 July). Relapse 5 months later which responded promptly to a short course of oral and rectal prednisolone. CRP and ESR were both high during the initial severe exacerbation. The rapid response to treatment was paralleled by a prompt fall in CRP whereas the ESR responded more slowly on a day-to-day basis. Despite clinical remission and a normal ESR the CRP remained slightly elevated and rose further during a subsequent relapse when the ESR did not change (Fagan et al., 1982).

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readily accessible to standard microbiological techniques, for example, in monitoring bacterial meningitis in neonates and infants (Sabel and Hansen, 1974; Sabel and Wadsworth, 1979; Corral1 et al., 1981; Peltola, 1982), in detection of infection following premature rupture of membranes (Evans et al., 1980), in female pelvic inflammatory disease, and in differential diagnosis of pelvic masses in women (Hajj et al., 1979; Angerman et al., 1980). It has been claimed that serum CRP levels may assist in identification of the level of urinary tract infection in girls (Jodal et al., 1975) but recent work does not support this view (Hellerstein et al., 1982). In malignancy, especially with metastases, the incidence of raised CRP levels is high and correlates well with other assessments of tumor load. I n some tumors, such as prostate or bladder carcinomas, the initial CRP values at presentation also correlate with prognosis. In those conditions which on their own do not provoke large rises in CRP concentration, the CRP response is a sensitive sign of intercurrent infection. This is the case in systemic lupus erythematosus (Figs. 6 and 7) and in leukaemia. Failure of the initial CRP response to subside or the occurrence of secondary elevation in patients recovering from active SLE (n -211

infection (n = 13) Y 0

: n

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: -i-

0 0

0

0

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-v-

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0 0

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FIG.6. Serum C-reactive protein (CRP) levels and erythrocyte sedimentation rates (ESR) in febrile patients with systemic lupus erythematosus in the presence and absence of intercurrent microbial infection (Pepys et a!., 1982e).

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M. B. PEPYS AND MARILYN L. BALTZ

cefurox irne gentamicin

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/->prednisolone abdominal pain diarrhoea E. coli septicemia

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FIG.7. Twelve-year-old girl with 2 year history of SLE characterized by recurrent febrile episodes associated with polyarthritis and cutaneous vasculitis. Two months before admission to Hammersmith she had been hospitalized elsewhere with an illness resembling Stevens-Johnson syndrome which had been ascribed to sensitivity to amoxycillin and/or to a Pseudomonas septicemia. On admission to Hammersmith Hospital she had a remittent pyrexia to 40"C,polyarthritis, erythematous skin rash, and three tender subcutaneous nodules. Her serum CRP was 10 mg/liter suggesting that the nodules were lupus profundus lesions rather than deep cellulitis or subcutaneous abscesses. Her dose of prednisolone was increased, azathioprine and plasma exchange were introduced, and, although no antimicrobial therapy was given, she went into remission. Over the next few months there were two episodes of asymptomatic E. coli bacteriuria which were treated with antibiotics. Six months after the first Hammersmith admission fever recurred in association with diarrhea and abdominal pain but without specific renal tenderness. All microbial cultures were negative except for the growth of E . coli from the urine. She received oral cephalexin but her condition deteriorated over the next 2 days and severe neutropenia, probably due to azathioprine, developed. Her CRP rose from 36 to 101 and then 137 mg/liter and at this stage her blood culture grew E. coli. lritravenous antibiotics were given and the serum CRP level fell rapidly, but there was little clinical improvement. Active SLE appeared then to be the sole cause of the fever and this was confirmed by the gradual development of a diffuse vasculitic rash and polyarthritis. Three pulse doses of 1 g of methylprednisolone were therefore given intravenously on successive days and produced a dramatic improvement in her clinical state with resolution of the fever (Pepys et al., 1982d).

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major surgery or myocardial infarction signal the presence of infective or other complications, such as thromboembolism. Since the CRP response is nonspecific a CRP value on its own can never be diagnostic, however, when interpreted in conjunction with the rest of the. clinical and laboratory investigations, and with access to the patient’s relevant history, it can make a valuable contribution to important clinical decisions. The concept of a nonspecific test for extent and activity of disease is, of course, not new and the erythrocyte sedimentation rate has long been and still is very widely used for this purpose (Kushner, 1981; Pepys, 1979b). To some extent, the sedimentation rate provides different information from the serum CRP level, for example, it can detect paraproteinaemias in the absence of an acute phase response. However, in the area where they overlap, CRP measurements have many advantages. Thus CRP levels respond much more rapidly than sedimentation rate to changes in disease activity and cover a much wider incremental range. The CRP concentration can increase from 100 pg/liter to 300 mg/liter in 2-3 days and with spontaneous recovery or appropriate therapy fall again at about the same rate. In contrast, the sedimentation rate changes within a much smaller range over a period of days or weeks. Furthermore, the sedimentation rate does not show the same sensitive response as the CRP to infection, nor the same discriminant difference between diseases like rheumatoid arthritis and SLE (Perreira da Silva et aZ., 1980; Pepys et d . , 1982d) (Fig. 8 ) or ulcerative colitis and Crohn’s disease (Fagan et d., 1982). Although measurement of sedimentation rate is simple, cheap, and readily accessible, it is often not performed correctly so that results may not be reproducible. There is also a significant diurnal variation in sedimentation rate associated with food intake, which has only recently been recognized, and should be considered if changes in sedimentation rate are to be taken as precise indicators of inflammatory activity (Mallya et aZ., 1982a). In contrast, serum CRP does not show this diurnal variation and it can be measured rapidly and precisely by sensitive immunochemical assays for which commercial kits are becoming generally available.

4 . Differential CRP Responses The finding that although the CRP response is nonspecific it nevertheless shows quantitative differences in different individuals and in different diseases is both clinically useful, as discussed above, and of considerable fundamental interest. It is possible that the different CRP levels seen in systemic lupus and rheumatoid arthritis or in ulcerative colitis and Crohn’s disease are due to differences in the intensity of

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150

ESR

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patients on steroids

CRP

0

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0

E

wl w

50

x 8

8

0.

9

11 o#

0 RA

SLE

RA

3

S LE

FIG.8. Serum C-reactive protein (CRP) levels and erythrocyte sedimentation rates (ESR) in patients with active arthritis due to rheumatoid arthritis or systemic lupus erythematosus. Each point represents the value in a different individual (Perreira da Silva et al., 1980).

stimulation provided by the underlying pathological process in each disease. However there is extensive tissue damage and inflammation in the “low responder” diseases, such as lupus and ulcerative colitis, and an alternative explanation may be that some mediator, or mediator-receptor or other component of the mechanism for inducing the acute phase response is inoperative in the affected individuals. They remain capable of mounting acute phase responses to microbial infection, so the basic synthetic and secretory apparatus is able to function in response to certain stimuli, though it is difficult to determine whether the level of response is the same as that mounted by other individuals suffering the same infection. Direct evidence in man of impaired responses to certain stimuli is provided by the observation that infusion of PGEl into patients with systemic sclerosis induces little or no increase in the level of CRP (or SAA) whereas there are major increases in control subjects (Whicher et al., 1980). Furthermore, in contrast to normal individuals and patients with rheumatoid arthritis, the peripheral leukocytes of the systemic sclerosis patients produced little if any IL- 1-like activity following incubationin vitro with heat-killed staphylococci (Whicheret al., 1982).

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These defects could be secondary to the on-going pathology of the diseases in question or they may be genetically determined. The normal and acute phase levels of SAP (Pepys et al., 1979a) and of C3 (Natsuume-Sakai et al., 1977) are under genetic control in inbred strains of mice and may reflect the capacity to respond to endogenous mediators such as prostaglandins, to exogenous stimulants such as microbial products or to produce and respond to IL-1. Further work is required to determine whether human CRP and other acute phase protein responses are also under genetic control and an ethical, standard acute phase stimulus would be desirable for this purpose. In view of the possible role of CRP and other acute phase proteins as modulators of inflammation, primary (genetic) or secondary processes which regulate the capacity to mount an acute phase response might contribute to pathogenesis of inflammation. Some recent experimental findings favor the concept of an intimate association between acute phase protein responsiveness and particular diseases. NZB/W mice which develop a spontaneous autoimmune disease closely resembling human lupus (Theofilopoulos and Dixon, 1981), fail to mount any acute phase response to progress of their disease despite being able to respond apparently normally to exogenous stimuli such as injections of LPS or casein (Rordorfet al., 1982). On the other hand MRL/l mice, which develop a spontaneous autoimmune disease more closely resembling human rheumatoid arthritis (Hang et al., 1982),mount a major acute phase response to increasing severity of their disease (Rordorf et al., 1982). Availability of these animal models should facilitate investigation of the underlying mechanisms and elucidation of their possible relevance to human disease. V. Serum Amyloid P Component

A. STRUCTURE Human SAP consists of ten identical glycosylated polypeptide subunits, each of molecular weight about 23,500, which are noncovalently associated in two pentameric discs interacting face-to-face (Osmand et al., 1977; Pinteric et al., 1976). SAP in the plaice has a very similar decameric structure (Pepys et al., 1978a, 1982c), but in rats and mice the SAP seems to be pentameric, corresponding more closely to the configuration of human CRP (de Beer et al., 1982a; Balk et al., 1982a). The SAP of all species studied so far is glycosylated (Baltz et al., 1982a). The circular dichroism spectrum of human SAP in the 200-240 nm

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range closely resembles that of human CRP but there are differences in the 240-320 nm range indicating differences in aromatic amino acid residues (Young and Williams, 1978).The almost complete amino acid sequence of human SAP which is available shows 50% strict, residuefor-residue identity with human CRP and taking conservative substitutions into account, the degree of homology approaches 70% (Anderson and Mole, 1982) (Fig. 2). In contrast to CRP, where no polymorphism has been described, the sequence of SAP derived from a pool of different individuals shows some microheterogeneity, glycine having been detected together with methionine, serine, and proline at residues 140, 143, and 146, respectively (Anderson and Mole, 1982). There may thus be different polymorphic or allotypic forms of SAP and, although this should be confirmed by further studies, it would be of considerable interest with respect to amyloidosis (see Section V,D). Another point of interest is that in the portion of SAP corresponding to the CRP sequence Phe-Tyr-Thr-Glu, which is part of the putative binding site for phosphorylcholine, there is a substitution of Asp for Glu-42 (Anderson and Mole, 1982). This may explain in part the failure of SAP to bind phosphorylcholine, other phosphate monoesters or CPS. It is interesting that rabbit CRP shows this same difference with respect to human CRP (Bach et al., 1977), that the precise binding specificity of rabbit CRP for phosphorylated ligands differs from that of human CRP (Oliveira et al., 1980), and that rabbit CRP shows a notably stronger binding affinity for agarose than does human CRP (M. B. Pepys, unpublished observations). In this last respect rabbit CRP resembles human SAP. It will be of great interest to know the sequence of other SAPSand CRPs, such as those of mouse and rat, respectively, to see if the residues in this region correlate with ligand specificity. Such sequencing studies are currently in progress (J. Taylor, C. Bruton, J. K. Anderson, J. E. Mole, M. L. Baltz, F. C. de Beer, D. Caspi, and M. B. Pepys, unpublished observations; see Note Added in Proof, A).

B. LIGANDBINDING Human SAP was first discovered to have calcium-dependent ligand binding properties with respect to agarose (Pepys et al., 1977a,b). SAP of other species has been isolated and identified on the basis of this same binding specificity (reviewed by Baltz et al., 1982a; Pepys et al., 1982a). Subsequently SAP was found also to undergo calciumdependent binding in uitro to amyloid fibrils of either AA or AL type (Pepys et al., 197913).More recently it has been shown that aggregated human SAP expresses new reactivities which are not expressed by

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native, nonaggregated SAP (de Beer et al., 1981; Pepys et al., 198213). These reactivities are directed toward two normal plasma proteins, fibronectin and CPbinding protein, and when suitably immobilized SAP is exposed to whole normal human serum it selectively binds these two proteins in a strictly calcium-dependent reaction. A pair of SAP 'molecules, for example, bound by an immobilized IgG anti-SAP molecule, binds a single molecule of fibronectin. SAP within the plasma or in serum is not bound to either fibronectin or C4-binding protein and apparently exists freely as single, uncomplexed molecules. When isolated, pure human SAP is exposed to free ionized calcium it rapidly aggregates and, if the protein concentration is sufficient, it precipitates out (Baltz et al., 198213).The mechanism of this phenomenon is not known but it does not seem to depend on the charge or sialation of the SAP since desialated SAP behaves in the same way (M. L. Baltz and M. B. Pepys, unpublished observations). Mouse and plaice SAP do not show the same effect (Baltz et al., 1982b),nor do human (de Beer et al., 1982f) or rat CRP (M. L. Baltz, F. C. de Beer, and M. B. Pepys, unpublished observations), but rabbit CRP does (Potempa et al., 1982) though to a lesser extent than human SAP. Calcium-dependent aggregation of the affected proteins during isolation or test procedures in uitro can be responsible for secondary interactions which may not occur under more physiological conditions or in uiuo. For example, isolated human SAP or SAP in whole serum bind to and agglutinate C3bi-coated sheep erythrocytes (Hutchcraft et al., 1981), but only in the presence of supraphysiological levels of ionized calcium (Baltz et al., 1982~).Native SAP in normal serum conditions does not bind to fixed C3bi (Baltz et al., 1982~). A similar phenomenon may underlie reports of apparent complces of rabbit CRP with LDL or VLDL in serum (Pontet et al., 1979; Cabana et al., 1982). The precise chemical nature of the ligand(s) for SAP is not known. Agarose is composed of repeating units of agarobiose, that is, 1,Slinked P-D-galactopyranose and 1P-linked 3,6-anhydro a-L-galactopyranose (Araki, 1956), but free galactose and lactose have no effect on SAP binding to agarose (M. L. Baltz and M. B. Pepys, unpublished observations). There are very marked differences between different lots of commercially available agarose, whether obtained in beaded or powder form, in their capacity for SAP binding (Pepys et al., 1979b), suggesting that a minor and variable constituent of the biopolymer provides the ligand for SAP. In view of the fact that SAP undergoes calcium-dependent binding, albeit at a low level, to sulfated polyacrylamide beads we previously investigated the possibility that the

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SAP might be binding to sulfate groups in agarose. However there was no correlation between capacity for SAP and the sulfate content of different agaroses, nor did deliberate introduction of sulfate groups into a low sulfate, low SAP-binding agarose increase its capacity. Another minor constituent of agarose is pyruvate and we have recently shown that the capacity of different agarose preparations for SAP correlates closely with their pyruvate content (C. R. K. Hind, M. B. Pepys, and D. Renn, unpublished observations) and further work is in progress to characterize more precisely the configuration of the ligand in agarose. The in vivo physiological or pathophysiological ligand for SAP is clearly not just any anionic group capable of electrostatic association with calcium, since many polyanions are not bound by SAP. A particular case in point is heparin and, despite claims to the contrary (Thompson and Enfield, 1978; Boxer et al., 1977), SAP does not bind significantly to heparin (Pepys e t al., 1980~). Furthermore under suitable conditions, as described above, SAP shows selective binding to particular specific plasma proteins. A hint of the possible nature of a ligand group for SAP in proteins is provided by some recent observations. First, amyloid fibrils consist of polypeptide chains arranged in antiparallel &pleated sheets and the conformation in which these chains fold back upon themselves are known as &turns (Glenner, 1980a,b; Cooper, 1983). The &turn forms when there are many glycine residues appropriately distributed among bulky and hydrophobic L-amino acids (Urry, 1974). It consists of a 10 atom hydrogen-bonded ring inserted into the polypeptide backbone such that the end peptide moiety has its peptide acyl oxygen directed nearly perpendicular to the plane of the other atoms (Urry, 1974).This is an exposed position in which it is capable of binding cations and in fact sequences which form @-turnsbind Ca2+with a highly selectivity over Na+, K+, or (Urry, 1974). Second, we have recently shown that in man normal elastic fibers throughout the body bear SAP on their surface and in association with their peripheral microfibrillar mantle (Breathnach et al., 1981a,b, 1983). Elastin was the first protein in which p-turns were described (Urry, 1974) and, just like amyloid fibrils, elastin is known to have an affinity for binding calcium. In view of the probability that pyruvate residues in agarose are the ligand for SAP and the fact that pyruvic acid has a carbonyl group which could bind calcium ions, it now seems possible that SAP is specifically equipped to recognize peptide sequences which include calcium-binding carbonyl groups in appropriate spatial orientation. The presence of such groups may be the feature in common between such apparently diverse protein

Me+

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ligands of SAP as amyloid fibers, elastic fibers, fibronectin, and C4binding protein. C. SERUMLEVELS The serum concentration of SAP is relatively stable in man, females (mean f SD, 33 2 10 mg/liter) having slightly but significantly lower values than males (43 2 14 mg/liter) (Pepys et al., 1978b). The levels in cord sera are much lower (4 & 2 mg/liter) (Pepys et al., 197813) but rise rapidly during the first weeks of life to reach the lower part of the adult range, around 10-20 mg/liter (R. F. Dyck, P. A. Davies, and M. B. Pepys, unpublished observations). In chronic inflammatory or neoplastic diseases in which CRP levels tend to be raised, the SAP concentration also rises but generally remains within the normal range and does not exceed 100 mg/liter (Pepys et al., 197813). Patients with macroglobulinemia may be exceptional in having higher SAP values than this even with modest rises in CRP (Jensson et al., 1982). In our experience acute stimuli such as major surgery are not followed by any significant or consistent change in SAP levels (Pepys et al., 1980a), however, Hashimoto and Migita (1979) have reported a doubling of SAP levels by the third postoperative day in Japanese subjects undergoing surgery. Among the other species in which SAP levels have been measured, mice are exceptional in two respects: first, there are marked genetically determined differences in normal levels between different inbred strains, and second, SAP is a major acute phase reactant in all strains (Pepys et al., 1979a). C57BL mice and lines with the same genetic background have low normal SAP levels of up to 10 mg/liter, while ostensibly healthy DBA/2 animals may be as high as 100 mg/ liter. Other strains such as C3H, BALB/c, CBA, N J , and SJL have intermediate levels between 20 and 80 mg/liter but crosses with C57BL mice always produce progeny with low levels (M. L. Baltz, K. Comer, A. J. S. Davies, K. P. W. J. McAdam, and M. B. Pepys, unpublished). Breeding experiments using these strains and also various recombinant lines are in progress in order to elucidate the genetic basis of this phenomenon. Preliminary results indicate that more than one locus is involved. Acute phase levels of mouse SAP are derived largely by d e novo synthesis of the protein, in the liver, rather than from release of preformed stocks (Baltz et al., 1980~). The tlI2of 12sI-labeledSAP measured both by whole body counting and by plasma sampling is about 7.5-9.5 hours in all strains studied (C57BL, CBA, BALB/c, DBA/2)

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regardless of whether the animals are normal and healthy or are mounting acute phase responses to acute or chronic stimuli, or have established amyloidosis (Baltz et al., 1982b). Changes in the plasma level are therefore apparently due to alteration in the rate of synthesis of SAP or of its secretion into the circulation. Mice deprived of T or B lymphocytes, either genetically or by experimental manipulations, have normal SAP levels and can mount normal acute phase responses to nonviable stimuli (Baltz et al., 1980~). The acute phase response is, however, affected in these immunologically deficient animals if the stimulus on which it depends results from immunologically induced inflammation. For example in experimental murine schistosomiasis mansoni the SAP level rises only when the T cell-dependent hepatic granulomata form (Pepys et al., 1980b). With regard to nonspecific mediators of inflammation, no necessary role in SAP production for either granulocytes (Baltz et al., 1 9 8 0 ~or ) the complement system (Pepys and Rogers, 1980) is demonstrable. Thus mice rendered agranulocytotic by irradiation or hypocomplementemic by cobra factor responded to injections of casein or croton oil with the same SAP levels as untreated controls. IL-1 or cruder products of activated macrophages stimulate SAP synthesis both by isolated hepatocytes in vitro and in the intact animal in uivo, though there is some evidence that the response is less marked than it is with SAA synthesis (Sipe et al., 1982; Tatsuta et al., 1982).

D. SAP AND AMYLOIDOSIS AP has been detected immunochemically using antisera to AP or SAP in deposits or extracts of all forms of amyloid in which it has been sought, regardless of the chemical nature of the fibril protein (reviewed by Pepys et al., 1982a) with the possible exception of intracerebral amyloid plaques (Shirahama et al., 1982). Immunohistochemical staining with anti-SAP therefore provides a valuable aid in the tissue diagnosis of all except the latter form of amyloid (Pepys et al., 1982a). Deposits of experimentally induced amyloid in mice (Baltz et al., 1980a) and guinea pigs (Skinner and Cohen, 1976) also contain AP as do naturally occurring amyloid deposits in cattle (F. C. de Beer, I. F. Rowe, E. Gruys, and M. B. Pepys, unpublished observations). The significance of this universal association is not known but a possible explanation may simply be that regardless of the chemistry of the subunit proteins, the formed amyloid fibrils all share a common structural feature which happens to be a ligand for SAP. The possible nature of this ligand has been discussed above (Section V,B). Alterna-

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tively, there may be a more intimate association between AP and the pathogenesis of amyloidosis. For example in man AP is a normal tissue protein located at sites where early amyloid deposition often occurs (see Section V,E), while in the mouse SAP is an acute phase reactant, levels of which are raised during experimental induction of amyloidosis and correlate more closely with amyloidogenesis than do those of SAA (Baltz et al., 1980b). It has been suggested that the calcium-dependent aggregation manifested by isolated human SAP may in some way contribute to amyloid fibril deposition (Pinteric and Painter, 1979; Painter et al., 1982), but it cannot be an essential feature of the pathogenesis since mouse SAP, which is also found in amyloid deposits, does not aggregate in the same way (Baltz et al., 1982b. The possible existence of polymorphism in the primary structure of SAP (see Section V,A) raises the question of whether some form of SAPIAP is particularly associated with the development of amyloidosis but there is no evidence on this point as yet. E. SAP-RELATEDMATERIAL IN NORMALHUMANTISSUES Normal human renal glomerular basement membrane (GBM) and the peripheral microfibrillar mantle of elastic fibers in blood vessels and tissues throughout the body contain a protein which binds antiSAP antibodies immunospecifically (Dyck et al., 1980a; Breathnach et al., 1981b). It is also present in some other, but not all, vascular basement membranes but is not detectable in renal tubular, dermoepidermal or other basement membranes (Breathnach et al., 1983). The normal tissue AP (TAP) is not eluted from tissue sections except by denaturing solvents which completely destroy tissue architecture but it can be released in soluble form from isolated GBM by digestion with collagenase. Solubilized TAP from GBM gives reactions of complete identity in gel double immunodiffusion analysis with SAP and anti-SAP. However it is heterogeneous in pZ, in molecular size, and in polypeptide chain composition. Some of the native material and of the subunits are the same size as SAP but the majority are heavier, suggesting that the TAP is covalently linked to collagen and/or other matrix proteins in the GBM (Dyck et ul., 1980a). SAP itself has no biochemical similarity or immunochemical cross-reactivity with either collagen or the elastic fiber microfibrillar proteins which have been characterized hitherto (Sear et al., 1981). The biological significance of TAP in normal tissues is not known but it is possible that the interaction between aggregated SAP and fibronectin (see Section V,B) may be relevant.

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Immunohistochemical studies with anti-SAP of renal biopsies in various forms of renal disease show distinctive abnormal staining patterns in the GBM and such staining is a simple and sensitive marker for glomerular pathology (Dyck et al., 1980b). When used routinely for examination of renal biopsies it also has the advantage of detecting small amounts of amyloid which may otherwise be missed unless Congo red staining is included. Anti-SAP is also a useful reagent for the demonstration of abnormalities in the structure and distribution of elastic fibers in skin biopsies (Breathnach et al., 1982). VI. Serum Amyloid A Protein

A. INTRODUCTION

SAA derives its name from its close biochemica, and immunocA.emical relationship to amyloid A (AA) protein which is the fibril protein in deposits of reactive systemic (secondary) amyloidosis (Glenner, 1980a,b). AA protein from isolated, dissociated amyloid fibrils is usually of molecular weight about 8000 and consists of 76 amino acid residues. AA subunits of smaller and occasionally of larger size have been characterized (Husby and Sletten, 1980; Isobe et al., 1980) but in all cases the peptides are not glycosylated. The amino acid sequence of AA is not related to any known protein other than SAA (Benditt et al., 1971; Levin et al., 1972; Ein et al., 1972; Sletten and Husby, 1974)and proteins homologous to human ANSAA have been identified in the monkey (Hermodson et al., 1972), mouse Eriksen et al., 1976), rabbit (Anders et al., 1977), mink (Anders et al., 1976; Waalen et al., 1980), guinea pig (Skinner et al., 1974), and duck (Corevic et al., 1977). Antisera raised against denatured AA protein cross-react with SAA and anti-SAA sera react with isolated AA and with AA-type amyloid fibrils. In its native state in serum SAA has an apparent molecular weight of 180,000 (Anders et al., 1975; Levin et al., 1973; Linke et aZ., 1975)but denaturation with acids, alkalis, chaotropic agents, or detergents yields a peptide of approximately 12,000 daltons, the N-terminal portion of which is almost completely homologous with AA (Marhaug et al., 1980). Proteolytic enzymes of leukocyte and serum origin can cleave SAA to yield AA-size or smaller fragments and it is generally believed that this mechanism is involved in the generation of AA during the pathogenesis of amyloidosis (Lavie et al., 1978, 1980; Zucker-Franklin et al., 1980; Silverman et al., 1980; Skogen and Natvig, 1981), although there is no direct evidence of the precursor-product relationship between SAA and AA.

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B. APoSAA In its native form, in undenatured serum, SAA (molecular weight 180,000) is associated with high-density lipoprotein (HDL), d = 1.12-1.21 g/ml, in rabbits (Skogen et al., 1979; Tobias et al., 1982) and mice (Benditt et al., 1979) as we11 as man (Benditt and Eriksen, 1977). It therefore seems to be an apolipoprotein and has been designated as apoSAA (Benditt and Eriksen, 1977). This is compatible with its primary structure which contains sequences capable of forming amphipathic helical domains similar to those characteristic of apolipoproteins such as apoA-I (Segrest et al., 1976; Segrest and Feldman, 1977). It has recently been claimed that some apoSAA in man may also be associated with LDL, IDL, and VLDL fractions (Marhaug et al., 1982a). Earlier reports that aIbumin is the “carrier-protein” for SAA in serum (Rosenthal and Franklin, 1977) and of the presence of complexes of SAA with albumin fragments, prealbumin, and pzmicroglobulin, (Marhaug and Husby, 1981; Marhaug et al., 1982b), are probably attributable to in vitro artifacts produced during denaturation of serum and subsequent chromatography under denaturing conditions. However a small portion of material with SAA immunoreactivity and large molecular weight does sediment in the ultracentrifuge with a density greater than that of lipoprotein (Marhaug et al., 1982a). SAA-rich HDL isolated from acute phase murine or human serum also contains the “normal” HDL apoproteins apoA-I and apoC (Benditt et al., 1982). The other apoproteins of apoSAA-containing human LDL, IDL, and VLDL are also the same as in non-apoSAA containing controls (Marhaug et al., 1982a). Furthermore, the proportions and absolute amounts of these “normal,” nonacute phase apoproteins is the same within each class of lipoproteins whether isolated from normal or acute phase serum (Benditt et al., 1982; Marhaug et al., 1982a). However in mice the HDL from acute phase serum includes a particular subset of apoSAA-bearing particles in which there are 2 moles of apoSAA for each mole of apoA-I (Benditt et al., 1982). These particles also contain about 10% more protein and 10% less phospholipid than non-apoSAA-HDL but in other respects their lipid composition and phospholipid profile are the same as control HDL (Benditt et al., 1982). It is not clear yet whether the binding of apoSAA ro HDL involves displacement of some phospholipid or whether these changes occurring with the acute phase response take place independently. Normal levels of apoSAA are very low indeed and this probably explains why it has not previously been widely remarked upon in studies of lipoprotein structure. There are however reports of HDL

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apoproteins which clearly correspond to apoSAA although they were not identified as such or indeed as acute phase proteins. (Shore et al., 1978; Malmendier et al., 1979). At the peak of the acute phase response apoSAA comprises up to 50% of the total apoproteins of HDL (Benditt et al., 1982; Marhaug et al., 1982a). C. POLYMORPHISM AA extracted from amyloid deposits shows microheterogeneity of amino acid sequence and variation in chain length, predominantly at the C-terminal end (Gorevic et al., 1978; Husby and Sletten, 1980). Polymorphism of apoSAA isolated from lipoprotein fractions of acute phase serum is also evident. In man 6 polymorphs of apoSAA have been demonstrated on the basis of differences in pZ (Bausserman et al., 1980; Marhaug et al., 1982a), while in the rabbit (Tobias et al., 1982) and mouse (Benditt et al., 1982) two forms have been identified. None of these proteins is glycosylated and the differences between forms within each species seem to be due to differences in amino acid composition and sequence. There is only one report so far of the complete amino acid sequence of apoSAA. This human protein, designated apoSAA,, consists of 104 residues of which the first 76 from the N-terminal are identical with AA from tissue amyloid deposits, though two variants with a curious double sustitution of alanine and valine at residues of 52 and 57 were detected (Benditt et al., 1982). Each of the two forms of rabbit apoSAA contains 103 residues but no sequence data have yet been reported (Tobias et al., 1982). Conventional polyclonal antisera to AA react with all the polymorphic forms of apoSAA in a given species but monoclonal antibodies which can differentiate between some of the forms in man have been prepared (McAdam et al., 1982). Further sequence information and the exchange between laboratories of proteins and antibodies will be required in the future to correlate the different nomenclatures and to establish the identities of the different polymorphic forms of apoSAA. An alternative approach to elucidation of the structure and genetic basis of this polymorphism is the application of recombinant DNA technology. There is already evidence at the DNA level for the existence in mice of a series of related but distinct apoSAA sequences (Stearman et al., 1982). D. SYNTHESIS Although claims have been made for synthesis of SAA by other cell types (Rosenthal and Sullivan, 1978; Linder et al., 1977), the only unequivocal evidence is for production by hepatocytes (Selinger et al.,

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1980a,b; Benson and Kleiner, 1980; Benson, 1982; McAdamet al., 1982; Tatsuta et al., 1982). These cells respond to IL-1 by increasing their rate of SAA synthesis in vivo or in vitro and this is associated with an increased rate of transcription of mRNA for SAA (Stearman et al., 1982). Murine apoSAA is secreted on its own by hepatocytes and not as a part of an already constituted lipoprotein particle (Benditt et al., 1982). If HDL is provided in vitro the newly secreted SAA rapidly binds to it and presumably the same thing happens in vivo in the plasma or intercellular fluid. Production of free apoSAA may explain the small amounts of apoSAA detected in human LDL, IDL, VLDL, and free of lipid (Marhaug et al., 1982a). The transcription of mRNA for SAA in liver cell nuclei of mice which have received lipopolysaccharide is increased from 3 to 12 hours afterward with a peak at 6.5-9 hours and a subsequent decline (Stearman et al., 1982). Similarly the synthesis of SAA protein and the increase in serum levels of SAA follow very rapidly after an acute stimulus. The rate of rise in circulating SAA concentration is rapid and the increment attained is enormous, reaching as high as 10,000 times normal values in some patients with acute infhmmatory disease (de Beer et al., 1982c,e). The absolute amounts of SAA in terms of weight or molarity which are present in the plasma are difficult to determine in view of the problems of obtaining suitable calibration standards for assays. ApoSAA concentrations can be measured after denaturation and dissociation from lipoprotein but where this has been done AA has generally been used for standardization and AA differs in terms of size and antigenicity (Rosenthal and Franklin, 1975; Sipe et al., 1976a,b; Ignaczak et al., 1977; Benditt and Eriksen, 1977; McAdam et al., 1978; Hijmans and Sipe, 1979). ApoSAA can also be measured in its native form (Benson and Cohen, 1979; de Beer et al., 198213; Chambers and Whicher, 1982) but a standard native lipoprotein calibrator which is both physicochemically and immunochemically stable is not available, quite apart from the heterogeneity of size and molar apoprotein content of lipoprotein particles. Despite these difficulties, it seems clear that peak acute phase levels of SAA in man and the mouse may be in the range of 1-5 mg/ml.

E. FUNCTIONS The function of apoSAA and of the apoSAA-rich HDL particles is not known but the latter is obviously quite different from normal “nonacute phase” HDL. Benditt et al. (1980) have reported that apoSAArich HDL is cleared from the plasma very rapidly, with tllz of 40-60 minutes in mice. This is much more rapid than the clearance of apoA-I

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or any other apoproteins for which information is available. They have therefore suggested that the function of apoSAA may be to confer a rapid clearance rate on HDL particles, possibly as a means of eliminating complexes of toxic or unwanted materials with HDL. Evidence in support of this hypothesis is provided by the observation that lipopolysaccharide added to rabbit serum forms a complex with HDL (Ulevitch et aE., 1979,1981) and that with acute phase rabbit serum the HDL in the complex contains apoSAA (Tobias et al., 1982). However 12sI-labeledlipopolysaccharide was cleared at the same rate by normal and acute phase rabbits (Tobias et al., 1982). Further work in this area will be of considerable interest, particularly with respect to possible differences in function of the different polymorphic forms of apoSAA. The limited data available so far indicate that individuals, either animals or men, are capable of making all the different forms in response to various different stimuli although they may not do so to an equal extent on different occasions (Gorevic et al., 1978; Tobias et al., 1982; Bausserman et d.,1982). This may be of particular interest with regard to the pathogenesis of amyloidosis, if for example there was an association between certain of the SAA types and the deposition of AA fibrils (Gorevic et al., 1982). However, no evidence for the existence of any such amyloid-prone form of SAA has yet been obtained (Westermark, 1982; Marhaug et al., 1982a). It has been reported that murine and human SAA, isolated by formic acid denaturation of acute phase serum and subsequent gel filtration chromatography in formic acid, can suppress the T cell-dependent in vitro antibody response to sheep erythrocytes of murine and human lymphocytes, respectively (Benson et al., 1975; Benson and AldoBenson, 1979; Aldo-Benson and Benson, 1982). No effect on T cellindependent B cell activation was demonstrated. This interesting observation is difficult to interpret in terms of a physiological role in vivo since the conformation of SAA isolated in formic acid may bear little relation to that of apoSAA in its native state in lipoprotein particles. Furthermore SAA prepared in this way is known to contain other proteins, including albumin fragments, prealbumin, and µglobulin which may be covalently linked to the SAA itself (Marhaug and Husby, 1981; Marhaug et al., 1983). In view of the extreme potency of lymphokines and monokines it is also not possible by conventional biochemical techniques to exclude the possibility of the presence of this type of biologically active peptide in the SAA preparations. In this regard it is of some interest that there are many reports of in vitro immunosuppressive or other cellular effects of a number of putatively pure acute phase proteins (reviewed by Sipe and Rosenstreich, 1981).

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Some of these observations have subsequently been shown not to be reproducible with more highly purified proteins or to have been due to low-molecular-weight contaminants (see Section IV,C,5) and interpretation of this type of study should therefore be approached with considerable caution.

F. MEASUREMENTIN CLINICALPRACTICE There is very much less experience with measurement of SAA levels in clinical practice than there is with CRP. This is largely because the only methods available have been rather laborious radioimmunoassays in some of which it has been necessary to denature serum samples before assay (see Section V1,D). In most cases antisera raised against AA have been used which cross-react with SAA but this is the case only with some antisera and denaturation of the SAA may be required to expose the cross-reactive determinants. Furthermore both AA and SAA are very poor immunogens in the common animals, such as rabbits, sheep, or goats, used to raise conventional antisera. The recent development of monoclonal anti-SAA reagents (McAdam et al., 1982; Marhaug et al., 1982b; Wood et al., 1982) and of more robust assay systems capable of detecting and quantitating SAA in its native lipoprotein form in serum (de Beer et al., 1982b; Chambers and Whicher, 1983) should extend the range of clinical and experimental characterization of the SAA response. The very low normal levels and the rate and extent of the acute phase rise in circulating SAA levels suggest that it may be clinically useful in monitoring the acute phase response (Rosenthal and Franklin, 1975; Gorevic et al., 1976; McAdam et al., 1978; Hijmans and Sipe, 1979; de Beer et al., 1982b,d). However, the information available so far indicates that SAA levels correlate very closely indeed with CRP levels (Van Rijswijk et al., 1980; Mallya et al., 1982d) and it is at present much easier to measure CRP. However there may be differences in the rate or sensitivity of the SAA response which could be of clinical interest and which should be revealed by future studies using suitable, generally accessible assay systems. On the other hand, excessive sensitivity of the SAA response may cause the serum levels to correIate less well with clinicalIy relevant pathological processes than is the case with CRP. A particular and unique reason for clinical interest in SAA levels is the likely role of SAA as the precursor of the AA-type amyloid fibrils. However raised SAA levels as part of the acute phase response apparently accompany all active, chronic inflammatory processes and it is not known why some individuals with such disorders develop

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amyloidosis and others do not, nor why some chronic inflammatory diseases have a much higher incidence of amyloidosis than others. The concept of differences in the amyloidogenic potential of different polymorphic forms of SAA has been discussed in Section VI,E, while the possible role of the proteolytic enzymes capable of cleaving SAA to AA and of their inhibitors has been suggested elsewhere (see Section V1,A). The possibility should also be considered of a contribution to the risk of amyloidosis simply from the peak levels or the overall production of SAA in different individuals or different diseases. In a first approach to this question substantial groups of very well characterized patients with rheumatoid arthritis, Crohn's disease, ulcerative colitis, or systemic lupus erythematosus (SLE) have recently been investigated (de Beer et al., 1982d). The reported incidence of amyloidosis in the fkst two disorders is relatively high (Glenner, 1980a; Bywaters, 1978; Rashid et al., 1980) but in the second two amyloid is really

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197

ACUTE PHASE PROTEINS

exceptional (Carstens et al., 1980; Sweet et al., 1981; Huston et al., 1981; Shorvon, 1977). It was found that with increasing disease activity, although SAA levels increased in all four diseases, the values attained were up to 10-fold higher in active, severe rheumatoid arthritis (Fig. 9),juvenile chronic arthritis (Fig. lo), or Crohn's disease (Fig. 11) than they were in active, severe ulcerative colitis (Fig. 11)or SLE (Fig. 12) (de Beer et al., 1982d). Patients with SLE who developed intercurrent microbial infection produced much higher SAA levels, comparable to those seen in active rheumatoid arthritis (Figs. 9 and 12). These results correspond closely with the CRP levels measured in the same patients and previously reported in these diseases by ourselves and others (see Section IV,E). They indicate that the risk of reactive systemic amyIoidosis may correlate with SAA levels and/or long-term SAA production and suggest that it may be valuable to monitor SAA levels serially in patients with diseases which predipose to amyloid in order to attempt confirmation of this hypothesis at the individual level. In addition it will be of interest to determine whether control of SAA

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levels, by appropriate use of antiinflammatory or other therapy, is able to reduce the incidence of amyloidosis. VII. Summary

The acute phase response among plasma proteins is a normal response to tissue injury and is therefore a fundamental aspect of many diverse disease processes. It probably usually has a beneficial net function in limiting damage and promoting repair but in some circumstances it may have pathological consequences. Sustained high levels of acute phase proteins and especially SAA are associated with the development of amyloidosis in some individuals. Increased concentrations of CRP may, by activating the complement system, contribute to inflammation and enhance tissue damage. Failure of the normal or appropriate CRP response may also possibly have deleterious effects. SAA is a polymorphic protein which is normally present only in trace

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199

amounts but which, during the acute phase response, becomes one of the major apolipoproteins associated with high-density lipoprotein particles. The function of apoSAA is not known but it must have considerable physiological significance apart from its role as the putative precursor of amyloid A protein fibrils. CRP and SAP have been very stably conserved throughout vertebrate evolution and homologous proteins are apparently present even in vertebrates. This strongly suggests that they have important functions although these have not yet been precisely delineated, The main role of CRP may be to provide for enhanced clearance of inappropriate materials from the plasma whether these are of extrinsic origin, such as microorganisms and their products, or the autologous products of cell damage and death. The interaction between aggregated CRP and plasma low-density lipoprotein may play a significant part in the normal function of CRP and may also have a role in lipoprotein metabolism, clearance, and deposition. SAP is a normal tissue protein as well as being a plasma protein. Aggregated SAP selectively binds fibronectin and this may represent an aspect of the normal function of SAP. The deposition of SAP in amyloid is evidently not a normal function but it is not known whether this deposition is involved in the pathogenesis of amyloid or whether it is merely an epiphenomenon. I n any case immunohistochemical staining for SAP is useful in the diagnosis of amyloid, in investigation of glomerulonephritis, and in studying disorders of elastic tissue. Regardless of its physiological or pathophysiological functions, the assay of serum CRP is a valuable aid to clinical management in a number of different situations and in different diseases provided results are interpreted in the light of full clinical information. It is a sensitive screening test for organic disease, it is useful for monitoring the activity of those infective, inflammatory, and neoplastic diseases which provoke major elevations, it is a sensitive test for detection of intercurrent or persistent infection, and it helps in monitoring resolution after acute events such as major surgery or myocardial infarction. ACKNOWLEDGMENT Supported in part by Medical Research Council Grant G979/51.

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Sipe, J. D., McAdam, K. P. W. J,, Torain, B. F., and Glenner, G. G. (1976b).Br. J . E x p . Pathol. 57, 582. Sipe, J. D., Vogel, S. N., Ryan, J. L., McAdam, K. P. W. J., and Rosenstreich, D. L. (1979). J . E x p . Med. 150, 597. Sipe, J. D., Vogel, S. N., Sztein, M. B., Skinner, M., and Cohen, A. S. (1982).Ann. N.Y. Acad. Sci. 389, 137. Skinner, M., and Cohen, A. S. (1976).In “Amyloidosis” (0.Wegelius and A. Pasternack, eds.), D. 339. Academic Press, New York. Skinner, M., Cathcart, E. S., Cohen, A. S., and Benson, M. D. (1974).J.Exp. Med. 140, 871. Skogen, B., and Natvig, J. B. (1981). Scand. J . Immunol. 14, 389. Skogen, B., Borresen, A. L., Natvig, J. B., Berg, K., and Michaelson, T. E. (1979).Scand. J . Immunol. 10, 39. Sletten, K., and Husby, G. (1974). Eur. J . Biochem. 41, 117. Smith, C. W., and Micks, D. W. (1970). Lab. Inoest. 22,90. Srivastava, L. M., Agarwal, D. P., Goedde, H. W., and Rohde, R. (1975). Tropenmed. Parasit. 26, 212. Starke, I. D., de Beer, F. C., Donnelly, J. P., Catovsky, D., Goldman, J. M., Galton, D. G., and Pepys, M. B. (1983).Submitted. Stearman, R. S., Lowell, C. A., Pearson, W. R., and Morrow, J. F. (1982).Ann. N.Y. Acad. Sci. 389, 106. Sweet, J., Bear, R. A., and Lang, A. P. (1981). Hum. Pathol. 12,853. Sztein, M. B., Vogel, S . N., Sipe, J. D., Murphy, P. A., Mizel, S. B., Oppenheim, J. J., and Rosenstreich, D. L. (1981). Cell. Immunol. 63, 164. Tatsuta, E., Shirahama, T., Sipe, J. D., Skinner, M., and Cohen, A. S. (1982).Ann. N.Y. Acad. Sci. 389, 467. te Velde, E. R., Berrens, L., Zegers, B. J. M., and Ballieux, R. E. (1979).Eur. J . Cancer 15, 893. Theofilopoulos, A. N., and Dixon, F. J. (1981). lmmunol. Reo. 55, 179. Thompson, A. R., and Enfield, D. L. (1978). Biochemistry 17,4304. Thompson, A. W., Fowler, E. F., and Pugh-Humphreys, R. G. P. (1979). lnt. J . lrnmunophamacol. 1,247. Tillett, W. S., and Francis, T. (1930).J.E x p . Med. 52, 561. Tillett, W. S., Goebel, W. F., and Avery, 0. T. (1930).]. Exp. Med. 52, 895. Tobias, P. S., McAdam, K. P. W. J., and Ulevitch, R. J. (1982).J.Immunol. 128, 1420. Tomasz, A. (1967). Science 157,694. Trautner, K., Cooper, E. H., Haworth, S., and Milford Ward, A. (1980).Scatad.]. Urol. Nephrol. 14, 143. Tsujimoto, M., Keizo, I., and Nojima, S. (198l).J.Biochem. 90, 1507. Turchik, J. B., and Bornstein, D. L. (1980). Infect. Immun. 30,439. Uhlenbruck, G., Karduck, D., Haupt, M., and Schwick, H. G. (1979). Z. Immunitaetsforsch. 155,262. Ulevitch, R. J., Johnston, A. R., and Weinstein, D. B. (1979).J. Clin. Inoest. 64, 1516. Ulevitch, R. J., Johnston, A. R., and Weinstein, D. B. (1981).j . Clin. Invest. 67, 827. Urry, D. W. (1974).In “Arterial Mesenchyme and Arteriosclerosis” (W. D. Wagner and T. B. Clarkson, eds.), p. 211. Plenum, New York. van Rijswijk, M. H., Ruinen, L., Marrink, J., and de Blecourt, J. J. (1980).In “Disease Evaluation and Patient Assessment in Rheumatoid Arthritis” (T. E. W. Feltkamp and J. K. van der Korst, eds.), p. 145. Staphleu, Alphen aan den Rijn, The Netherlands.

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Voelkel, E. F., Levine, L., Alper, C. A., and Tashjian, A. H., Jr. (1978).J.E x p . Med. 148, 1078. Volanakis, J. E. (1982a).Ann. N.Y. Acad. Sci. 389, 235. Volanakis, J. E. (1982b).J.Immunol. 128, 2745. Volanakis, J. E., and Kaplan, M. H. (1971).Proc. Soc. E x p . Biol. Med. 236, 612. Volanakis, J. E., and Kaplan, M. H. (1974).J.Zmmunol. 113, 9. Volanakis, J. E., and Kearney, J. F. (198l).J.E x p . Med. 153, 1604. Volanakis, J. E., and Narkates, A. J. (1981).J.Immunol. 126, 1820. Volanakis, J. E., and Wirtz, K. W. A. (1979). Nature (London) 281, 155. Waalen, K., Sletten, K., Husby, G., and Nordstoga, K. (1980).Eur. J. Biochem. 104,407. Walsh, L., Davies, P., and McConkey, B. (1979).Ann. Rheum. Dis. 38, 362. Wannemacher, R. W., Jr., Pekarek, R. S., Thompson, W. L., Curnow, R. T., Beall, F. A., Zenser, T. V., D e Rubertis, F. R., and Beisel, W. R. (1975). Endocrinology 96, 651. Werner, M. (1969). Clin. Chim. Acta 25,299. Westermark, P. (1982).Biochim. Biophys. Acta 701, 19. Whaley, K. (198O).J.Erp. Med. 151, 501. Whicher, J. T., Martin, M. F. R., and Dieppe, P. A. (1980).Lancet 2, 1187. Wicher, J., Bell, A., Unwin, J., Martin, M., and Dieppe, P. (1982). Ann. N.Y. Acad. Sci. 389,474. White, A., Fletcher, T. C., Pepys, M. B., and Baldo, B. A. (1981). Comp. Biochem. Physiol. 69C, 325. White, A., Fletcher, T. C., and Pepys, M. B. (1982).Comp. Biochem. Physiol. 74B, 453. Williams, R. C., Jr. (1982).Ann. N.Y. Acad. Sci. 389, 395. Williams, R. C., Jr., and Quie, P. G. (1968).J. Zmmunol. 101,426. Williams, R. C., Jr., Kilpatrick, K. A., Kassaby, M., and Abdin, Z. H. (1978).J . Clin. Invest. 61, 1384. Williams, R. C., Jr., Husby, G., Wedege, E., Walker, L. C., and Tung, K. S . K. (1980). In “Streptococcal Diseases and the Immune Response” (S. E. Read and J. B. Zabriskie, eds.), p. 695. Academic Press, New York. Wood, D. D., Gammon, M., and Staruch, M. J. (1982).J.Zmmunol. Methods. 55, 19. Wood, H. F. (1963).Yale]. Biol. Med. 36,241. Wood, H. F., McCarty, M., and Slater, R. J. (1955).J.E x p . Med. 100, 71. Wood, H. F., Diamond, H. D., Craver, L. F., Pader, E., and Elster, S. K. (1958).Ann. Intern. Med. 48, 823. Yother, J., Volanakis, J. E., and Briles, D. E. (1982).J.Zmmunol. 128, 2374. Young, N. M., and Williams, R. E. (1978).J.Zmmunol. 121, 1893. Ziccardi, R. J., and Cooper, N. R. (1977).J.Immunol. 118, 2047. Zucker-Franklin, D., Lavie, G., and Franklin, E. C. (1980). In “Amyloid and Amyloidosis” (G. G. Glenner, P. Pinho e Costa, and F. de Freitas, eds.), p. 456. Excerpta Medica, Amsterdam.

NOTE ADDED IN PROOF

A. Recently approximately 30% of the amino acid sequence of rat CRP has been defined and shows 79% strict residue-for-residue identity with human CRP [Anderson, J. K., Taylor, J. A., Pepys, M. B., Bruton, C. J., and Mole, J. E. (1983). Znimunobiology 164,2041, This establishes that the protein isolated and designated as rat CRP (de Beer et al., 1982a) is indeed the counterpart of human CRP.

2 12

M. B. PEPYS AND MARILYN L . BALTZ

B. Further work on the interaction between rabbit CRP and plasma lipoproteins has established that native CRP in acute phase rabbit serum does form complexes with very low-density lipoprotein (VLDL) and apoB-containing lipoproteins in general. The existence of these complexes is, however, absolutely dependent on the concentration of apoB-containing lipoprotein in the serum or plasma. Serum samples from most normal rabbits fed a normal diet do not contain complexed CRP but in hyperlipidemic serum from cholesterol-fed animals all the CRP tends to be complexed with apoBcontaining lipoprotein (Rowe, I. F., Soutar, A. K., Trayner, I. M., Balk, M. L., d e Beer, F. C., Walker, L., Bowyer, D., and Pepys, M. B. Submitted).

ADVANCES IN IMMUNOLOGY, VOL. 34

Lectin Receptors as Lymphocyte Surface Markers NATHAN SHARON Deporfment of Biophysics, The Weizmonn Institute of Science, Rehovoth, Israel

.................................

.........

11. Methodology ................................ A. Detection and Enumeration of Lectin-Bindi B. Techniques for Cell Separation . . . . . . . .

.......... ........................

111. Murine Lymphocyte Subpopulations . . . B. Receptors for Other Lectins

.................................

B. Receptors for Other Lectins V. Lymphocytes of Other Animals A. Rat

............

E. Chicken ....

.............................. ........................... ............................ ............................

2 13 215 218 22 1 223 223 225 229 230 230 260 265 265 277 28 1 28 1 282 283 285 285 287 29 1

I . Introduction

One of the most significant advances in immunology in the past two decades has been the realization of the enormous functional heterogeneity of the cells of the immune system and the demonstration that the operation of this system requires interaction between different types of such cells. Another notable advance has been the identification of various stages of differentiation of B and T cells. Progress in these and related areas was critically dependent on the use of cell surface markers for the identification, enumeration, and separation of different lymphocyte subpopulations. Such markers can be viewed as molecular identity tags expressed on the surfaces of cells. I n most cases it is impossible to identify or separate lymphocytes by other means, since even when they differ in their functional properties or stage of differentiation, they are often morphologically and biochemically 213 Copyright 0 1983 by Academic Press, Inc. All rights of reproductiml in any form reserved. ISBN 0-12-022434-8

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indistinguishable, and are present in the same lymphoid tissue. Interest in cell surface markers of lymphocytes has also been greatly stimulated by the findings that they control and determine many of the cell-molecule and cell-cell interactions characteristic of immune function. Cell surface markers are of two major types-surface antigens detectable by antibodies, and surface saccharides, detectable by lectins. The most thoroughly investigated of the former are the antigens expressed on the surface of murine lymphocytes, such as the €I-2 antigens, the Thy 1 antigen characteristic of T cells, TLa antigens present only on thymocytes and leukemic cells of certain mouse strains, Lyt antigens that are markers for T lymphocyte subsets, and surface membrane immunoglobulins (SmIg)’ that are confined to B cells (Katz, 1977; Loor and Roelants, 1977; McKenzie and Potter, 1979). Antigenic surface markers have been identified on lymphocytes of other animals, particularly of man. Thus, human B lymphocytes express SmIg and the HLA-D related Ia-like antigens, whereas human T lymphocytes react with T cell-specific heteroantisera. [Other well established markers for human Iymphocyte subpopulations, such as receptors for the Fc portion of human immunoglobulin and the receptor for sheep erythrocytes (E-rosette), are not detected routinely by their corresponding antibodies, and cannot therefore be considered strictly as “antigenic.”] Recently a large number of human surface antigens have been identified by monoclonal antibodies generated against human lymphocytes (Haynes, 1981; Nadler et al., 1981; Reinherz and Schlossman, 1980). Using such antibodies, it is possible to identify and separate functional subclasses of T cells, e.g., helperlinducer and suppressorlkiller cells. There is no doubt that with the aid of monoclonal antibodies many more surface antigens characteristic of specific lymphocyte subpopulations-whether of man or of other animals-will be identified. The other class of surface markers that is gaining increasing importance in immunological research is of lectin receptors. Although direct experimental evidence is still lacking, it is quite certain that the expression of these receptors, like that of the surface antigens, is

Abbreviations used: CFC, colony-forming cells; CTL, cytotoxic T lymphocytes; FACS, fluorescence activated cell sorter; FITC, fluorescein isothiocyanate; GVH, graft-versus-host; HRP, horseradish peroxidase; IL-2, interleukin 2; LPS, lipopolysaccharide; MLR, mixed lymphocyte reaction; SmIg, surface membrane immunoglobulin; TCGF, T cell growth factor;TdT, terminal deoxynucleotidyltransferase. All sugars are of the D-configuration unless otherwise noted. For abbreviations of lectins see Table I.

LECTIN RECEPTORS

215

controlled by specific genes. However, since lectin receptors are carbohydrate in nature, residing in the oligosaccharide sequences of membrane glycoproteins or glycolipids, they must be secondary gene products, just as the ABO human blood group determinants are. Because of the great complexity of the cell surface, the ability to detect the lectin receptors depends not only on the presence of these gene products on the surface, but also on the configuration and relative disposition of the membrane constituents, which may, for example, mask such receptors. A. LECTINS-A BRIEF SURVEY Lectins (Latin legere, to pick out or choose) were originally defined as blood group specific agglutinins found in plant extracts (Boyd and Shapleigh, 1954). They are now known as sugar binding proteins (or glycoproteins) that agglutinate cells or precipitate glycoconjugates (Goldstein et d.,1980).Table I lists some of the lectins most widely used in immunological research. (For more information on these and other lectins, see the reviews by Goldstein and Hayes, 1978; Lis and Sharon, 1981; Pereira and Kabat, 1979a; the applications of lectins in immunology have been reviewed by Lis and Sharon, 1977, and by Sharon, 197913, 1980.) All lectins are oligomeric proteins with several sugar-binding sites per molecule, i.e., they are multivalent. They combine noncovalently with mono- and oligosaccharides, both simple and complex, in the same way that antibodies bind antigens. Binding may involve several forces, mostly hydrophobic and hydrogen bonds, but only rarely electrostatic ones since most monosaccharides with which lectins interact are devoid of electrical charge. Precipitation of glycoproteins (and polysaccharides) by lectins is similar in many respects to the well known precipitation reaction between antibody and antigen: it is specific, depends on the concentration of each of the reactants, exhibits a maximum at an optimal ratio of the two, and may be inhibited specifically by low-molecular-weight “haptens”-compounds identical with or derived from the sugar(s) for which the lectin is specific. The same sugars will also inhibit the agglutinating activity of the lectins. There are, however, several important differences between lectins and antibodies, in addition to lectins being of nonimmune origin (many of them are found in plants and bacteria, organisms which d o not possess the capacity of immunologic response). Thus, whereas antibodies are structurally similar, lectins are structurally diverse, their only common feature being that they are all proteins (Sharon et

TABLE I LECTINSUSED IN IMMUNOLOGY" Specificity Number Abbreviated name Con A DBA

HPA LCA LBL LPA LTL

Human blood

of Source Jack bean (Canaualia ensifomis) Horse gram (Dolichos bijlorus) Garden snail (Helix pomatia) Lentil (Lens culinaris) Lima bean (Phaseolus limensisc) Horseshoe crab (Limulus polyphemus) Winged pea (Lotus tetragonolobus)

subunits

sugar

tVpe

Mitogenic activity

108,000

4

Man, Glc

-

+

110,000

4

aGalNAc

A1

79,000

6

aGalNAc

A

-

42-63,000

4

-

+

I1 138,000 1269,000 335,000

4 8 18

Man, Clc, ~L-Fuc~ a-GalNAc a-GalNAc NeuNAc

A A

f

-

+ +

120,000

4

~L-FUC

0

-

MW

-

PHA PNA P W S BA UEA WGA

WL a

Red kidney bean (Phaseolus oulgaris) Peanut (Arachis hypogaea) Pokeweed (Phytolacca americana) Soybean (Glycine maz) Gorse (Uler europeus) Wheat germ (Triticun vulgare) Vicia villosa

120,000

4

GlcNAcpl+2Mand

-

+

110,Ooo

4

Gal, Galp1+3GalNAc

-

(+P

-

+

GalNAc

-

(+)'

I ~L-FUC I1 GlcNAcpl+4GlcNAc GlcNAc, NeuAcP GlcNAcpl+4GlcNAc GalNAm 1+3Galh

0 -

+

-

-

120,000

4

(31-65,000)

n.d.

36,000

2 4

-

-

References are given only for studies not cited in Goldstein and Hayes (1978) and Lis and Sharon (1981).

* Kornfeld et al. (1981). 4 E

Also known as Phaseolus lunatus. Lonngren et al. (1981). Active apparently only in polymerized form, on sialidase-treated cells of a limited number of animal species. 'Active only in polymerized form on sialidase-treated cells; pig lymphocytes are stimulated without sialidase-treatment of the cells. Bhavanandan and Katlic (1979), Monsigny et al. (1979), and Peters et al. (1979). * Kaladas et al. (1981).

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NATHAN SHARON

al., 1974). From this point of view, lectins are similar to enzymes, although they are devoid of catalytic activity. Another difference is in the range of specificities, since it is possible to obtain antibodies specific against virtually any organic compound, whereas the specificity of lectins is restricted (by definition) to carbohydrates. Interestingly, anti-carbohydrate antibodies with specificities similar to those of lectins are known and there are many similarities between the combining sites of the two classes of compound (Kabat, 1978). In the context of the present article, perhaps the most significant difference is that antibodies bind to cells so strongly that they cannot be readily removed from the cells by the corresponding haptens. Lectins, on the other hand, can be easily removed under mild conditions by competing sugars, with nearly full recovery of the cells with which the lectin had interacted.

B. LECTINSIN IMMUNOLOGY Lectins were first employed in immunology by Paul Ehrlich in the early 1890s simply as antigens. This was a short while after the discovery of the occurrence of hemagglutinating substances in plant extracts-ricin in the castor bean, Ricinus communis, (1888) and abrin in extracts of the jequirity bean, Abrus precatorius, (1891). Ehrlich realized that ricin and abrin would be more useful antigens than the bacterial toxins, such as that of diphtheria, which were popular research tools at the time. Although the preparations used by him were very crude by present day criteria (we now know that both the “ricin” and “abrin” were mixtures of a nonagglutinating protein toxin and a nontoxic agglutinin, all of which specifically bind galactose), he was able with their aid to establish some of the fundamental principles of immunology. He found that immunization by ricin and abrin resulted in the formation of serum proteins capable of specifically precipitating and neutralizing these substances, and that there was a quantitative relationship between the amount of antiserum and that of antigen it could neutralize. Moreover, antiricin did not protect the animal against the toxic effects of abrin nor vice versa, which provided clear evidence for the specificity of the immune response. Ehrlich also demonstrated that, during pregnancy, immunity to the toxins is transferred from the mother to the offspring b y the blood and that after birth it may be transferred through the milk. In 1908, Landsteiner and Raubitschek established that the relative hemagglutinating activities of various seed extracts were quite different when tested with red blood cells from different animals. In spite of this demonstration of species specificity, it was presumed for

LECTIN RECEPTORS

219

several decades that plant agglutinins were nonspecific. It was not until the end of the 1940s that Boyd and Reguera (1949) and Renkonen (1948) independently reported that certain seeds contain agglutinins specific for some human blood group antigens. Thus, a crude extract of the lima bean, Phaseolus limensis, and of the tufted vetch, Vicia C M C C U , both specific for a-N-acetylgalactosamine, were found to agglutinate only blood type A erythrocytes and the lectin from Lotus tetragonolobus, specific for L-fucose, to agglutinate only type O(H) erythrocytes. Additional lectins specific for blood types A, B, O(H), M, N, and other groups are now known (Bird, 1978; Judd, 1980). In this context it should be recalled that the first evidence that sugars are the immunodeterminants of human blood types was obtained by Winifred M. Watkins and Walter T. J. Morgan in 1952 with the aid of blood type specific Iectins (for a recent review see Watkins et al., 1981). At present some lectins commonly serve in blood banks as an aid in blood typing, mainly because of the unavailability of a natural anti-O(H) antibody and because certain of them can distinguish Al and Az subgroups (Bird, 1978; Judd, 1980; Race and Sanger, 1975). Occasionally they are employed for separation of mixed erythrocyte populations, for example, in the rare cases of blood group mosaicism resulting from chimerism, somatic mutation, or bone marrow transplantation. The Dolichos biflorus agglutinin (DBA) is particularly useful in separating Al cells from mixtures of Al and 0 blood (Booth et aZ., 1957). Lectins also serve for the identification of “secretors,” individuals who secrete blood group substances in their saliva or other body fluids. The interest of immunologists in lectins was greatly increased as a result of the discovery that lymphocytes can be stimulated to grow and divide by phytohemagglutinin (PHA, a lectin from the red kidney bean Phaseolus vulgaris) (Nowell, 1960; for a recent comprehensive review see Hume and Weidemann, 1980). Many other lectins are now known to possess mitogenic activity (Lis and Sharon, 1981). Since certain lectins (e.g., PHA and concanavalin A) stimulate only T cells, while others [e.g., pokeweed mitogen (PWM)] stimulate B cells as well, they also serve as an aid in identifying the major lymphocyte subpopulations. The gross morphological changes and biochemical events occurring in lectin-stimulated lymphocytes in vitro resemble many of the antigen-induced immune reactions that take place in uivo. Furthermore, B cells stimulated by suitable lectins are capable of synthesizing immunoglobulins in a fashion similar to cells stimulated by antigens i n vitro. However, while any particular antigen will stimulate only a small proportion of the lymphocytes (usually 0.02-0.2%), lectins

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NATHANSHARON

stimulate a high proportion (up to 70 or 80%)of the cells, regardless of the antigenic specificity of the lymphocyte receptors. Lectins thus belong to the large group of polyclonal activators or polyclonal ligands. They serve as an important aid in studies of the mechanism by which an antigen, acting at the surface of the lymphoid cell, specifically triggers or specifically inhibits clonal expansion and immunoglobulin synthesis. Other products synthesized by lectin stimulated lymphocytes include a variety of lymphokines such as interleukin 2 (IL-2) (Mizel and Farrar, 1979; Watson and Mochizuki, 1980) and interferon (Van Damme et al., 1981). Mitogenic stimulation by lectins (especially by PHA) is also used as a diagnostic tool to detect congenital and acquired immunologic deficiencies, to detect sensitization caused by infectious agents or in some autoimmune diseases, and to monitor the effects of various immunosuppressive and immunotherapeutic treatments (Oppenheim et al., 1975). Another most useful application of lymphocyte stimulation by lectins has been for cytogenetic studies of chromosomes of man and other animals (Robbins, 1964). This has led to an increased understanding of relationships between chromosome abnormalities and human diseases. Lectins are also widely employed by immunologists to study the properties and constitution of lymphocyte membranes (Nicolson, 1976a,b; Sharon and Lis, 1975). With suitable lectin derivatives, it has been possible to examine not only the distribution of lectin receptors on the surface of lymphocytes but of the mobility of the receptors in the membrane as well, and to demonstrate, for example, that redistribution and capping of the receptors on lymphocyte surfaces occurs as a result of lectin binding. Lectin receptors, i.e., membrane constituents that react with lectins, can be detected on electrophoretograms or isolated in purified form by the same methods as used for the isolation of surface antigens with the corresponding antibodies. Detection is best done by lectin staining of electrophoretograms of membrane preparations. The receptors can be isolated from the solubilized membranes either by precipitation with the lectin alone, or in combination with antilectin antibody. Isolation on a preparative scale is best achieved by affinity chromatography on immobilized lectins (Lotan and Nicolson, 1979). Lymphocyte membrane constituents isolated by lectin affinity chromatography include a host of interesting and important glycoproteins such as histocompatability antigens, Thy 1 antigens, B cell surface immunoglobulins, and various other lectin binding glycoproteins. Recent examples are the isolation of the human analog of murine Thy 1 (Ades et al., 1980) and the human T cell leukemia antigens (Seon et al.,

LECTIN RECEPTORS

22 1

198l),in both cases on columns of lentil lectin (LCA), and the isolation of glycoprotein receptors for peanut agglutinin (PNA), using immobilized PNA, from human peripheral blood lymphocytes which had been treated with sialidase (neuraminidase) (Farrar et al., 1981), and for Ulex europeus agglutinin (UEA) from the membranes of human lymphocyte cell lines (Giirtler, 1981). Techniques for receptor isolation which require prior solubilization of the lymphocyte membrane by detergents may reveal cryptic receptors that are inaccessible to the lectin on the intact cell. To overcome this disadvantage, we have recently developed a new approach for the isolation of lectin receptors in their native form, without the use of detergents, by plucking them from the cell surface (Jakobovits et al., 1981, and unpublished data). In this technique cells are bound to lectin-coated beads; upon mechanical disruption the cells are sheared off the beads leaving behind lectin receptors which can be examined while still on the beads. The receptors can also be released from the beads by specific sugars, permitting their further characterization. C. IDENTIFICATION AND SEPARATION OF CELLS

The application of lectins for the identification and separation of cells can be considered as an extension of their use for purification of glycoproteins and glycopeptides on the one hand, and for histochemical characterization of cells in tissues on the other. Although lectins have been widely employed since the early 1960s for blood typing, and in isolated cases also for the separation of erythrocytes of individuals of mixed blood types, there were only scattered reports prior to 1975 on their use for the identification and separation of other types of cell. As early as 1949, Li and Osgood developed a method for the separation of leukocytes from erythrocytes in human blood by agglutination of the latter with PHA. The erythrocytes could be removed from the mixture by centrifugation but could not be recovered in suspension. It was while using this method for the preparation of leukemic cells for culturing that Nowell (1960) discovered by chance the mitogenic properties of PHA. Edelman et al. (1971) demonstrated that it was possible to separate murine thymocytes from murine erythrocytes by concanavalin A bound to nylon fibers; only the thymocytes bound to fibers with low lectin density (both types of cell bind to heavily derivatized fibers). The binding was highly specific, since it could be almost completely inhibited by methyl a-mannoside, but not by galactose. The bound

222

NATHAN SHARON

cells could not be removed by the specific hapten, presumably because of the formation of strong nonspecific interactions with the surface of the fibers. Instead, the cells could be rapidly and quantitatively released by plucking the taut fibers with a needle. Unlike lymphocytes bound to immobilized concanavalin A, HeLa cells bound to immobilized LCA could be removed from the matrix in high yield and in viable form upon addition of specific sugars (Kinzel et al., 1976). Although LCA has the same sugar specificity as concanavalin A, the ease of removal may b e the result of its lower sugar-binding affinity, and possibly also because, under the experimental conditions used, no secondary interactions occurred in the system. Schnebli and Dukor (1972) were the first to demonstrate that lectins may specifically bind to and agglutinate certain murine lymphocyte subpopulations. They found that splenocytes of nude mice (devoid of T cells) and cortisone resistant thymocytes (T cells) were agglutinated equally well by soybean agglutinin (SBA), whereas thoracic duct cells (mainly T cells) were not agglutinated. They also showed that splenocytes of nude mice were agglutinated at considerably lower concentrations of wheat germ agglutinin (WGA) than either cortisone resistant thymocytes or thoracic duct cells, and suggested that the receptor for this lectin may serve as a new specific marker for B cells. Little or no agglutination of lymphocytes was observed with pokeweed mitogen, Robinia pseudoacacia agglutinin, and Ulex europeus agglutinins I and 11, whereas waxbean agglutinin and FHA agglutinated B and T cells almost equally well, and the thoracic duct cells slightly less so. The different behavior with SBA of the two T cell preparations used was taken as further evidence for the existence of subpopulations of thymus-derived lymphocytes. The authors made the important suggestion that SBA could “prove to b e useful for the separation of subpopulations of peripheral T cells” but did not present any experimental results on this point. Hammarstrom et al. (1973)foundHeZix pomatia agglutinin (HPA) to be a selective marker for sialidase-treated T lymphocytes of human peripheral blood; only a small proportion of the B lymphocytes was stained by the fluorescein-labeled lectin. The area started to gain momentum only some 5 years ago, as a result of findings made independently in our laboratory and elsewhere: (1) the discovery by Reisner et al. (1976a) that murine thymocytes can be separated into cortical and medullary cells b y FNA, which led to the proposal that the PNA receptor is a marker of immature lymphoid cells; (2) the demonstration that murine T and B splenocytes can be

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223

separated b y SBA (Reisner e t d., 1976b); and (3) the finding that sialidase treated human blood lymphocytes can be separated into B and T cells by HPA (Hellstrom e t aZ., 1976a). In the short span of half a dozen years PNA, SBA, HPA, and several other lectins have become extremely important tools for identification and separation of lymphocyte subpopulations. There are also strong indications that their use may soon be extended to clinical medicine, as evidenced by the recent application of SBA for isolation of stem cells from human bone marrow for transplantation across major histocompatibility barriers (Reisner et al., 1981b, 1983). Considering that there are now at least 100 purified lectins and that the number of such lectins is growing rapidly (over 1000 have been detected in crude extracts of plants, microorganisms and animals), there is little doubt that their potential for cell identification and separation has barely been touched. I I . Methodology

A. DETECTION AND ENUMERATION OF LECTIN-BINDING CELLS Examination of suspended cells by staining is usually the first step in the development of a method for fractionation of the cells with lectins. Lectin-binding cells are most readily detected under the microscope with the aid of fluorescent (fluorescein or rhodaminelabeled) lectin derivatives. Other derivatives, such as those commonly employed for the detection of cell surface antigens by antibodies, either in the light microscope or electron microscope, may be used (Schrevel et al., 1981; Sharon and Lis, 1975) (Table 11). For example, ferritin-labeled PNA has been employed by E. Skutelsky (see Lis and Sharon, 1977) to demonstrate by electron microscopy the presence of PNA receptors on immature thymocytes and their absence on the mature cells. It is also possible to detect cells to which lectins are bound by suitable derivatives of antibodies to the lectins (e.g., fluoresceinated or ferritin-labeled antibodies). In another modification, biotin derivatives of lectins (e.g., biotinyl-PNA) were detected by fluorescein-labeled avidin (Maccario e t al., 1981). Receptors for N-acetylgalactosamine specific lectins (e.g., SBA) have been visualized by gold-labeled porcine blood group A substance known to have a terminal N-acetylgalactosamine residue which can bind to the unoccupied combining site of such lectins when on cell surfaces (Fath et al., 1980; Fath and Ackerman, 1981). Radioactive derivatives of lectins are useful both for quantitating the overall binding of the lectins

224

NATHAN SHARON

TABLE I1 METHODSFOR DETECTIONO F LECTINRECEPTORSON LYMPHOCYTES AND FRACTIONATION OF LYMPHOCYTES BY LECTINS Method Detection Direct visualization

Indirect visualization With enzymes With fluorescent or ferritin labeled antibodies With ferritin-avidin Au toradiography Fractionation Selective agglutination Mixed rosetting Affinity chromatography

Flow microfluorimetry

FOR

Form of lectin

Comments on technique

Fluoresceinated derivative Femtin conjugate Gold complex

Light microscopy, FACS Electron microscopy Electron microscopy

HRP derivative Native

Light microscopy Light microscopy or electron microscopy

Biotin derivative Radioactively labeled (e.g., with InsI)

Electron microscopy Light microscopy or electron microscopy

Native Native Immobilized Native Fluoresceinated derivative

Immobilized anti-lectin antibodies FACS

to cells or to examine the binding patterns of individual lymphocytes in various cell preparations by quantitative autoradiography. By this technique, for example, the binding of seven 12sII-labeledlectins to small lymphocytes from mouse thymus, spleen, and bone marrow has been examined (Saveriano et al., 1981). In all cases, it is essential to test for the specificity of the reaction by including controls in which a suitable inhibitory sugar is present. It is sometimes desirable to assess the distribution of lectin receptors on lymphoid cells in tissues, and not in suspensions as is usually done. This is because studies on cell suspensions suffer from the disadvantage that the spatial relationships between cells are lost; thus valuable additional information on the properties of cells in situ may be gained by using frozen or paraffin-embedded tissue sections. Important information on the distribution of PNA binding (PNA+) cells in lymphoid tissues from man, mouse, rat, hamster, guinea pig, rabbit, sheep, and chicken has thus been obtained by Rose and her co-workers (Rose and Malchiodi, 1981; Rose et al., 1980) using horseradish

LECTIN RECEPTORS

225

peroxidase (HRP) conjugated to PNA; the lectin that was bound to the cells was visualized by oxidation with a tetraaminobiphenyl dye. Enumeration of the lectin-positive cells in suspension requires visual counting of the stained cells. Such counting, however, is laborious, especially when the incidence Of lectin-positive cells is very low. Furthermore, accurate quantitative discrimination of the amount of fluorescein on the cell surface is not possible using a fluorescence microscope. To analyze labeled cells on the basis of the amount of cell fluorescence they display, it is best to use a fluorescence-activated cell sorter (FACS). Results obtained from the examination of the binding patterns to lymphocytes, although useful for development of procedures for cell separation, may be misleading, particularly since there is often no correlation between the binding of a lectin to cells and agglutination (Lis and Sharon, 1977; Sharon and Lis, 1975).

B. TECHNIQUES FOR

C E L L SEPARATION

Several techniques are now available for cell separation by lectins (Table 11), of which selective agglutination is the simplest and most popular. It is the technique of choice when working with mixtures of cells that differ markedly in their lectin-binding properties and when a high proportion of the cells is agglutinated, as is the case with murine thymocytes and PNA (Fig. l),or murine splenocytes and SBA (Reisner et al., 1976a,b). The agglutinated cells are separated from the unagglutinated ones by sedimentation at unit gravity in a viscous medium (50%fetal calf serum or 5% bovine serum albumin) and are then dissociated into single cells by a sugar for which the lectin is specific (galactose or lactose in the case of PNA and SBA). Both the unagglutinated and agglutinated cells are recovered in very good yield (up to 80% combined), and the cells are fully viable. In addition to its simplicity, this method has the advantage that it can easily be scaled up to large numbers of cells (1O'O or more). Only poor separation by this technique can be achieved, if at all, when the number of cells is relatively small (less than 1@),the percentage of lectin-positive cells is low (less than 10-20%), or the density of lectin receptors on the cells is low (
226

NATHAN SHARON

---aThymus

Thymocytes

-

\

30 min

fetal Calf Serum 20% in PBS

cells

D -Galactose,OlM

t)

cells

I

.,:.

. I . .

PNA+

PNA-

FIG. 1. Fractionation of mouse thymocytes by peanut agglutinin.

Hypaque density gradient; the rosettes can then be dissociated by a specific sugar, and the erythrocytes separated by a second centrifugation on Ficoll-Hypaque. Alternatively, the erythrocytes are removed from the rosettes by osmotic shock. For example, separation of PNA+ cells from murine spleen, an organ in which their level is low (5-15%), is best achieved with the aid of rabbit erythrocytes (Reisner et al., 1978, 1980a). Rosetting with sialidase-treated sheep erythrocytes was used by Berrih et al. (1981a,b) for the fractionation (as well as visualization) by PNA of murine thymocytes and splenocytes. For cell separation by affinity chromatography, lectins immobilized on a solid support, either by covalent or noncovalent attachment, are employed. The same affinity adsorbent can be used repeatedly, giving economical and reproducible separations. Generally, separation is done on columns of the immobilized lectin. A suspension of cells is applied to the column; upon washing the column

LECTIN RECEPTORS

227

with a buffer, the unbound cells are eluted. The lectin-bound cells are then eluted with a specific sugar. The whole procedure is a gentle one, it does not impair cell viability to any significant extent, and the total yields are high. Using affinity chromatography on immobilized HPA, separation of sialidase treated B and T lymphocytes of mouse, man, and rat has been achieved (Hammarstrom et al., 1978; Hellstrom et al., 1976a); more recently, human cord blood lymphocytes have been separated on immobilized PNA into two fractions that differ in their immunological activity (Fig. 2) (Rosenberg et al., 1983). A method for the preparation of columns with different sugar specificities, by simply adsorbing lectins to a single glycoprotein conjugated to Sepharose, has been described in which the need for the separate coupling of each lectin to Sepharose is obviated (Pereira and Kabat, 1979b). The blood type A specific lectins from Dolichos biflorus, Helix pomatia, and Phaseolus lunatus, and the type O(H) specific lectins from Lotus tetragonolobus and Ulex europeus, were each adsorbed to individual columns of hog gastric mucin blood group A+ H substance, coupled to Sepharose. Such columns retained erythrocytes of blood group specificity corresponding to that of the adsorbed lectin, and the retained cells could be eluted together with the lectin by specific sugars. Furthermore, mixtures of cells of two blood types could be separated when the proportion of the retained cells was not too low. However, this method has not been tested with lymphocytes. Cells which bind a lectin specifically can be isolated by affinity chromatography on columns to which antibodies to the lectin are covalently bound. Thus, murine thymocytes which had been incubated with a subagglutinating concentration of PNA were fractionated on a column of anti-PNA-Sepharose (Irle et al., 1978). The unbound (mostly PNA-) thymocytes were recovered in the column wash, and the bound (PNA+)cells by elution with galactose. It has however been reported that although the thymocytes that were specifically eluted from the column consisted of over 98% PNA+ cells, as judged by examination with FITC-PNA, the unbound thymocytes also contained high levels (30-50%) of PNA+ cells (Hardt et al., 1980; Wagner et al., 1980~). Affinity chromatography can also be performed in tubes or dishes to which the lectin is attached. Thus, separation of human peripheral lymphocytes into fractions that differ in binding to lentil lectin has been achieved by incubation of the cells in plastic tubes or petri dishes coated with a gelatin layer to which the lectin had been coupled (Boldt

FIG.2. Binding of mouse thymocytes to PNA covalently linked to Sepharose beads (200-300 pm in diameter):right, control; left. beads with bound PNA+ thvmocvtes. The cells can be eluted with 0.2 M galactose. (Rosenbergand Sharon, unpublished.)

LECTIN RECEPTORS

229

and Lyons, 1979). The adherent cells (approximately 25-50% of the total) were recovered by melting the gelatin, and the cell-bound lectin was removed by treatment with mannose. Occasionally, combinations of different methods are used. For example, separation of PNA+ and PNA- thymocytes has been achieved with the aid of PNA-coated rabbit erythrocytes. The erythrocytes were fixed to plates with poly-L-lysine. PNA+ cells adhered to the monolayer of the rabbit erythrocytes, leaving the PNA- cells in suspension; the adhering cells were specifically eluted by galactose (Cayre et al., 1981). Flow microfluorimetry is useful, especially for analytical purposes, and is usually done in a FACS (Herzenberg et al., 1976). In this technique, a suspension of single cells, labeled with a fluorescent lectin, is hydrodynamically focused into a narrow jet and passed through a beam of light generated from a laser. The laser operates at a wavelength selected to excite fluorescence in the labeled cells. The signal emerging from each cell is electronically processed to produce electric pulses which serve to charge the liquid stream exactly when the droplet containing a desired cell is forming. The charged droplets that separate from the stream are directed into the space between two charged plates which deflect them to a collecting reservoir; uncharged droplets continue on their original course to another reservoir. In this way, cells can be sorted one by one according to the amount of fluorescent lectin bound. Sorting may also be done according to other parameters, such as size. The major disadvantages of flow microfluorimetry are the length of time (hours) required to separate large numbers (106-107 cells) of cells and the high cost of the equipment. Whichever technique is used, care should be taken to avoid prolonged contact (over 30 minutes) between the lectin and the cells, since this may result in uptake of the lectins by the cells, or in the formation of nonspecific bonds that cannot be dissociated by specific sugars. Also, when working with mitogenic lectins, prolonged contact may lead to lymphocyte stimulation. It should be noted, however, that many of the lectins used-for cell separation are nonmitogenic, or mitogenic only under special conditions (e.g., when polymerized or when the cells have been treated by sialidase; cf. Table I). C. How PUREARE “PURE”CELL PREPARATIONS? For cells, the terms “pure” and “purity” are not the same as when customarily applied to chemical substances. Such a substance is considered to b e pure when all its molecules are identical. Opera-

230

NATHAN SHARON

tionally, purity is determined b y demonstrating homogeneity and absence of impurities, usually by physicochemical criteria. The complexity of cells is such that a truly homogeneous preparation, consisting of cells that are identical in all respects, cannot be obtained. This means that no cell population or subpopulation isolated from a living organism or grown in vitro is biologically homogeneous, nor is it chemically pure. Even with cloned lines, the cells remain heterogeneous with respect to a variety of metabolic, mitogenic, and shape-related criteria. It is therefore best to define purity in terms of a single phenotypic trait, typically one that is expressed on the cell surface. In any case, the isolation of cells having even one property in common greatly facilitates the characterization of that property and its relationship to other aspects of the cell phenotype., Fractionation of cells is, however, not without risks. Purification procedures designed to recover a representative culture of lymphocytes from blood or other tissues may lead to the removal of less well-defined subpopulations of these cells (Boldt et al., 1972; Durkin et al., 1975; Eisen et al., 1972). In addition, cell fractionation may lead to alterations in cellular constituents, as shown by the finding of a marked increase in potassium and a decrease in sodium ion content in isolated rat thymus cells, as compared to the levels of these ions in rapidly excised whole thymus (Lichtman et al., 1972). Even such a simple and commonly used procedure as cooling cells to 4°C will disrupt their microtubules (Olmsted and Borisy, 1973), altering the cells structurally and possibly metabolically. The above problems must be borne in mind when attempting to purify cell subpopulations, whether by lectins or by other means. Ill. Murine Lymphocyte Subpopulations

A. RECEPTORS FOR PEANUT AND SOYBEAN AGGLUTININS The first clear-cut demonstration that subpopulations of T cells carry distinct lectin receptors was made in the course of our studies on the interaction of PNA with mouse thymocytes (Reisneret al., 1976a). Two lymphocyte subpopulations are easily distinguished in the thymus, one located in the cortex and the other in the medulla (Fig. 3). The medullary thymocytes, which in the past could be isolated in sufficient quantities only from mice that had been treated with cortisone or radiation, comprise approximately 10% of the total number of thymus cells. They exhibit cell-mediated reactivities [e.g., respond to PHA, react in-the mixed lymphocyte reaction (MLR) and possess the ability to induce graft-versus-host (GVH) disease], showing that they are

231

LECTIN RECEPTORS

LARY THYMOCYTES (40%) LOW THY-I ,HIGH

H-2,

GVH', MLR' ,PHA',

CORTISONE , RADIATION RESISTANT

CORTICAL THYMOCYTES (-90%) HIGH THY -I ,LOW H-2

GVH-,MLR',

,

PHA-

CORTISONE, RADIATION

S

FIG.3. Schematic representation of the anatomical location of the lymphocyte subpopulations in a thymus of an adult mouse. The plus and minus signs denote cells reactive or nonreactive, respectively, in the various tests: GVH, MLR, PHA.

immunologically mature or immunocompetent. I n addition, the medullary thymocytes lack detectable TL antigen and express a low density of the Thy 1 antigen and a high density of the H-2 antigen. I n contrast, the cortical thymocytes, which comprise the majority of the thymus cells (-90%) and which are eliminated by treatment of the animal with cortisone or radiation, are immunologically immature. Thus, they are inactive in cell-mediated immune reactions (MLR and GVH) a n d are not stimulated by PHA. They also differ from the medullary cells by a high level of Thy 1 and T L antigens and a low level of H-2 antigen, and include cells that are smaller in size and more dense. The medullary thymocytes are believed to be the maturation product of the cortical ones, although it is possible that the two subpopulations develop independently (Cantor and Weisman, 1976; Droege and Zucker, 1975; Goldschneider, 1980; Shortman et al.,

1975). Examination by fluorescence microscopy of thymocytes treated with fluorescein isothiocyanate-labeled (FITC) PNA revealed that the majority of the cells were stained, whereas a small portion (- 10%)was not; galactose, a sugar which binds to €"A, completely inhibited staining. I t was further found that whereas the cortisone-resistant thymocytes were not agglutinated by PNA, most of the untreated

232

NATHAN SHARON

thymus cells were; the clumps formed were separated from the unagglutinated single cells by sedimentation at unit gravity and recovered in suspension, free of lectins, by the addition of galactose (see Fig. 1). The separated cells, which were obtained in good yield (up to 80%), were fully viable (>95% in each fraction). In all the properties tested b y us (level of Thy 1 and H-2 surface antigens, stimulation by PHA and GVH activity), the cells agglutinated by PNA (PNA+ cells) were essentially identical with the cortical thymocytes, whereas the unagglutinated fraction (PNA-) consisted of cells which were similar to the cortisone-resistant medullary thymocytes, as well as to spleen T cells (Reisner et aZ., 1976a). To illustrate the efficiency of the fractionation, results of the MLR of the thymocytes, before and after fractionation by PNA are given in Fig. 4. We also demonstrated that mouse B lymphocytes are more susceptible to agglutination by SBA than T lymphocytes. Under suitable conditions only B splenocytes were agglutinated by this lectin (Reisner et aZ., 1976b). Using the same procedure developed for the isolation of PNA+ and PNA- thymocytes, splenocytes were fractionated into SBA+ and SBA- subpopulations which were characterized by their surface antigens (IgG, IgM, and Thy l),the GVH reaction and the mitogenic response of the cells to PHA and concanavalin A (T mitogens) and to lipopolysaccharide (LPS, a B cell mitogen). The results showed that

T

0 X

I

-

-THYMOCYTES~+SPLENOCYTES+

FIG. 4. Activity in the mixed lymphocyte reaction of C57BL mouse thymocytes C57BL; (a) fractionated by PNA and of splenocytes. Irradiated stimulator cells: (0) CBA.

233

LECTIN RECEPTORS

the SBA+ fraction consisted mainly of B cells and the SBA- fraction of T cells, with some cross contamination (Reisner et al., 1976b). This method gives better results than the conventional ones for the isolation of T and B splenocytes (treatment with nylon fibers to yield nonadherent T cells; lysis with anti-T antiserum and complement to give B cells) (Rosenfelder et al., 1979; van Eijket al., 1979)(Table 111). It was used, for example, in studies of the mitogenic response of dinitrophenyl (DNP)-modified splenocytes to anti-DNP antibody (Wilchek et al., 1979). DNP-modified SBA- cells responded extremely well to the antibody (stimulation index loo), whereas the SBA+ cells were not stimulated (stimulation index 1.2). Effective separation by SBA of lymph node B and T cells could also be achieved (van Eijk et al., 1979). The difference between the lectin binding properties of the lymphocyte subpopulations examined could be rationalized on the basis of the specificity of the lectins used, and the limited information available on the structure of carbohydrates present on lymphocyte surfaces. PNA is specific for Gal/31~3GalNAc,but may also combine with nonreducing terminal galactose residues in other compounds (Lotan et al., 1975; Pereira et al., 1976). The disaccharide Galp 1+3GalNAc is present in membrane glycoproteins (e.g., in glycophorin), in membrane glycolipids (e.g., ganglio-N-tetraosylceramide or asialo-GM,), and on lymphocytes (Sharon and Lis, 1982). In general, however, sialic acid residues are attached to the disacTABLE 111 ['*c]CALACTOSE INTO ACID-PRECIPITABLE MATERIAL FROM ISOLATED CBNJ SPLEEN CELL FRACTIONS STIMULATED B Y VARIOUS MITOGENS" INCORPORATION OF

~~~

~~

T cell mitogens* Splenocytes T cells Nylon nonadherent SBAB cells SBA+ Purified using antiserum to T cells

Concanav- Phytohemalin A agglutinin

B cell mitogensb Lipopolysaccharide

Lipoprotein

Control cellsb

11133 26487

13324 21453

656 58 1

2232 1361

218 287

1201

745

5025

12077

471

99 1

1022

2916

10802

692

Data from Rosenfelder et al. (1979).

* Radioactivity incorporated (cpm).

234

NATHAN SHARON

charide, so that its interaction with PNA is precluded. SBA, in addition to interacting with galactose, binds more strongly to N-acetylgalactosamine residues (Lis et al., 1970; Pereira et al., 1974), so that the two lectins do not necessarily combine with the same carbohydrate structures in glycoconjugates. It has also been reported that cortisoneresistant thymocytes and spleen T cells are more negatively charged and have a higher content of sialic acid than unfractionated thymocytes (Despont et al., 1975). We have further found that treatment with sialidase of cells devoid of PNA or SBA receptors, resulted in the appearance of such receptors. We have therefore proposed a simple model which incorporates our findings (Fig. 5) and have made the following tentative assumptions (Sharon, 1979a; Sharon and Reisner, 1979): 1. Cell surface receptors for PNA and SBA change in an orderly manner during murine lymphocyte differentiation and maturation. 2. The change invoIves the attachment of sialic acid residues to galactose and N-acetylgalactosamine residues on the cell surface. 3. The PNA receptor is a marker for immunologically immature lymphoid cells. 4. Hemopoietic stem cells that are devoid of GVH activity may be PNA+SBA+;such cells may be isolated with the aid of the lectins. 5. Results of experiments in mice may be applicable to man. 7 Thymus 7 r Spleen 1

T lymphocytes

-

/’ Immature

Stem cell

SBA+ Mature M a G re

\

B lymphocytes

\

\

,-- - - - - - - - - -

FIG.5. Sequential changes in lectin receptors during lymphocyte differentiation.0, Receptor for SBA and PNA; M, receptor for SBA only; 1 0 , sialic acid. (Modified from Sharon and Reisner, 1979.)

235

LECTIN RECEPTORS

Further work carried out in our laboratory and elsewhere has largely supported most of the above assumptions, and has established the use of PNA and SBA as important tools for the study of murine lymphocytes. Moreover, the new metho s have permitted better characterization of lymphocyte subpopulations in terms of their surface properties, effector functions, and differentiation patterns.

9

1 . Distribution Data from several laboratories on the quantitative distribution of PNA+ cells in different mouse organs, as examined mostly by staining of cells in suspension with FITC-PNA, are summarized in Table IV. It can be seen that the agreement between the results is remarkable. In cortisone-resistant thymocytes, some staining with FITC-PNA was reported (Dumont and Nardelli, 1979; Fowlkes et al., 1980; London et aE., 1978; see also Fig. 6). PNA+ lymphocytes in mice are not confined to the thymus; as expected, their level in most other organs is much lower. The proportion of PNA+ cells found in some organs may, however, depend on the technique and concentration of PNA used. Thus, about 40% of PNA rosetting lymphocytes were found in the spleens of adult mice at a PNA concentration of 20 pgIml, whereas at the same concentration of the fluorescent lectin, the IeveI of PNA+ cells was 10-15% (Fig. 7 ) (Berrih et al., 1981b). In the thymus, both techniques gave the same percentage of PNA+ lymphocytes at a TABLE IV DISTFUBUTION OF PNA+ CELLS IN MURINEORGANS PNA+ cells (%)

Thymus Peripheral lymph node Spleen Bone marrow Peyer’s patch Peripheral blood lymphocytes Fetal liver

90 15 20

Reisner et al. (1976a, 1979).

85 16 6 19 36

* London et al. (1978) and Roelants et al. (1979). Rose et al. (1980). Newman and Boss (1980). Weak intensity of staining by FITC-PNA.

82

3 5 4 24

86 13 20 25c

19

236

NATHAN SHARON

4

-

N

‘z Y 2

Y 8 2 K

w

m

f2

1

0

8

4

16

12

FLUORESCENCE UNITS o(lO-zI

FIG.6. Fluorescence profiles, obtained in the FACS, of thymocytes from B6 mice: and cortisone resistant (---) after staining with FITC-PNA; unstained normal (-) (-.- .-). The cortisone treatment enriches for PNA dull staining cells. (From Fowlkes et al., 1980.)

v)

-

~ 1 0 0

--

:so-

m

,p---

_---A

- c -

-

-4-

W

a

2

10

-Q---,

20

---

- /o

-0-

- - - - - ,------

30

I

1

40

50

PNA CONCENTRATION (pg/rnll

FIG. 7 . PNA+ murine lymphocytes assessed by fluorescent microscopy (0-0, thymus; 0---0, spleen) or by the PNA rosetting technique (A-A, thymus; A---A, spleen). (From Berrih et al., 1981a.)

LECTIN RECEPTORS

237

wide lectin concentration. Although in one study peripheral blood was reported to have a high level of PNA+ cells, the intensity of the fluorescence of the FITC-PNA stained cells was much weaker than that of the thymocytes, suggesting that the number of PNA receptors on these cells is low (Newman and Boss, 1980).In the spleens of aging mice the percentage of PNA+ cells found by selective agglutination was somewhat higher than that in the young animals (20-40%, as compared to 14-23%, respectively; cf. also Table IV) (Globerson et aZ., 1981). No change in the percentage of FITC-PNA cells was observed on thymus or spleen cultures incubated for up to 72 hours with either LPS (a B cell mitogen) or PHA (a T cell mitogen) (Rose et al., 1980). The distribution of PNA+ cells in various murine organs has also been assessed by examination of the binding of FITC-PNA and HRP-PNA to cryostat or paraffin sections of the organs (London et al., 1978; Raedler et aZ., 1981a,b; Rose and Malchiodi, 1981; Watanabe et al., 1981). In thymus, the PNA+ cells were located almost exclusively in the cortex (Fig. 8) and they comprised 80-90% of the total number of thymocytes, in agreement with the data obtained b y selective agglutination with PNA (London et al., 1978). In adult thymus, a corticomedullary gradient of the PNA-binding capacity of the thymic

FIG.8. Binding of HRP-PNA to mouse thymus. C, Cortex; M, medulla. x288. (From Rose and Malchiodi, 1981.)

238

NATHAN SHARON

cells was detected, which led to the suggestion that this phenomenon may be correlated with the maturational degree of the thymocytes (Raedler et al., 1981a,b). To the extent that staining was observed in the medulla, it was confined to the cytoplasm of the large and medium-sized mononuclear cells, but was absent from the membranes of the thymocytes, whereas in the cortex the membranes of the thymocytes were stained (Watanabe et aE., 1981).In cortisone-treated mice, all remaining PNA+ cells were located in the cortex, whereas PNA- cells were found both in the cortex and the medulla. London et al. (1978) have examined, by staining with FITC-PNA, the quantitative changes in the number of PNA+ cells present in suspensions of the thymocytes following treatment of mice with cortisone or with irradiation. Within 2-3 days after injection of hydrocortisone hemisuccinate, the number of PNA+ thymocytes decreased by a factor of 210, whereas that of the PNA- cells decreased by only 5.7. Similar results were obtained in irradiated mice (Fig. 9). 10‘

m

= 10’

-L

r

b

Y

n L

Y 0

5c

-

=

V

106

10’

I

l

l

l

1

2

3

4

l

5

.

,

6

,

7

8

Days a l t e r t r e a t m e n t

FIG.9. Changes in mouse thymocyte subpopulations following irradiation. Cells: total; 0-0,PNA+; 0 - .-0,PNA-. (From London et al., 1978.)

A-A,

LECTIN RECEPTORS

239

There was a rapid increase in the number of PNA+ cells upon thymic regeneration, accompanied by a much smaller rise in the PNA- cells. An inverse relationship between the PNA-binding ability and the electrophoretic mobility of thymocytes from normal and cortisonetreated mice was found by Dumont and Nardelli (1979) (Fig. 10). Thymocytes recovered from the fractions with the lowest electrophoretic mobility were all strongly PNA+, whereas those with the highest electrophoretic mobility were all PNA-. Of the thymocytes recovered 2 days after treatment of the mice with. cortisone, only a small proportion ( 5 % ) where PNA+. Eight days after the treatment, the thymus started to regenerate and the majority of the thymocytes exhibited dull staining with FITC-PNA. By day 14 most of the cells were strongly PNA+, as in normal thymus. Reciprocal experiments, in which normal thymocytes were separated by selective agglutination with PNA, definitely established that the PNA+ cells have a lower electrophoretic mobility than the PNA- cells, and that these two cell types also differed in size. These findings further support the suggestion that the difference in PNA binding between the cell subpopulations is the result of masking of the corresponding receptors by attachment of sialic acid residues. It has indeed been shown that T lymphocyte differentiation is accompanied by an increase in the sialic acid content of the Thy 1antigen (Hoessli et al., 1980). However, the

Electrophoretic fraction number

FIG.10. PNA binding and electrophoretic mobility of cells from adult C3H mice (A) before and (B) 2 days after treatment with cortisone acetate, 200 m g k g body weight. (Data from Dumont and Nardelli, 1979.)

240

NATHANSHARON

increased sialylation of this antigen cannot by itself account for the increased sialic acid content of mature (peripheral) T cells, mainly because they carry less Thy 1 than do immature thymocytes. Therefore changes in other glycoproteins and/or glycolipids must also occur. A new class of specialized cells recently found in murine thymus, designated as thymic nurse cells and identified as epithelial cells, has been shown to be PNA- (Born and Wekerle, 1982; Wekerle et al., 1980). Although these cells express products of the K/D regions as well as of the I A and I-E/C regions, they lack lymphocyte differentiation markers such as Thy 1, Lyt 1, and Lyt 2, as well as SmIg. On the basis of these and other findings, it has been postulated that an intraepithelial differentiation cycle is an essential step in the intrathymic T lymphocyte generation. Although the presence of PNA+ cells in spleen is well established, Watanabe et aZ. (1981) reported that HRP-PNA did not stain any splenic regions [in contrast to London et al. (1978),who found 3-6% of weakly stained cells in spleen, and to Rose and Malchiodi (1981) who found staining of spleen germinal centers], while FITC-PNA stained some fibrillar structures among lymphocytes in the peripheral regions of white pulp. The reason for this discrepancy is not clear. PNA+ cells were detected by staining sections of lymph nodes (7-16%, depending on mouse strain examined) and bone marrow (6-19%) (London et aZ., 1978). Rose et al. (1980) have reported that PNA bound to cells in murine germinal centers (Peyer’s patches) but not to those in other areas containing activated lymphocytes (Fig. 11). There was a good correlation between the presence of PNA+ cells in germinal centers in sections of lymphoid organs, and in cell suspensions from the same organs. Since no increase in the number of PNA+ cells was found in splenocytes which had been stimulated in vitro with either LPS or PHA, it was concluded that PNA does not bind to activated lymphocytes per se but only to cells in particular anatomic locations. The anatomic origin of PNA+ germinal center cells is not clear, but there is preliminary evidence that some or all of them are B lymphocytes. These observations, together with the suggestion that the PNA receptor is a marker for immature lymphocytes, support the possibility that germinal centers harbour a population of immature B cells (Rose et al., 1980). The use of PNA to separate germinal center cells should allow further characterization of their properties and functions. Massive binding of PNA to cells within germinal centers has also been reported by Raedler et al. (1981a,b). There were however some cells weakly stained by PNA in unstimulated lymph nodes; these cells

+

LECTIN RECEPTORS

241

were scattered uniformily throughout the entire organ, with the exception of primary follicles. The PNA- cells were mature B cells, plasmocytes, T lymphocytes, dendritic and histiocytic reticulum cells, as well as endothelial cells. By ultrastructural analysis, evidence was obtained that centroblasts and centrocytes are the cellular elements within the germinal centers displaying PNA receptors on their surface. Since centrocytes and centroblasts are considered to be B lymphocyte progenitors, proliferating after antigenic challenge, Raedler et al. (1981a,b) concluded that the expression of PNA receptors (i.e,, galactosyl residues) may be essential for maturation of both T and B lymphocytes. Spleen lymphocytes or lymph node cells from 4- to 5-day-old mice differed from adult peripheral T lymphocytes, and resembled cortical thymocytes, either young or old, in having high levels of PNA+ cells (up to 40% in lymph node cells). Peripheral T lymphocytes from newborn mice may therefore represent a peculiar stage in the T lymphocyte maturation, intermediate between immature cortical thymocytes and peripheral T cells of the adult mouse (Piguet et al., 1981). A study of PNA binding during ontogenesis revealed that PNA+ cells appear very early during the development of liver, thymus, and spleen (London et al., 1978, 1979a). In thymus, the appearance of PNA+ cells correlated well with that of Thy 1+ cells; on day 14 of gestation, both PNA+ and PNA- celIs were present but, in contrast to the mature thymus, the proportion of PNA- cells was considerably higher than that of PNA+ cells (Fig. 12). Some PNA+ cells were found in fetal liver before the thymic rudiment became fully colonized. The early onset of PNA+ cells in embryonic organs, and in particular the finding of such cells in fetal liver before their appearance in the thymus, would suggest that PNA binds to prothymocytes or lymphoid stem cells. A similar conclusion was presented by Reisner et al. (1980a) who found that prothymocytes are agglutinated by PNA and SBA. However, the presence of high levels of PNA- cells in fetal liver, coupled with other observations, has led London et al. (1979a) to postulate that two different pathways exist for the formation of functional T lymphocytes: in thymus, immunoincompetent thymocytes and immunocompetent thymocytes may develop from separate lineages, without maturation from PNA+ to PNA- cells; in the periphery, adult functional T cells (mostly PNA- cells) could arise from nonfunctional PNA+ cells present in large amounts in the prenatal period. Data supporting the existence of two independent pathways of lymphocyte maturation in the thymus were also obtained upon

242

NATHAN SHARON

FIG.11. Binding of HRP-PNA on frozen sections of murine Peyer’s patches (a) and section from the same block conventionally stained with hematoxylin and eosin (b). X 170. (From Rose et al., 1980.)

examination of the changes in PNA receptors of thymocytes during T cell differentiation in lethally irradiated and reconstituted mice (Kraal et al., 1981b). In such mice, there is a 6-day lag before the thymus starts to regenerate; during this period a relative enrichment was observed of PNA- cells which are Lyt 1+23-, extremely radioresistant, and of host origin. The responses to mitogens of the PNA- cells were found to recover earlier than those of the PNA+ cells, suggesting that maturation of the PNA- subpopulation precedes that of the cortical PNA+ subpopulation. This difference in maturation clearly indicates that at least part of the PNA- fraction develops independently.

LECTIN RECEPTORS

243

Some of the above conclusions have been challenged b y Raedler et al. (1981a,b) who found that in cryostat sections of prenatal mouse thymus, all lymphocytic cells were PNA+ [in contrast to the data of London et al. (1979a) obtained by FITC-PNA]. They considered this finding, in conjunction with the observation of a corticomedullary PNA binding gradient in thymus of adult mice, as supporting the thesis of thymic lymphocyte differentiation along a capsular-medullary axis. It has been known for a long time that during pregnancy, thymic involution occurs in various mammalian species, especially in mice and humans. Using FITC-PNA, Phuc et al. (1981) have found that in

244

NATHAN SHARON

JOO'

DAYS OF GESTATION

FIG.12. PNA+ cells in murine lymphoid organs during ontogeny, drawn from data of London et ol. (1979a).

pregnant mice, the number of cortical thymocytes was greatly decreased, whereas the pool of steroid-resistant medullary thymocytes (as assayed by a specific heteroantiserum) appeared to be unchanged. Therefore, pregnancy-induced thymic atrophy in mice is linked to the decrease in the steroid-sensitive cortical cells, which is in accord with earlier histological observations that the atrophy occurs in the cortical zone of the thymus, while the medullary area remains unaffected. PNA has been employed to distinguish preleukemic cells from end stage leukemia cells in mice. Preleukemic bone marrow and spleen cells of C57BL/6 mice, that had been inoculated with the radiation leukemia virus D-RadLV, were PNA+ whereas the end stage leukemia cells were PNA- (Reisner et al., 1980b). This observation provides further evidence that preleukemic cells possess surface markers similar to those of the prothymocyte. Both in vivo and in vitro, the cells in the thymus susceptible to the viral transformation were present mainly among the PNA+ thymocytes. Studies of thymocytes in AKR mice during leukemogenesis revealed a decrease in the number of PNA+ cells at the late preleukemic and at the leukemic stages of the disease (Zielinski et al., 1980, 1981). Concomitantly, the expression of murine virus leukemia antigen was increased, and there were changes in phenotypic markers (such as Lyt

LECTIN RECEPTORS

245

1, Lyt 2, Thy 1, and H-2 antigens), indicating the disappearance of immature thymocytes and the appearance of more mature cells. The results confirm and extend previous observations that a shift to a mature thymocyte phenotype occurs during the leukemogenic process, and focus attention on the parallels between normal and neoplastic differentiation. 2. Surface Markers Results of measurements of the binding of PNA and SBA to mouse lymphocytes, both before and after treatment with sialidase, are summarized in Fig. 13 and corroborate the model presented by us (Fig. 5 ) . In general, whenever receptors for these lectins were absent, they appeared upon treatment of the cells with the enzyme. For example, Rose and MaIchiodi (1981) found that treatment with sialidase of lymphocytes (including B cells) either in suspension or in frozen sections, makes all of them bind PNA strongly, although the effect depends on the concentration of enzyme used. Perhaps the only exception is the report by Newman and Boss (1980) that the majority of B splenocytes after sialidase treatment did not bind PNA. The reason for this discrepancy with other results is not clear. There is little doubt, therefore, that all lymphocytes bear the sugars that bind PNA, but in the majority of cases these sugars are not accessible to the lectin because of substitution by sialic acid.

FIG.13. Binding of 'P61-labeledPNA and SBA to mouse lymphocytes before and after the cells have been treated with sialidase: T, and T2,immature and mature thymocytes, respectively; T, and B,, T and B splenocytes, respectively. Columns indicate total binding before (W) and after (0) sialidase treatment; (0),a small proportion of PNA+ cells without sialidase treatment. (Data from Prujansky, 1977.)

246

NATHAN SHARON

The distribution and quantitative expression of various surface antigens was most thoroughly examined in PNA+ and PNA- thymocytes using fluorescent antibodies or differential cytotoxicity caused by specific antisera and complement. As an illustration, the distribution of the Thy 1 antigen on murine thymocytes, before and after fractionation by PNA, is given in Fig. 14 (Betel et al., 1979). Results obtained by this and other techniques are summarized in Table V. Clearly, PNA+ and PNA- cells differ significantly in their surface phenotyes, although most differences are quantitative rather than qualitative. Such studies have also led to the identification of two new distinct subsets of thymocytes; cells that are PNA- and Lyt 1+23- (Betel et al., 1979, 1980; Zeicher et al., 1979), and those that are PNA+ and Lyt 6.2+ (London and Horton, 1980).Recently, separation by the FACS of PNAthymocytes into Lyt 2.2+ and Lyt 2.2- subpopulations has been reported (Mage et al., 1981). It has been suggested that the latter cell type may represent an intermediate form along one of the T cell lineages within the thymus (presumably for the generation of Lyt 1+23- cells). Accordingly, PNA binding is progressively, though

0

32

64

96

fluorescence units x

128

IO-*

FIG. 14. Fluorescence profiles in a FACS of B6 mouse thymocytes separated by selective agglutination with PNA and then stained with anti-Thy 1.2 (AKR anti-C3H) serum and FITC goat anti-mouse Ig. (From Betel et al., 1979.)

160

247

LECTIN RECEPTORS TABLE V SURFACEANTIGENSOF MUFXNELYMPHOCYTES

Cells and markers Thymocytes Thy 1 H-2 TLa Lyt 1+23+

PNA+

PNA-

References

High Low

Low High

+

+,-

Reisner et al. (1976a) Reisner et al. (1976a) Roelants et al. (1979); Zeicher et al. (1979) Betel et al. (1980); Wagner et al. ( 1 9 8 0 ~ ) Betel et al. (1980); Wagner et al. ( 1 9 8 0 ~ ) London and Horton (1980)

+

+

Lyt 1+23Lyt 6.2 Splenocytes Thy 1 Ig

+

+,-

+ +,-

Roelants et al. (1979) Roelants et al. (1979)

incompletely, lost during maturation and Lyt 1+23-cells gain the Lyt 6 antigen before their migration from the thymus (London and Horton, 1980). By means of double immunofluorescence labeling with FITC-PNA and rhodamine-labeled polyvalent goat antimouse immunoglobulin (for the detection of antisera to Lyt 6.2, Thy 1.2, and SmIg), three T lymphocyte subsets were further identified: PNA+Lyt 6.2+, PNA+Lyt 6.2-, and PNA-Lyt 6.2+,having distinct distribution in adult thymus and spleen (London, 1980). In the thymus, most of the lymphocytes were PNA+Thy 1.2+and PNA+Lyt 6.2-. In spleen, however, there was almost the same distribution of PNA+Thy 1.2+ and PNA+Lyt 6.2+ subsets. The three subsets (PNA+Lyt 6.2+, PNA+Lyt 6.2-, and PNA-Lyt 6.2+) were also found in fetal liver, thymus, and spleen, although at different levels, which changed during ontogenesis. The PNA+Lyt 6.2+subset was present in all three organs during fetal life at a low level (12%at most) similar to that found in the adult animals; the PNA+Lyt 6.2- subset was also present in the fetal organs examined, being very low in the liver (4-8%)and spleen (1-7%)and high in the fetal thymus (50-70%). In contrast, the percentage of PNA-Lyt 6.2+ spleen cells was very low in fetal and neonatal life (1-5%) and increased significantly between 1 and 2 weeks of age (to 18%), whereas in the other two organs it remained low (0-4% prenatal, 2%in adult). These results demonstrate that during fetal life, lymphocyte subsets are phenotypically differentiated as in adult life. Since the

248

NATHAN SHARON

phenotypes identified with PNA and anti-Lyt 6.2 are expressed so early during ontogenesis, it was concluded that the assumption of an i n situ maturation taking place either through the thymic or peripheral environment is unlikely, and that the findings obtained are consistent with the view that differentiation of T lymphocytes occurs preferentially through various‘ lineages present during early ontogenesis. Moreover, the quantitative changes that occur in some of these subsets from fetal to adult life, may explain certain immunologically immature features of the young murine spleen. Thus, in prenatal life a large proportion of Lyt 6.2+ spleen cells are PNA+ which may account, at least partially, for the suppressive function of this organ, whereas more “mature” T lymphocytes of the PNA-Lyt 6.2+ phenotype occur after only 1week of age, expressing most of the mature properties of T lymphocytes. 3. Biochemical Characteristics With the availability of a simple and effective method for the fractionation by selective agglutination with PNA of murine thymocytes into immature (cortical) and mature (medullary) cells, it became possible to compare directly the biochemical properties of the two subpopulations. Such a comparison has revealed marked differences in the levels of several enzymes and metabolites examined (Table VI). Whenever tested, the results obtained with the PNA- cells were the same as those for cortisone-resistant thymocytes. Thus, the level of 5’-nucleotidase was about 10 times lower in the PNA+ than in the PNA- cells, and it was suggested on the basis of these and other data that the absence of 5’-nucleotidase activity may be considered as a marker for selective maturation arrest (Dornand et al., 1980). Compared to unfractionated thymocytes, the PNA- thymocytes incorporated much less iododeoxyuridine after short-term (4 hour) incubation, showing that the latter subpopulation consists largely of nondividing cells (Madyastha et al., 1980). The level of (2’-5’)oligoisoadenylate synthetase (an enzyme characteristic of differentiated and nonproliferating cells) was three times higher in the PNA- than in the PNA+ thymocytes (Kimchi, 1981). The low level of the enzyme in the latter subpopulation was correlated with the inability of the cells to produce interferon (y-type), rather than to their inability to respond to interferon; it increased upon stimulation of the cells with concanavalin A. PNA- thymocytes, on the other hand, produced the same levels of y-interferon as T splenocytes. Of special significance are the studies of terminal deoxynucleotidyltransferase (TdT), since this enzyme is considered as a marker of

TABLE Vl BIOCHEMICAL DIFFERENCESBETWEEN MURINE THYMOCYTESUBPOPULATIONS SEPARATED BY PNA Thymocytes Strain of mice Prostaglandins E (pg/loB cells) 5'-Nucleotidase (nmole P,/mg proteidhour) 20-Hydroxysteroid deh ydrogenase (pmole/4 x 10B cells/hour) Adenosine deaminase (nmoleslmg proteidhour) Purine nucleoside phosphorylase (nmoleslmg proteinhour) (2'-5') Oligo-isoadenylate synthetase ( c p d 8 hoursl2O mg protein) Interferon synthesis in concanavalin A-stimulated cells (U/ml) Thymidine incorporated in unstimulated cells (cpmll hour)

C57BL/6J

Unfractionated

PNA+

PNA-

14

7

82

C3Hleb DBA2 Swiss B/W (m) C57BL/6 BALBIc (DBA x C57BL)F,

30 30 15-35 17 35 41 9500

18 20 8.3-25 12 15 14 14500

130 150 90-210 72 131 211 2770

(DBA x C57BL)F,

1350

2280 1400

BALBIc

BALBlc

<1

BALB/c

200,000

T splenocytes

Reference" 1

2 (320 ? 70)

5

1950

4

920

5370

4

4100

4050

3

128-256

128-256

3

25,000

15,000

3

References: (1)Bauminger (1978); (2) Dornand et al. (1980); (3)Kimchi (1981); (4) Sidi et al. (1982); (5)Weinstein and Berkovich ( 1981).

250

NATHANSHARON

immature cells. Indeed, Rothenberg (1980) has found that relative to total protein synthesis, the PNA+ thymocytes synthesized 5 to 10-fold more TdT (as well as 10- to 20-fold more TL and 10- to 20-fold less H-2) than the PNA- cells. The biosynthetic rates of the surface markers examined, as well as that of TdT, were consistent with the steady-state phenotypes of the cortical and medullary thymocytes. This implies that changes in the expression of differentiation antigens during T lymphocyte development are controlled, at least in part, at a translational or pretranslational level. However, some synthesis of TdT by the PNA- thymocytes, even after two cycles of separation of the cells by selective agglutination with the lectin, was observed (Rothenberg, 1980). In a subsequent study (Cayre et al., 1981)this was shown to be due to contamination of the PNA- fraction by PNA+ cells, since repeated fractionation of the PNA- cells on immobilized PNA (three successive cycles of affinity chromatography) resulted in almost complete removal of the TdT-producing cells. Incubation of the rigorously purified PNA- thymocytes with TP5, a synthetic pentapeptide with biological activity similar to that of thymopoietin, resulted in the synthesis of TdT in a large number of cells in the fraction, and the appearance of TL on the surface of about 20% of the cells. Cayre et al. (1981) concluded that if indeed there was no significant contamination of these PNA- preparations by PNA+ cells, the results appear to contradict the proposed sequence according to which PNA+ cells mature to PNA- cells in the thymus. It was postulated that either immature cells in the thymus are PNA- (and cortisone sensitive) therefore representing a third class as suggested by others (Weissman et al., 1978) but not yet identified in earlier experiments with the lectin, or that “mature” cells may indeed be induced, under special conditions, to re-express TdT and even TL. A marked difference was observed between PNA+ and PNAthymocytes in the time course of response to stimulation by concanavalin A, as assessed by the labeling of their membrane components (phospholipids, glycolipids, and glycoproteins) and of their DNA with radioactive precursors (van Eijk and Muhlradt, 1979). PNA+ thymocytes responded early, with a peak of stimulated biosynthetic activity at about 15 hours after the start of cultivation, whereas with the PNAthymocytes, the peak was about 25 hours later. Upon polyacrylamide gel electrophoresis, in the presence of sodium dodecyl sulfate, of the glycoproteins labeled with [3Hlfucose or PHIgalactose from PNA+, PNA-, and cortisone-resistant cells, the PNA- and cortisone-resistant cells gave indistinguishable patterns, which differed significantly from those of the PNA+ cells. It was concluded that the two peaks of

LECTIN RECEPTORS

251

biosynthetic activity and blast transformation observed in the course of concanavalin A stimulation of thymocytes are caused by two distinct cell populations, PNA+ and PNA- (or cortisone resistant), which require different times for maximal response to the mitogen, and react independently of one another. These findings may also account for the contradictory results obtained by different workers regarding the response to concanavalin A of murine thymocytes, It is possible that the time at which the response is measured is rather critical and that the response may further depend on the concentration of cells in the assay system.

4 . Functional Properties Following our initial report that PNA+ and PNA- mouse thymocytes differ in their response to PHA and in their GVH activity (Reisner et al., 1976a) and that SBA+ and SBA- splenocytes exhibit similar differences, it was found that PNA+ thymocytes (Umiel et al., 1978; Fig. 3), as well as PNA+ and SBA+ splenocytes (Reisner et al., 1980a; Fig. 15) are nonresponsive in the MLR. When splenocytes were separated by mixed rosetting with low PNA concentrations (2.5 pglml), the PNA+ cells (10% of total) consisted chiefly of T lymphocytes that did not respond to T mitogens and suppressed antigen-specific responses. However, when spleen cells were separated at high PNA concentration (10 pg/ml), both T and B cells (50% of total) were obtained in the PNA+ fraction, and these cells Stimulation index:

FIG. 15. MLR response of fractionated mouse splenocytes: 0, C57BLJ6 spleen cells as stimulator; @, C3H/HeJ spleen cells as stimulator. Cells: a, unseparated; b, SBA-; c, SBA+; d, SBA+ PNA-; e, SBA+ PNA+. (From Reisner et al., 1980a.)

252

NATHAN SHARON

did not differ in their functional properties from the PNA- cells (Berrih et at., 1981a). The percentage of PNA+ T splenocytes was almost independent of the PNA concentration used, whereas that of the B splenocytes increased markedly with the increase in lectin concentration (Fig. 5).These results suggest that B lymphocytes too have receptors for PNA, but that these are considerably fewer in number than those on T cells; alternatively, their affinity for PNA may be lower. The PNA+ splenocytes isolated by rosetting at low PNA concentration apparently belong to an early T subpopulation which lacks at least some of the T cell functions. Indeed, it is known that the response to mitogen stimulation is related to the stage of lymphocyte differentiation (Stobo, 1972). The T splenocytes responding to T mitogens express the PNA-Thy 1.2+Lyt6.2+Ig- phenotype, while the suppressor T lymphocytes have the PNA+Thy 1.2+Lyt 6.2+Ig- phenotype (Berrih et al., 1981a,b; cf. also Wagner et al., 1980~). We have shown that the two thymocyte subpopulations have different effects on tumor growth in mice (Umiel et al., 1978). The PNA+ cells accelerated the growth rate and increased the number of takes of 3LL tumors in mice more than the unfractionated thymocytes, thus acting as suppressor cells. The PNA- cells, on the other hand, caused pronounced inhibition of tumor growth and a decrease in the number of tumor takes, similar to that observed with spleen cells, indicating that they act as helper cells, In another study, we have found that PNA+ cells from embryonic mouse liver were enriched in suppressor activity, as evidenced by their effect on the MLR of adult murine spleen cells, and the response of the latter to different mitogens (Globerson et al., 1979; Rabinovichet al., 1979) (Fig. 16).When spleen cells from aging (24- to 30-month-old) mice, manifesting low response to concanavalin A and PHA, were fractionated by PNA, it was found that the PNA+ cells suppressed the response of splenocytes of young mice to the above mitogens (Globerson et al., 1981). The PNA- cells were not suppressive, but they responded to concanavalin A and PHA better than the unseparated cells. It was suggested that at least part of the decrease in lymphoid cell function in aging may be caused by an increase in the activity of suppressor cells, although this may not be true for all mouse strains. Natural killer (NK) cells in murine spleen are PNA-, suggesting that these cells and prothymocytes are distinct splenic subpopulations (Koo et al., 1981). PNA+ cells isolated b y fractionation in the FACS or by selective agglutination from concanavalin A-activated murine splenocytes were found to exert a marked suppressive effect on the primary antibody response in vitro to sheep red blood cells, whereas the PNA- cells did

253

LECTIN RECEPTORS

200

-

R

I

0 150 X Y

E

P c 0

100

5

50

L CD

ABCD

ICD

ABCD

0,S FIG.16. Suppression of adult spleen cell response to mitogens by embryonic liver cell preparations. 0,f Mitogen; a, - mitogen; A, spleen; B, spleen and liver, unfractionated; C, spIeen and liver PNA- cells; D, spleen and liver PNA+ cells. LPS, ConA

FHA

LFS

Lipopolysaccharide; DxS, dextran sulfate. Ordinate: ['Hlthymidine incorporation expressed by mean cpm. (From Globerson et al., 1979.)

not affect the antibody response (Imai et al., 1979; Nakano et al., 1980a,b). I t is noteworthy that in this system too the suppressor cells were in the PNA+ fraction. It was found that the PNA+ suppressor splenocytes were Thy 1.2+ and Lyt 2.2+, showing that they were T cells. Antigen-specific suppressor cells could also be separated by PNA. The suppressor T cell activity was eliminated by treatment of the concanavalin A-activated T cells either with antibodies to the ganglioside asialo-GM, [Gal/31+3Gal~1+4(NeuNAccw2+3)Glc/3Cerl and complement, with anti-Forssman antiserum and complement, or with antigloboside antiserum and complement, but not by treatment

254

NATHAN SHARON

with anti-GM, antiserum and complement. Conversely, helper T activity induced by LCA and separated by Limulus polyphemus agglutinin (LPA), a lectin specific for sialic acid, was eliminated by treatment of the cells with anti-GM, and complement, but not by the treatment with the other antiglycolipid antisera and complement. It appears, therefore, that asialo-GM, (or related sugar sequences devoid of sialic acid) is abundant on the cell surface of suppressor T cells, and GM, (or related sugar sequences containing sialic acid) is present on the surface of helper T cells. The level of PNA+ T cells in concanavalin A-activated splenocytes, as monitored in the FACS, markedly decreased with age in New Zealand black (NZB) mice but not in nonautoimmune strains (Imai et al., 1980). On the other hand, LPA+ T cells in splenocytes activated by LCA were found to gradually decrease both in NZB and nonautoimmune strains. Since PNA selectively binds to concanavalin A-induced suppressor T cells, these results support observations by other investigators that suppressor T cell function declines with age in NZB mice (Barthold et al., 1967; Gerber et al., 1974; Krakauer et al., 1976). Fractionation of thymocytes by PNA has been used in several studies to examine in vitro developmental and functional relationships between cortical and medullary thymocytes. Both PNA+ and PNAthymocytes were shown to be required for in vitro generation of suppressor cells, as assayed by their effect on the MLR (Eisenthal et al., 1979,1982).Although there were earlier indications for the requirement of cell cooperation in the generation of suppressor cells, direct evidence for such a requirement was only possible using the PNA fractionation technique. The PNA- thymocytes were shown to be responsible for the production of a series of murine interleukins (lymphocyte stimulating factors that are not H-2 or species restricted) by mitogenstimulated thymus and thymus-derived cells (Basham et al., 1981). When PNA+ thymocytes obtained by affinity chromatography with PNA on a column of anti-PNA were cultured in the presence of concanavalin A and a supernatant from lymphoid cultures treated for 24 hours with concanavalin A or with irradiated lymph node cells, they acquired new cell membrane markers characteristic of mature thymocytes, including the loss of the PNA receptor ( I r k et al., 1978). The treated cells also acquired other properties characteristic of mature, immunocompetent thymocytes, such as the ability to respond to PHA. These findings were interpreted as providing support to the idea of a differentiation pathway in which PNA+ thymocytes are precursors of the PNA- cells. Maturation of PNA+ thymocytes, as evidenced by increase in the re-

255

LECTIN RECEPTORS

sponse to PHA or changes in surface markers, could also be induced by thymic epithelial supernatants (Kruisbeek, 1979; Kruisbeek and Astaldi, 1979) (Fig. 17), by THF, a thymic hormone known to potentiate T cell differentiation (Trainin et al., 1980), by the synthetic pentapeptide thymopoietin3,_,, (TP5) (Nash et aZ., 1981), and by tumor necrosis factor-positive serum (Abbott et al., 1981). It should be noted, however, that no maturation of PNA+ to PNA- cells, as measured by increase in thymidine incorporation in concanavalin A-stimulated cells, was observed upon preincubation of the PNA+ cells with partially purified IL-2 (Bodeker and Muhlradt, 1980; Bodeker et al., 1980). Also, the PNA+ cells could not be induced to release IL-2, thereby excluding an IL-2 mediated maturation. These authors have further pointed out that whenever maturation of PNA+ to PNA- cells has been observed, it may be ascribed to the presence of contaminating PNA- cells which selectively proliferate in a population of dying PNA+ cells. Several groups have studied independently the generation in vitro of cytotoxic T lymphocytes (CTL) by thymocyte subpopulations, and

THYMOCYTES UNTREATED

pl

PNA+

PNA-

PHA per culture

FIG. 17. Effect of thymic epithelial culture supernatant on P4C1thymidine incorporation into mouse thymocytes stimulated with PHA, before and after separation with peanut agglutinin. -, Thymic epithelial culture supernatant; ---,control. (Modified from Kruisbeek and Astaldi, 1979.)

256

NATHAN SHARON

have obtained essentially the same results and reached similar conclusions (Cooley and Schmitt-Verhulst, 1979; Hardt et al., 1980; Kruisbeek et al., 1980; Mathieson et al., 1979; Wagner et al., 1980a,b,c). Thymocytes are apparently poor in helper T cell precursors, as indicated by the observation that their induction into CTL can be greatly enhanced by addition of antigen-specific T helper cells (Baum and Pilarski, 1977). Upon separation of the thymocytes by selective agglutination with PNA, it was found that only the PNA- fraction was able to mount an autonomous cytotoxic response to allogenic cells (Kruisbeek et al., 1980) or to self-modified syngeneic cells, i.e., cells modified by trinitrobenzyl sulfonate (TNBS) (Cooley and SchmittVerhulst, 1979), trinitrophenol (TNP) (Hardt et al., 1980; Kruisbeek et al., 1980), or Sendai virus (Hardt et al., 1980). The presence of T cell growth factor (Kruisbeek et al., 1980), of IL-2 (Hardt et al., 1980; Wagner et al., 1 9 8 0 ~or ) ~of lymph node or spleen helper cells from mice sensitized to trinitrochlorobenzene (Cooley and SchmittVerhulst, 1979), permitted the differentiation of both alloreactive (anti-self TNP and TNBS) and H-2 restricted CTL responses in the PNA+ thymocyte subpopulation and enhanced the level of cytotoxic cells generated in the PNA- thymocyte cultures. Kruisbeek et al. (1980) have, however, concluded that the thymic epithelial supernatant does not act b y inducing maturation per se, but via induction of helper T cells within the PNA+ population; the newly formed helper T cells help the already present precursor CTL to develop into effector CTL. To determine whether the CTL precursors within thymocytes were derived from the medullary or cortical cells, Wagner and his co-workers (Hardt et al., 1980; Wagner et al., 1980c) separated the two subpopulations with the aid of PNA by affinity chromatography on a column of anti-PNA, according to the method of Irle et al. (1978). The PNA+ thymocytes obtained by specific elution from the column were all Lyt 123+. The thymocytes that were not bound to the column, which contained 30-50% PNA+ cells, were treated with anti-PNA antibodies and complement, to yield a PNA- subpopulation essentially devoid of contaminating PNA+ cells; it consisted of 90% Lyt 1+23- and 10% Lyt 123+ cells. Although the PNA+ thymocytes were unable by themselves to mount alloreactive CTL, in the presence of IL-2 both alloreactive and H-2 restricted TNP-specific CTL high responses were observed, comparable in magnitude to that of peripheral T cells (Fig, 18).On the other hand, the highly purified PNA- cells, like cortisoneresistant thymocytes, were able to mount autonomously both

257

LECTIN RECEPTORS PNA- thymxyres

PNA' t h y m y r e s

I

I'l

I

51

I

251

Ratio effector to target cells

FIG.18. Cytotoxic T Iymphocyte (CTL) immune responsiveness of PNA+ and PNAthymocytes. Thymocytes of 4- to 5-week-old BG/Lyt 1.1mice were fractionated by cell affinity chromatography on immobilized PNA. PNA+ thymocytes (3 x lo6)and PNAthymocytes (3 x lo6) were cultured together with 2 x lo6 BALB/c stimulator cells (A-A) or with TNP-conjugated syngeneic spleen cells (0-0) in the absence (solid symbols) or presence (open symbols) of 70 p1 of helper factor. After 5 days the alloreactivity induced was tested against TNP-conjugated EL, cells. Background lysis during the 3 hour SICr test did not exceed 24%. (From Wagner et al., 1980a.)

alloreactive and H-2-restricted TNP-specific CTL; that is, their CTL responsiveness was not absolutely dependent on exogenously added IL-2, although IL-2 enhanced the magnitude of CTL responses generated. The ability of the PNA- cells to mount autonomous CTL responses was ascribed to the presence in this subpopuIation of Lyt 1+23- T helper cells. Results along similar lines have been reported by Hamaoka et al. (1981), who showed that among the various thymocyte subsets, the PNA+ cells obtained by selective agglutination with the lectin were able to generate CTL responses, contingent on the presence of killedhelper factor produced from stimulated T cells that had been primed by Mycobacterium tuberculosis. These data, as well as similar ones obtained in other laboratories, provide direct evidence that Lyt 123+cells represent a common source of alloreactive and H-2 restricted CTL precursors in unprimed lymphocyte populations, and that addition of IL-2 (T cell growth factor, TCGF) bypasses the need for helper T cells and their solubIe products(s) in these PNA+ precursor CTL and allows their development into effector CTL. They also show that along the differentiation pathway from Lyt 123+to Lyt 1-23+ cytotoxic effector cells, cytolytic activity is carried out by T cells that still express the Lyt 123+ phenotype.

258

NATHAN SHARON

Since the apparent immunoincompetence of cortical PNA+ thymocytes is probably due to the lack of T helper cells, it is possible that the thymus-dependent, rate-limiting step for the generation of CTL immunocompetence is operating on the level of Lyt 1+23- T-helper (inducer) cells, whereas CTL precursors already exist on a prethymic level. If, on the other hand, the thymic major histocompatibility complex is selecting self-restricted CTL precursors, the PNA+Lyt 123+ thymocytes that are endowed, in the presence of IL-2, with a CTL repertoire similar to that of peripheral T cells must have already passed the postulated intrathymic selection process (Wagner et at., 1980a,b ,c). Further work (Draber and Kisielow, 1981)has shown that the majority of the PNA+ cells did not respond to concanavalin A even in the presence of TCGF, and that the responsive population constitutes a minor fraction (about 15%) of PNA+ high-density thymocytes with surface antigenic phenotypes similar to those of mature T cells (high levels of H-2 and low levels of Thy 1).Such a unique combination of properties and the presence of the TL antigen on some members of the responding population, suggests that it contains cells at intermediate stages of differentiation between immature thymocytes and mature T cells. Marked differences were observed in the migration patterns of PNA+ and PNA- thymocytes after their injection into syngeneic hosts (Kraal et al., 1981a; Madyastha et al., 1980). The PNA+ cells migrated preferentially to the spleen, whereas the PNA- cells were lymph node seeking. Incubation with anti-Lyt sera revealed that PNA-Lyt 1+ cells homed in popliteal lymph nodes and Peyer’s patches, but not in mesenteric lymph nodes (Kraal et al., 1981a). Upon treatment of the PNA+ cells with galactose oxidase, they were no longer agglutinated by PNA and their propensity to localize in the spleen of syngeneic animals was drastically curtailed (Madyastha et al., 1980). Structures resembling the PNA receptor are known to be involved in phenomena related to biological recognition. Most importantly, Ashwell and Morel1 (1974) have demonstrated that sialidase treatment of certain serum glycoproteins, which leads to the exposure of galactosyl residues, caused their prompt removal from the circulatory system via a specific receptor on the surface membrane of hepatocytes. Treatment with sialidase of rabbit and human erythrocytes also caused their rapid disappearance from the blood. The PNA receptor present on most thymocytes may thus be responsible for their prompt removal from circulation and sequestration in the liver.

259

LECTIN RECEPTORS

5. Application to Bone Marrow Transplantation A key prediction of our model (Fig. 5) is that murine hemopoietic stem cells are PNA+SBA+.Since GVH disease is the major obstacle encountered in attempts to achieve bone marrow transplantation across histocompatibility barriers, the isolation of such PNA+SBA+cells seemed highly desirable. We have indeed found that sequential fractionation of mouse spleen cells by SBA and PNA afforded a fraction enriched in stem cells as measured by spleen colony assay in vivo. The PNA+SBA+ cell fraction was also devoid of GVH activity as evidenced by its ability to reconstitute lethally irradiated allogeneic mice (Reisner et al., 1978). The results of a typical experiment are shown in Fig. 19. Grafting lethally irradiated allogeneic mice with unfractionated splenocytes or with cells unagglutinated by SBA resulted in high mortality (13/15 and 15/15, respectively) within the first 30 days after irradiation. The two

0

5

10

15

20

25

30

90

Time (Days)

FIG.19. Cumulative mortality of irradiated (BALB/c X C57BL/6)F, mice after t r a n s plantation with splenocytes (lo' cells per animal) from SWR mice, starting with 15 mice unfractionated splenocytes; 0-0, splenocytes sein each group. Grafts: A-A, quentially agglutinated by SBA and PNA; 0-0, splenocytes unagglutinated by SBA; O - - - O , control without graft. (From Reisner et al., 1978.)

260

NATHAN SHARON

mice surviving the first 4 weeks were suffering from wasting disease (delayed GVH reaction) and died within the second month after transplantation. In a parallel experiment, the few surviving mice were sacrificed 4 and 5 weeks after transplantation. Spleen histology and bone marrow differential count revealed typical GVH symptoms. Among the mice grafted with the twice-agglutinated fraction, only 1of 15 died. The remaining mice survived more than 6 months. Spleen histology and bone marrow differential count taken 4 and 5 weeks after transplantation in a parallel experiment revealed that in the reconstituted mice both spleen and bone marrow were completely restored. These findings led to the studies on the application of lectins to the separation of bone marrow cells for transplantation in humans, as will be discussed in a later part of this article (Section IV,A,4).

B. RECEPTORS FOR OTHER LECTINS Many other lectins have been tested for differential binding to or agglutination of murine lymphocyte subpopulations, and in attempts to identify new subpopulations. Satisfactory results were obtained only in a limited number of cases. The lectin from the snail Helix pomatia (HPA) specific for N-acetylgalactosamine has been shown to be a useful reagent for the identification and isolation of T cells in several species, including mouse and man (Hammarstrom et al., 1978). Treatment of the cells with sialidase is required to unmask the receptor sites for this lectin. For certain purposes, however, it may not be advisable to use this method, since removal of sialic acid from cells is known to change markedly many of their biological properties, such as response to certain mitqgens, recognition b y viruses and mycoplasma, rate of clearance from the circulatory system, and homing to target organs (for review see Flowers and Sharon, 1979). Sialidase-treated mouse splenocytes were separated on a column of HPA covalently bound to Sepharose, to yield an unbound fraction highly enriched in B cells, and a fraction eluted with 1 mg/ml N-acetylgalactosamine that was highly enriched in T cells (Fig. 20). The intermediate fraction contained both B and T cells, in a ratio similar to that of the unfractionated splenocytes. The total cell yield was typically between 60 and 70%,and the fractionated cells exhibited functional integrity as shown by their response to mitogens and in the assay for antibody-dependent cell-mediated cytotoxicity. Affinity chromatography on HPA-Sepharose has permitted the isolation for the first time of a cell fraction highly enriched in mouse natural killer (NK) cells, for which no conventional surface marker was

26 1

LECTIN RECEPTORS

a

b

c

d

FIG. 20. Fractionation of mouse spleen cells (treated by sialidase) by affinity chromatography on Sepharose bound HPA: a, unfractionated cells; b, unadsorbed to column; c, eluted with 0.1 mg/ml N-acetylgalactosamine; d, eluted with 1 mg/ml N-acetylgalactosamine. Unshaded bars indicate HPA+ cells; shaded bars indicate cells with surface immunoglobulin. (From Hammarstrom et al., 1978.)

previously known (Haller et al., 1978). NK activity was highest in the fraction eluted from HPA-Sepharose with 0.1 mg/ml of N-acetylgalactosamine, and was somewhat enriched in the last fraction eluted with 1 mg/ml of the sugar. Separation between NK activity and alloreactive cytotoxic activity was also achieved, showing that while NK cells are HPA,+ (the S subscript denotes that the HPA receptors are cells which have been pretreated with sialidase) their lectin binding properties seem to differ from those of alloreactive cytotoxic T cells. It was suggested that HPA receptors may represent a simple and reliable marker of NK cells, and may be useful for purification of this cell type. Using a sensitive mixed rosetting assay (with human type A erythrocytes) combined with immunofluorescence, HPA receptors were found on the surface of approximately 90% of sialidase-treated peripheral T lymphocytes, 75% of thymocytes, 30% of bone marrow cells, 20% of nude spleen cells, 15-50% of peritoneal exudate macrophages, and a subpopulation of peritoneal exudate mast cells (Mattes and Holden, 1981). The Thy 1+ nude spleen cells were predominantly HPAs-. Approximately 5% of B lymphocytes were weakly positive, and neutrophils were negative. HPA receptors were partially detected also on cells which had not been treated with sialidase. Two methods for the fractionation of lymphocytes by HPA were employed, which gave significantly different results. Thus, when HPAs+ cells were removed by mixed rosetting from sialidase-treated splenocytes, T cell mitogen responses were abolished, while B cell mitogen responses, T cell cytotoxicity, and natural killer cytotoxicity were only slightly affected, if at all. Upon fractionation by affinity chromatography on HPA-agarose, all natural killer cells bound to the

262

NATHAN SHARON

column, as well as a considerable number of B lymphocytes. Cytotoxic T cells were heterogeneous: roughly half were not bound, but the remainder were bound and eluted. Since in this study, elution of the HPAs+ cells from the column was in a single step with 5.0 mM N-acetylgalactosamine, it is difficult to compare the results with those of Haller et al. (1978). A single membrane glycoprotein, with an apparent MW of 130,000, appeared to be responsible for the bulk of the binding of HPA to sialidase-treated lymphocytes (Axelsson et al., 1978). This glycoprotein was expressed on mouse T lymphocytes, both normal and malignant, but not on B cells. Binding of surface labeled glycoproteins from mouse CTL to a panel of immobilized lectins has been examined (Kimura et al., 1979). Evidence was obtained for specific interaction of a glycoprotein (designated as T145, MW 145,000) with the Vicia villosa lectin ( W L ) , specific for GalNAccr1+3 Gal (Kaladas et al., 1981). CTL bearing the T145 marker were isolated by adsorption to columns of Sepharosebound W L , and eluted with the inhibitory sugar N-acetylgalactosamine. Several recent reports, however, have questioned the relationship between the expression of T145, of cytolytic activity, and of binding of W L . Conzelmann et al. (1980) selected WL-resistant mutants which expressed T145 or a T145-like glycoprotein with altered carbohydrate. Although the mutants were cytolytically active, they bound up to 100-fold less of the lectin than the parental lines. In a later study (Braciale et al., 1981), W L coupled to sheep erythrocytes via chromic chloride was employed to quantitate the population of cells capable of binding the lectin by the number of rosettes formed. Using Isopaque-Ficoll centrifugation to separate rosetting from nonrosetting cells, fractionation was achieved of cells from an in vitro primary mixed lymphocyte culture, into cytotoxic and noncytotoxic populations. Binding of W L did not closely parallel the presence of cytotoxic activity, raising the possibility that the cytotoxic cells may only be a subset of the W L binding lymphocytes. In another study the relationship between the binding of W L and the expression of cytolytic function in murine lymphoblasts was examined (MacDonald et al., 1981). T cell blast populations activated against H-2 or parasite antigens, all had comparable levels of fluorescence after treatment with W L , followed by antibodies to W L and fluorescent sheep antirabbit IgA, although the cytolytic activity of these cells varied widely. Furthermore, when lymphoblasts binding large or small amounts of W L were sorted on the basis of their relative fluorescence intensity and tested for cytolytic activity, no appreciable difference

LECTIN RECEPTORS

263

in this activity between the two populations was observed. These results are inconsistent with the suggestion that W L binds selectively to CTL. By selective agglutination with LPA, specific for sialic acid, separation was achieved of helper T cells from other cells found in murine spleen (Nakano et al., 1980a). Preliminary results on the interaction of a partially purified lectin from the Maine lobster (Homarus americanus) with murine lymphocytes have been reported (Hartman et al., 1976, 1977). The lectin, specific for sialic acid residues, agglutinated cortisone-resistant thymocytes and spleen T cells; however only a small percentage of normal thymocytes was agglutinated. It was suggested that the lobster agglutinin covalently linked to Sepharose could be used for the separation and isolation of cortical lymphocytes. Such separations, if successful, would be complementary to the separation with PNA. In any event, the above results provide further support to the findings that mature and immature mouse thymocytes differ in their content of surface sialic acid. As mentioned earlier, mouse B cells are agglutinated more strongly by WGA than T cells (Schnebli and Dukor, 1972). Bourguignon et al. (1979) have reported the separation of mouse spleen B and T cells by selective agglutination of the B cells with this lectin. The aggregates formed upon treatment of the splenocytes with WGA were separated from the monodisperse T cells by gravity sedimentation and subsequently dissociated into single cells by treatment with the inhibitory monosaccharide, N-acetylglucosamine, to yield a B cell-enriched fraction. Approximately 10- 15% cross-contamination of the resultant T and B cell fractions was observed, which is higher than that obtained when splenocyte separation is done with SBA (Reisner et al., 1976b; Rosenfelder et al., 1979). Immobilized WGA has been used to fractionate mouse bone marrow cells; elution of the bound cells was achieved with N acetylglucosamine (Nicola et al., 1978). Because of the complexity of bone marrow samples, which contain a great variety of cells at different stages of maturation, a complete separation of different types of cells was not obtained. However, the distribution profiles of the various cells suggest that this type of column separation can give valuable purification of several of the cell types present. Labeling experiments with FITC-WGA (specific both for N-acetylglucosamine and N-acetylneuraminic acid) as well as other results have indicated that the stem cells are among bone marrow cells with the highest surface density of N-acetylneuraminic acid (Visser et al., 1981). More than 90% of the cells from embryonic thymus (13 days

264

NATHAN SHARON

gestation) of mice of different strains, were brightly stained with fluorescein-labeled Dolichos bijlorus lectin (FITC-DBA) (Kasai et al., 1980). No cells were observed in fetal liver and adult lymphoid organs such as thymus, spleen, lymph nodes, and bone marrow. The proportion of DBA+ embryonic thymocytes declined sharply with the development of gestation (Fig. 21), and such cells were completely absent in the adult thymus. On the other hand, Thy 1+cells increased in reciprocal proportion. The DBA receptor seems to appear when the stem cells have reached the thymus, and it was proposed that this receptor may be considered as a fetal differentiation marker. DBA also bound to spontaneous leukemia cells of GRS/A mice, but not to lymphoid cells of the host (Muramatsu et al., 1980). Ten different lectins were screened for selective interaction with mouse hemopoietic colony-forming cells (CFCs) using agglutination or quantitative analysis of the number of fluoresceinated lectin molecules bound per cells (as measured in the FACS) (Nicola et al., 1980b). PWM, HPA, SBA, and PNA preferentially bound to CFCs which permitted 4- to 10-fold enrichment for these progenitor cells by sorting for the highly fluorescent cells. Further analysis of the low- and high-angle light scattering characteristics of the CFCs indicated that these cells were polydisperse, but could be enriched 10-fold by selecting for cells with high intensity low-angle (0') scatter and low intensity high-angle (90") scatter. PWM gave the best enrichment (10-

Gestation ( d a y s ) FIG.21. Change in percentage of cells binding the Dolichos bijlorus lectin (DBA+) and Thy 1+ cells in the thymus of CBA mouse embryos. CBA mouse fetal thymocytes were stained with FITC-DBA and monoclonal anti-Thy 1.2. The percentage of positive cells was counted by a fluorescence microscope. (Modified from Kasai et al., 1980.)

LECTIN RECEPTORS

265

to 15-fold) for CFCs from adult bone marrow, from fetal sources (liver and blood), and from the spleens of mice injected previously with the outer membrane lipoprotein from Escherichia coli. Three parameter sorting for CFCs using the FACS (low-angle scatter, high-angle scatter, and cell bound FITC-PWM) resulted in large enrichment factors (16to 50-fold) for CFCs from all the above sources. Over 7% of the cells sorted from bone marrow and 28% of the cells sorted from fetal peripheral blood were hemopoietic CFCs. Ninety percent of the cells in these fractions had the morphology of blast cells or myelocytes. It was concluded that screening of other developmental systems using quantitation of fluorescence with lectins should prove of general value for the purification of cells at selected differentiation states. In a recent attempt to identify new lymphocyte subpopulations in murine thymus, the interaction of over 30 FITC-labeled lectins with the thymocytes as well as with the cortisone-resistant thymocytes was examined by flow microfluorimetry (Fowlkes et al., 1980). Only preliminary indications were obtained for the differential binding of several of the lectins, to two or possibly three thymocyte subsets. Although it was concluded that in addition to PNA, certain other lectins (e.g., thwe of Maclura pomifera or Sophora japonica, both specific for galactosyl residues) could probably be used to detect, define, and fractionate thymocyte subpopulations, no procedures were presented for this purpose. In another study of the binding patterns of several lectins to small lymphocytes of mouse thymus, spleen, and bone marrow, major and minor populations were distinguished in each organ, but no correlation with functional properties was given (Saveriano et al.,

1981). IV. Human Lymphocyte Subpopulations

A. RECEPTORS FOR PEANUT AND SOYBEAN AGGLUTININS 1 . Thymus Because of the many similarities between the lymphoid systems of mouse and man, it seemed to us reasonable to assume that the findings described above with PNA and SBA should be applicable also to human lymphocytes. We did indeed find that the majority of human thymocytes (60-80%) bound FITC-PNA, and that all the sialidasetreated cells bound the lectin (Reisner et al., 1979). The PNA+ subpopulation was separated from the PNA- cells by differential

266

NATHANSHARON

agglutination with the lectin (Reisner et al., 1979). The former cells responded poorly to the mitogenic stimulation by PHA and in the MLR, whereas the latter subpopulation responded strongly to both stimuli. Thus it seems that in humans, as in mice, the PNA+ thymocytes are functionally immature. In the mouse this subpopulation is sensitive to cortisone treatment and can be selectively eliminated. Indeed, the cortisone-resistant, immunologically competent thymocytes were found in the thymus of a child treated with high doses of corticosteroids (Galili and Schlesinger, 1975). Normally, however, no such manipulations are possible in humans; PNA thus provides a unique tool for the isolation of both human cortical (immature) and medullary (mature) thymocytes. Examination of FITC-PNA of isolated human thymocytes carried out in several laboratories generally gave values of 50-80% of PNA+ cells, both in children and in fetuses (Table VII). After fractionation by the lectin, the purity of the PNA+ and PNA- subpopulations, as assayed by FITC-PNA, was 95 and 99%,respectively (Umiel et al., 1982). Whenever tested, the PNA+ thymocytes were considerably less immunoreactive than the PNA- thymocytes. Thus, upon separation of human thymocytes on a discontinubus Ficoll gradient, the PNA+ cells were found mainly in the layers containing immunoincompetent cells (London et al., 1979b). TABLE VII DISTRIBUTION OF PNA' CELLSIN HUMAN TISSUES ~

PNA+ cells (%)

a Peripheral blood lymphocytes Thymus (fetuses) Thymus (children) Spleen Tonsils Adenoids Cord blood

b

1

5

70

52

- -

'

d

1 6 72 50 51 - 13 -

e

f

4 27 54

-

4

75

- 5 5 - 14 18 - - - - 9 18 - 24 - - 21

Reisner et al. (1979) and Lis et al. (1979). (1979b). Ballet et al. (1980). Levin et al. (1980). Richard et al. (1981). Maccario et al. (1981).

* London et al.

c

LECTIN RECEPTORS

267

Evidence has since been obtained that human thymus contains a small subpopulation of PNA+ cortical thymocytes which express mature T cell antigens ( T l , T3, and T12) as well as a medullary subpopulation expressing the T6 antigen (Umiel et al., 1982). Neither the PNA+Tl+ nor the PNA+T1- cells were stimulated by PHA, although stimulation of the former cells occurred in the presence of IL-2. It should be recalled that the presence of PNA+ thymocytes with an immunologically functional repertoire has been previously documented in the murine system. The relation between the expression of the PNA receptor and the receptor for the Fc portion of IgM ( T , or T,) on human thymocytes in short-term culture was investigated (Musiani et al., 1981). Approximately 80% of the thymocytes were stained by FITC-PNA (PNA+ cells), but only 65% were agglutinated by the lectin. After 24 hours of incubation, 23% of the PNA+ cells and 33% of the PNA- cells were T,+. Longer incubation resulted in an increase of the T , + cells and a decrease of PNA+ cells. After 96 hours incubation, 60% of the unfractionated thymocytes were TxI+.Within the PNA+ subpopulation the proportion of T,,+ cells was then 6076, and 70% in the PNA- subpopulation; concomitantly, the proportion of PNA+ cells in the thymus decreased to 40%. It was suggested that increased expression of the receptor for Fc-IgM, which is associated with the disappearance of the PNA receptor, could be considered a step in the maturation of thymocytes to peripheral T lymphocytes. Studies on the distribution of PNA+ and PNA- cells in sections of human thymus treated with HRP-PNA or FITC-PNA gave results supporting the conclusions obtained by examination of the thymocytes in suspension: the PNA+ lymphocytes were found in the cortex, and the PNA- thymocytes in the medullary regions (Christensson et al., 1982; Raedler et al., 1981a,b; Rose and Malchiodi, 1981; Umiel et al., 1982) as is the case in mice. Fractionation of thymocytes by selective agglutination with PNA, as well as with SBA, and staining of the cells with the fluoresceinated lectins, have been employed in a study aimed at following the changes in human thymus during ontogeny (Richard et al., 1981). In the thymus of children, approximately 50% of the cells react with both PNA and SBA (i.e., they are PNA+SBA+),approximately 23% are PNA-SBA-, and 23% are PNA-SBA+. This pattern of distribution of lectin receptors was compared with the reactivity of the same cells with monoclonal antibodies recognizing the T cell differentiation antigens A50 and a series (T3, T6, and T8) that defines three discrete stages of T cell differentiation. A clear-cut correspondence has been

268

NATHAN SHARON

found between the PNA+SBA+ cells and the common cortical T3-T6+A50- thymocytes, between PNA-SBA+ cells and late (medullary) T3+T6-A50+ thymocytes, and between PNA-SBA- cells and early T3-Y6- thymocytes. Strikingly, in the fetus a majority of thymocytes are PNA- (80%), most of them being PNA-SBA+. During ontogeny, the percentage of the PNA-SBA+ phenotype gradually decreases, while in turn that of the PNA+SBA+ phenotype increases. In addition, some PNA+SBA- cells appear late during ontogeny; the proportion of SBA+ cells is constant throughout. These changes are in sharp contrast to the results obtained from examination of the surface antigens since no major modifications in the proportions of T6+, T8+, and A50+ cells occur during ontogeny. Thus, in fetal thymus, in addition to late thymocytes, many common thymocytes are PNA-SBA'. It is also possible that prothymocytes are PNA-, as suggested by Galili et

al. (1980). Although the above findings demonstrate that there is a good correlation between PNA and SBA receptors on thymocytes and certain surface antigens defined by monoclonal antibodies on these cells, direct evidence for maturational or ontogenic affiliation between cortical PNA+ and medullary PNA- T cells is still lacking in humans as in mice. Human PNA+ thymocytes, obtained by selective agglutination with the lectin, contained per cell about half the number of glucocorticoid receptors in comparison with the PNA- thymocytes (Ranelletti et al., 1981), whereas the PNA+ cells had a similar number of receptors as the unfractionated thymocytes. Despite the lower number of glucocorticoid receptor sites, the PNA+ thymocytes did not differ from the PNA- cells in their sensitivity to the inhibitory effects of glucocorticoid (triamcinolone acetonide) on protein and DNA synthesis by the cells. However, the PNA+ cells appeared to be less resistantin vitro to the steroid-induced cell lysis as compared with the PNA- cells. It was suggested that glucocorticoid receptor density and corticosensitivity are not directly related, and that the number of glucocorticoid receptor sites may be dependent on the degree of immunologic maturation in the thymus. Earlier it was reported that prothymocytes are highly sensitive to glucocorticoids in uitro, whereas the bulk of the cortical thymocytes (as well as the medullary thymocytes) are highly resistant to the drug (Galili et al., 1980). The PNA- thymocytes were responsible for the marked enhancement caused by all-truns-retinoic acid in the response of human thymocyte cultures to mitogens or allogenic cells (Side11et al., 1981). Retinoic acid increased the number of mitogen-stimulated thymocyte colonies developing in soft agar. It was suggested that the target of

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269

retinoic acid must be lymphoid cells at a later stage of maturation than those that express the receptor for PNA. 2 . Other Lymphoid Organs In preparations of mononuclear cells isolated from human tonsils, about 10-15% of the cells were PNA+ (Ballet et al., 1980; London et al., 1979b; see also Table VII). In contrast to peripheral blood, most of these PNA+ cells did not ingest latex particles and thus are not monocytes but lymphocytes. Only a small percentage of the PNA+ tonsil lymphocytes formed E rosettes, suggesting that the PNA+ cells are B lymphocytes, possibly germinal center cells as proposed for the mouse by Rose and Malchiodi (1981). The difference in PNA receptors between thymocytes, tonsil lymphocytes, and peripheral blood lymphocytes may reflect their degree of maturation, as also indicated by other surface markers (Chess and Shlossman, 1977) and by the presence of the enzyme TdT. Examination of cryostat sections of human tonsils, lymphoma lymph nodes, reactive lymph nodes, and miscellaneous tumors with HRP-PNA demonstrated selective binding of the lectin to lymphocytes in germinal centers (Rose and Malchiodi, 1981; Rose et al., 1981) (Fig. 22). Ultrastructurally, these cells could be identified as centrocytes and centroblasts (Raedler et al., 1981a,b). Cell suspensions from lymph nodes from 21 patients with non-Hodgkin’s lymphomas were phenotyped for PNA receptors (with the aid of FITC-PNA), and a variety of surface markers (Rose et al., 1981). Four out of five cases of centroblastic/centrocytic follicular lymphoma had a PNA+SmIg+C3d+ phenotype. Both of two cases of centroblastic-centrocytic diffuse lymphoma were PNA-. There was no correlation between PNA binding and the presence of heavy or light chain Ig class, nor was there a correlation between PNA binding and the presence of C3d receptors. The binding pattern of PNA to one case of thymic hyperplasia and two cases of malignant lymphoblastic T type lymphoma suggested that some, but not all, cortical thymocytes bind PNA. The results confirm the restricted binding of PNA to subsets of B and T lymphocytes, and illustrate its potential use as a marker in follicular lymphoma in situ or in cell suspension.

3 . Peripheral and Umbilical Cord Blood Examination of the peripheral blood lymphocytes of normal donors showed that usually less than 2% of lymphocytes bound the lectin (Reisner et al., 1979; for values from other laboratories see Table VII).

270

NATHAN SHARON

FIG.22. Binding of HRP-PNA to human tonsil. GC, Germinal centre; CO, corona. x 198. (From Rose and Malchiodi, 1981.)

This residual binding, or the high levels (up to 6%) sometimes observed, is due largely to monocytes, most of which are PNA+ (see also Haimovitz et al., 1982). Upon treatment of the cells with sialidase, binding occurred to all the cells; both B and T lymphocytes had on the average 3.8 x 106 PNA binding sites per cell (Newman e t al., 1978). Elevated levels of PNA+ nonadherent mononuclear cells, considered as immature (up to 69%), were found with the aid of FITC-PNA and the FACS, in the blood of nearly half out of a total of 38 human patients early after allogenic bone marrow transplantation (Elfenbein et al., 1981; Griend et al., 1981). In concanavalin A-stimulated peripheral blood T lymphocytes, high levels of PNA+ cells (35%) were found (Sakane et aE., 1981). Separation of the stimulated T cells by the autologous rosette technique afforded two fractions that differed markedly in the level of PNA+ cells and helper and suppressor activities, The autorosetting fraction that acted as suppressor cells contained 94% PNA+ cells, whereas the nonrosetting fraction, which exhibited helper activity, contained only 7% PNA+ cells. These results are analogous to the finding in the murine system, where suppressor cells are always present in the PNA+ subpopulation. As mentioned earlier, mouse B splenocytes are SBA+and can readily

271

LECTIN RECEPTORS

be separated from the T splenocytes that are SBA- by selective agglutination of the former cells with SBA. No separation of human peripheral blood lymphocytes could however be achieved by SBA, since the lectin agglutinated both the B cells and a major fraction of the T cells. Monocytes were also agglutinated. Upon separation by mixed rosetting with autologous erythrocytes and SBA, the SBA+ fraction contained T helper cells for antibody production whereas the SBAfraction was enriched with T suppressor cells (Reisner et al., 1980c) (Fig. 23). Similar observations were made in tests of the proliferative response to mumps antigen. A monoclonal antibody, anti-Leu2a, which recognizes the same T cell antigen previously identified by a heterologous anti-human T cell serum, was found to define by indirect immunofluorescence a subpopulation of SBA- cells of intermediate staining intensity which was not detectable in the SBA+ population. The results obtained clearly demonstrate that human suppressor and helper T cells can be separated by SBA. Preliminary studies also indicated that FITC-SBA could be used to stain helper T cells. Such cell staining, as well as the separation by agglutination of the two T cell subpopulations, could be used for clinical evaluation of immunodeficient patients to assess whether the deficiency is in antibody-producing cells or in the accessory cells. Peripheral blood lymphocytes of patients with different types of

I,

a

b

c

a

b

c

FIG.23. Antibody production in peripheral blood mononuclear cells fractionated by SBA. Columns: a, unseparated; b, unagglutinated; c, agglutinated. Left: Polyclonal stimulation of antibody production by PWM. Right: Specific assay after incubation with SRBC (PFC, plaque-forming cells). Results are shown as mean f SD. (From Reisner et al., 1980c.)

272

NATHAN SHARON

leukemia varied in their PNA binding properties (Fig. 24) (Barzilay et al., 1982; Reisner et al., 1979). A considerable proportion of patients out of a total of 220 examined, had a high level (220%)of PNA+ cells and were classified as “lectin-positive.” About half of the patients with acute lymphocytic leukemia or with myeloid leukemia were lectinpositive, as were over two-thirds of the Burkitt’s leukemia and lymphoma patients examined. Of special significance is the finding that no more than 11% of patients with acute leukemia in remission were lectin-positive. Contrary to the patients with acute leukemia, only 9%of the chronic lymphocytic leukemia patients examined had >80% of PNA+ cells. The presence of high levels of PNA+ cells (215%) in the peripheral blood of 13 out of 25 patients with childhood acute lymphocytic leukemia was reported by Levin et al. (1980). It was further suggested that a high level of PNA+ cells may serve as an indication for a poorer prognosis. In patients with chronic myeloid leukemia, neutrophils were less readily agglutinated by PNA than in normal individuals, whereas they were more readily agglutinated by LPA (Taub et al.,

1980). Attempts to find a correlation between the level of PNA+ cells and Normal

Acute Leukemias ALL

ALL AML Burkitt Remission

CLL 0 0 0

0

0

‘0°[ 80 o n

0

0

3

0 0

1

0

o n

0

D 0 0

0

nnon

l5maK

no no n o 0

0 0 0 0 0 0

0

I 0 0 0 0 0 0

0

0

I

I A

68 N= 24 85 29 14 FIG. 24. PNA+ cells in peripheral blood of healthy human donors and leukemic patients. ALL, Acute lymphocyte leukemia; AML, acyte myelocytic leukemia; CLL, chronic lymphocytic leukemia. N denotes the number of subjects.

LECTIN RECEPTORS

273

the expression of B or T cell surface markers did not give clear results (Barzilay et al., 1982). Thus, the percentage of lectin-positive cells among acute T lymphocytic leukemia patients did not differ significantly from the unclassified acute lymphocytic leukemia. Thus, while the pattern of PNA binding to normal human peripheral blood lymphocytes and to human thymocytes is in line with the proposal that the receptor for this lectin may be a marker for immature cells, this relation holds only partially when cells of leukemic patients are examined. The finding that a considerably larger percentage of patients with acute lymphocytic leukemia are lectin-positive, than are patients with chronic lymphocytic leukemia, is in agreement with the notion that cells in the former leukemias are more immature than those of the latter ones; moreover, as expected, upon remission the percentage of €"A+ cells decreases. There is, however, a significant number of acute lymphocytic leukemia cases whose mononuclear cells do not bind the lectin, whereas cells of chronic lymphocytic leukemia patients are lectin-positive. It would appear that, contrary to what was expected from a survey of a small number of patients, PNA binding to cells cannot serve, at least for the time being, as a diagnostic aid for the classification of leukemias. Similar conclusions were reached by Galili et al. (1981) who examined the interaction of FITC-PNA with normal and malignant lymphoid cells in humans. No mature peripheral cells in any of the lymphoid organs bound PNA. In contrast to the normal differentiation pathway, expression of PNA receptors did not seem to coincide with that of T cell characteristics in the various malignant lymphoid cells studied. Evidently, more information is needed about the properties of the PNA receptor before it can be used as a differentiation marker of malignant lymphoid cells. The finding that the PNA receptor may be a marker for immature human lymphocytes made it likely that fetal cells could be detected and isolated with this lectin. Isolation of such cells from maternal blood is most desirable since they may be used for prenatal diagnosis, thus obviating the need to perform amniocentesis (see for example Herzenberg et aZ., 1979). Since the immune system of the newborn is immature in several respects, as best demonstrated in studies of umbilical cord blood, we first tried to isolate PNA+ lymphocytes from cord blood. In preliminary experiments it was found that 15-25% of cord blood mononuclear cells were stained by FITC-PNA and that the staining was inhibited b y galactose (Lis et al., 1979). Separation of PNA- and PNA+ subpopulations from individual donors could be achieved by selective agglutination with PNA. However, the

274

NATHAN SHARON

reproducibility of the separations was poor, mainly due to the small number of cells (30-80 x los) available from each individual donor, and the low percentage of PNA+ cells. Using immobilized PNA, instead of the lectin in solution, resulted in good yields of both fractions, even when cord blood of a single donor is used and the starting number of cells is low (Rosenberg et al., 1983). The two subpopulations, unbound (PNA-) cells and bound (PNA+) that were specifically eluted, were found to respond differently to mitogens and in the MLR. Although there were variations between individual experiments, a consistent pattern was observed. The average ratios of responses of the PNA+ and PNA- cells were 0.25, 0.15, and 0.15 for PHA, concanavalin A, and pokeweed mitogen, respectively; in the MLR the ratio was 0.15 (Fig. 25). The number of T cells determined by sheep red blood cell rosette formation was much smaller in the PNA+ than in the PNA- fraction. The low response of PNA+ cells to mitogenic stimulation may therefore be due to the presence of a large number of immature pre-T or pre-B cells that cannot be triggered by the lectin.

60

X

40

20

Exp I

Exp 2

FIG.25. MLR of human umbilical cord blood mononuclear cells from two individual donors before and after fractionation on immobilized PNA. Stimulation index is the ratio of the response in the presence of allogeneic cells to the response in the presence of syngeneic cells (Rosenberg and Sharon, unpublished).

275

LECTIN RECEPTORS

Human cord blood lymphocytes fractionated by PNA were also studied by Maccario et al. (1981).The percentage of PNA+ cells found by these workers (20.5 8.5) was similar to our results (Rosenberg et al., 1983), as was the relative number of E rosette-forming cells in the PNA+ fraction, but the number of SmIg+ cells was considerably higher. However, no immunological functions were reported, probably because the number of PNA+ cells is insufficient when cord blood from single donors is fractionated by selective agglutination.

*

4 . Application to Bone Marrow Transptantation The demonstration that cells producing GVH disease can be removed from mouse bone marrow by selective agglutination with lectins, to yield a fraction that is suitable for bone marrow transplantation across histocompatibility barriers (Reisner et al., 1978; see also Section 111,A75),raised the possibility that the same approach may also be applicable to humans. Experiments carried out along these lines have however shown that the distribution of receptors for PNA and SBA in human bone marrow is markedly different from that of the murine bone marrow. It was further found that 20-80% of the human bone marrow cells are agglutinated by SBA, and that most of the pluripotential stem cells (colony-forming cells) are retained in the SBA- cell fractionation. This fraction was also depleted of T cell alloreactivity in vitro (Reisner et aE., 1980d, 1982) (Fig. 26). A minor population of T cells (suppressor/killer) still remaining in the SBA-

Monkey

Human

a

b

c

d

e

a

b

c

d

e

FIG.26. CFCs in human and monkey bone marrow cells fractionated by sheep red blood cells and SBA. Cell fractions: (a) leucocyte-rich; (b) E+; (c) E-; (d) E-SBA-; (e) E-SBA+. (From Reisner et al., 1980d.)

276

NATHAN SHARON

fraction could be removed by rosetting with sheep red blood cells to yield a SBA-E- nonrosetting fraction. A second rosetting with sialidase-treated sheep red blood cells ensured complete removal of the T cells. The use of SBA in the first step of fractionation is advantageous in that, without significant loss of the stem cells, it greatly decreases the total number of bone marrow cells to b e processed by the rosetting method, which cannot be efficiently applied to large volumes of cells. As with human cells, the majority of monkey bone marrow cells (80-90%) are agglutinated by SBA, and the SBA- fraction is markedly enriched with colony-forming units relative to the unseparated cells (Reisner et al., 1980d) (Fig. 26). The unagglutinated fraction of both human and monkey marrow cells is also highly enriched with spontaneously dividing cells, as shown by uptake of tritiated thymidine by the unstimulated cells. Transplantation with lectinseparated cells were first tested in Cynomolgus monkeys, as a model for bone marrow transplantation across histocompatibility barriers in man. Female Cynomolgus monkeys were prepared for transplantation with marrow from unrelated, allogeneic males, by total body irradiation (850-1000 r) and a cytotoxic drug (cyclophosphamide). Of six monkeys transplanted with SBA-fractionated marrow, five achieved sustained engraftment with complete conversion to donor karyotype (Reisner et al., 198la). Follow up was adequate to assess GVH disease in four of the animals, none of which developed clinical or pathological evidence of the disease. In an experiment in humans the same procedure was used to fractionate marrow cells from an HLA-A,B,Dr nonidentical, MLR nonreactive, paternal donor for transplantation into an infant with acute leukemia (Reisner et al., 1981b). This transplant became completely engrafted and resulted in full recovery of normal, donor-derived hemopoietic function without GVH disease, sustained for 11weeks after transplantation, at which time the patient’s leukemia recurred. Subsequently the patient received chemotherapy and achieved a remission with regeneration of normal marrow cells of donor origin. More recently, the same technique of depletion of immunoreactive T cells by SBA agglutination and E-rosetting has been used in bone marrow transplantation from haploidentical, MLR reactive parents into three children with severe combined immune deficiency (Reisner et al., 1983). Two patients achieved durable engraftment with reconstitution of both humoral and cell-mediated immunity. Neither of these children developed GVH disease. The third

277

LECTIN RECEPTORS

child achieved only a transient engraftment with concomitant development of mitogen-responsive lymphocytes of paternal origin. The above results demonstrate that histoincompatible bone marrow depleted of T cells by agglutination with SBA and E-rosetting can be transplanted into lethally irradiated patients, or patients with severe immune deficiencies, without risk of GVH disease, and that the transplanted cells are capable of reconstituting durable hemopoietic and lymphoid functions in the recipients of the transplant.

B. RECEPTORS FOR OTHERLECTINS Treatment of human peripheral blood lymphocytes with sialidase has been shown to uncover receptors for HPA (Hammarstrom et al., 1973; Hellstrom et al., 1976a). Since the receptors appeared mainly on T lymphocytes, these cells could be separated from B cells using HPA-Sepharose (Fig. 27). The cell fraction which was not retained by the column was highly enriched in B lymphocytes, as judged b y the percentage of cells having SmIg or receptors for complement-treated sheep erythrocytes. Elution with 0.1 mg/ml N-acetylgalactosamine afforded a fraction similar in composition to that of the starting material. The cell fraction eluted at 1.0 mg/ml N-acetylgalactosamine, however,

80 la -s 60

20

a

b

C

d

FIG. 27. Fractionation of human peripheral blood lymphocytes (after treatment with sialidase) on HPA-Sepharose 6MB. a, Unfractionated cells (100%);b, unabsorbed cells (yield 6%); c, cells eluted with 0.1 mg/ml N-acetylgalactosamine (yield 27%); and d, cells eluted with 1.0 mg/ml N-acetylgalactosamine (yield 48%). Black columns, cells with receptors for HPA (HP+);dotted columns, cells forming spontaneous rosettes with sheep erythrocytes; hatched columns, cells carrying surface Ig; circled columns, cells forming rosettes with complement-treated sheep erythrocytes. (Modified from Hellstrom et al., 1976a.)

278

NATHAN SHARON

contained almost no cells with surface markers characteristic of B cells and constituted a preparation of practically pure T lymphocytes; the total yield of these cells was about 80%. In human cord blood, the majority of B cells are HPAs+, as they are in adult peripheral blood (Hellstrom et al., 1978). Chromatography on immobilized HPA has also been used to isolate a minor subpopulation of sialidase-treated B lymphocytes from human peripheral blood. This fraction constituted about 10% of the total number of HP&+ lymphocytes in the blood (Hellstrom e t at., 1978). These cells were only weakly bound by the immobilized lectin and thus appeared in the intermediate fraction eluted at 0.1 mg/ml N-acetylgalactosamine. They express both SmIg and the lectin receptor, but their functions have not been defined. The HPA receptors on these B cells were different from those on the majority of T cells, and indications were obtained that similar structures were expressed on immature B cells. Therefore, the HPA receptor seems to fall into the category of differentiation markers, and may constitute a useful tool for characterization and separation of human lymphocytes within both the T and B compartments. The procedure of Hellstrom et al. (1976a) has been modified to permit the routine purification of human peripheral blood B lymphocytes for use in tissue typing (Schrempf-Decker et al., 1980). It afforded an 80% pure B lymphocyte population within 3 hours so that typing for HLA-A,B,C antigens and for HLA-DR alloantigens could be done on the same day. A comparison with two rosetting procedures for the isolation of T and B lymphocytes showed that the HPA fractionation method gave better results in terms of yield and viability of the cells, in particular of B cells. Because of its high yield, the HPA fractionation method has the further advantage for clinical use that it requires less blood for complete B cell typing. It should be recalled, however, that the cells have to be treated with sialidase before fractionation by HPA, and that for certain other purposes, the use of such cells may pose distinct disadvantages, as discussed earlier in connection with the separation of murine lymphocytes by HPA. A major cell surface glycoprotein (apparent MW 150,000 under reducing conditions) was responsible for almost all the binding of HPA to sialidase-treated human lymphocytes, as has been found with mouse T cells (Axelsson et aZ., 1978). The glycoprotein was present on normal and malignant T lymphocytes and on chronic lymphocytic leukemia cells, but not on various B cells. In 13 patients with chronic lymphocytic leukemia, binding of FITC-HPA to 90-100% of the sialidase-treated peripheral blood lym-

LECTIN RECEPTORS

279

phocytes was observed (Hellstrom et al., 1976~). Almost all the SmIg+ (B) cells were also HPA,+, in contrast to normal individuals in which the HPA receptor is present predominantly on the sialidase-treated T lymphocytes. Patients in remission with low numbers of leukemic cells had low numbers of blood lymphocytes carrying both SmIg and HPA receptors. Leukemic cells appear thus to be SmIg+HPA,+. It was suggested that testing for this combination may provide a valuable tool for monitoring patients with chronic lymphocytic leukemia. Data on a large number of patients are required, however, before the validity of this suggestion is widely proven. Expression of HPA-binding surface glycoproteins, HLA-DR antigens, and common acute lymphocytic leukemia antigens, was investigated in seven histiocytic lymphoma cell lines and in a panel of human hemopoietic and nonhemopoietic normal and neoplastic cell lines (Nilsson et al., 1981). The histiocytic lymphoma cell lines differed markedly in the binding. Among the panel of human cell lines tested, all T leukemia lines expressed only a 150,000-dalton HPA receptor, while all myeloma lines expressed only a 210,000-dalton receptor. The remaining types of lymphoid cell lines displayed heterogeneity with respect to the major HPA-binding surface glycoproteins. Erythroleukemia and nonhemopoietic cell lines did not bind HPA. Human peripheral blood T cells were separated by affinity chromatography on Sepharose-bound WGA into two discrete subpopulations; elution of the WGA+ cells was accomplished using N acetylglucosamine (Hellstrom et al., 1976b). The two subpopulations differed in their responses to concanavalin A and PHA, the strongly responding cells being found among the cells that were retained by the column. Both fractions contained progenitors of alloreactive T cells, proliferating in the MLR and acting as effector cells in cellmediated lympholysis (Lehtinen et al., 1980). The proliferation and cell-mediated lympholysis activities of the two fractions were equal and were similar to those of the unfractionated cells. However, when the lymphocytes were fractionated after 5 days MLR, most of the proliferating and cytolytic cells were found in the WGA+ subpopulation. It was also shown that the low level of proliferating and cytolytic activity in the WGA- subpopulation was not caused by suppressor cells present in this subpopulation. When peripheral blood lymphocytes were fractionated by affinity chromatography on WGA, the cells retained by the lectin responded less well to PHA than those that were not retained (Boldt and Lyons, 1980), a result completely different from that obtained by fractionation of the T lymphocytes alone (Hellstrom et al., 1976b).

280

NATHAN SHARON

Separation of human peripheral lymphocytes into fractions that differ in binding to LCA has been described (Boldt and Lyons, 1979). However, no functional differences were observed between the two fractions, except that the LCA+ cells responded better to stimulation by this lectin than the LCA- cells. Lima bean lectin (LBL) was found to bind to human peripheral B lymphocytes and monocytes, and to approximately half of the T lymphocytes (Munske et al., 1981). It was suggested that this selectivity may permit the fractionation of the T lymphocytes into LBL+ and LBL- subpopulations. The L-fucose-binding lectin from Lotus tetragonolobus (LTL) binds to human neutrophils and eosinophils, but not to lymphocytes, monocytes, platelets, or fibroblasts (Nicola et al., 1980a; Tung and Van Epps, 1979). Furthermore, binding showed species specificity and was not observed with lymphocytes, granulocytes, or macrophages from guinea pigs or mice. Leukemic cells from patients with chronic or acute granulocytic leukemia bound the lectin, whereas cells from patients with lymphocytic or monocytic leukemia failed to bind the lectin. It was concluded that LTL identifies a species and cell-specific marker on human granulocytes, and that this surface marker may be useful in differentiating various types of acute leukemias (Tung and Van Epps, 1979). Studies in the FACS of the interaction of FITC-LTL with human bone marrow cells showed that the degree of binding within the granulocytic series increased with progressive differentiation (Nicola et al., 1980a; Morstyn et al., 1980). Marrow monocytes and nucleated erythroid cells bound LTL, in contrast to monocytes and nonnucleated red cells in the peripheral blood which showed negligible binding. Lymphocytes, both in marrow and in blood, displayed negligible binding of LTL. These properties allowed an enrichment of hemopoietic progenitor cells (CFC) from human marrow cell suspensions and a depletion of colony-inhibiting cells, when present, by selection of cells with the appropriate fluorescence intensity (Fig. 28). By a combination of three parameter cell sorting (low-angle and high-angle scatter characteristics in addition to fluorescence intensity), a 36-fold enrichment of the granulocyte-macrophage progenitor cells was achieved. The most enriched fraction was composed of 23%progenitor cells (colony- and cluster-forming cells), with a yield of 36%. In populations most enriched for granulocyte-macrophage colonyforming cells, immature cells (blast cells, promyelocytes, and myelocytes) made up 95% of the cells present.

LECTIN RECEPTORS

90

c,

60

-

28 1

r

4 U +

2 U I

Zi

r Y A

U w

v

0

c z

U w

a Ly n

90 -1+z+-

70 60 -

80

50

-

-

3 4 COLLECTION WINOOWS ,

+

NUCLEAlEO ERYTHROIO

FIG.28. Distribution of human bone marrow cells labeled with FITC-LTL according to fluorescence intensity. (From Morstyn et al., 1980.)

V. Lymphocytes of Other Animals

Studies of the immune system of animals other than mouse or man have been greatly hampered by the lack of suitable lymphocyte surface markers. From the limited amount of work done on the interaction of lectins with lymphocytes of rats, guinea pigs, cattle, sheep, monkeys, and chicken, it is quite apparent that lectins may provide useful aids for the investigation of the immune system of these animals as well. A. RAT Using FITC-PNA it was found that most of the thymocytes (87%) and bone marrow cells (83%)of Sprague-Dawley rats have receptors for PNA; in spleen and lymph nodes around 40% PNA+ cells were present (London et al., 1981).Except for the thymus, the distribution of

282

NATHAN SHARON

PNA+ cells in the rat is thus markedly different from that in the mouse (cf. Table IV). Double labeling experiments, with goat anti-rat IgG F(ab’)2 coupled to rhodamine isothiocyanate, and with FITC-PNA, showed that very few of the PNA+ cells were SmIg+, so that in the rat the presence of PNA receptors correlates either with T cell lineage or with null cell (non Thon B) lineage. Based on this and other findings, it was further suggested that in the rat the PNA receptor is a cell surface marker for immature T cells in thymus and bone marrow, and for more mature stages of the T cell lineage in spleen and lymph nodes. A distribution of PNA receptors markedly different from that of the mouse was also observed upon examination of the binding of HRPPNA on frozen sections of organs of Wistar rats (Rose and Malchiodi, 1981). Binding to rat thymic cortex was very weak, which is in disagreement with the findings of London et al. (1981) mentioned above, perhaps because different rat strains were used. The binding of PNA to rat germinal centers was very weak, compared with that found with germinal centers of mouse, man, and sheep. In rats of strain DA, 32% of the sialidase-treated splenocytes and 48% of sialidase-treated lymph node lymphocytes possess receptors for HPA, as revealed b y staining of the cells with fluorescence derivatives of the lectin (Swanborg et al., 1977). Separation of the HPAs+ cells from the HPAs- cells was achieved by affinity chromatography on columns of the immobilized lectin. The finding that the HP&+ cells were devoid of SmIg and the close correlation with the reported T cell content of rat spleen and lymph nodes suggested that these are probably T cells. Although no details were given, it was also noted that 81-94% of sialidase-treated Lewis rat thymocytes bound HPA, whereas only 1%of the cells expressed SmIg.

B. SYRIAN HAMSTER No binding of HRP-PNA to frozen tissues of different lymphoid sections (thymus, Peyer’s patches, spleen, or mesenteric lymph nodes) of Syrian hamsters was observed (Rose and Malchiodi, 1981). However, upon treatment with sialidase, all these tissues became PNA+, suggesting that in the hamster lymphoid cells the PNA receptor is always masked by sialic acid residues. Using a procedure essentially identical to that developed for the fractionation of murine splenocytes by SBA (Reisner et aZ., 1976b), T and B lymphocytes of the Golden hamster were separated (Weppner and Adkison, 1980). Only the B splenocytes were agglutinated by the lectin, and they could be separated from the unagglutinated T cells by

LECTIN RECEPTORS

283

sedimentation through 50% heat-inactivated fetal calf serum at unit gravity. About 80% of the cells were recovered in fully viable form, of which 64% were in the top fraction containing unagglutinated (SBA-) cells, 22% in the agglutinated and sedimented cells (SBA+)and the rest in a middle fraction. More than 80% of the SBA+cells and only 5% of the SBA- cells were SmIg+. These results are in agreement with earlier observations that 39% of hamster splenocytes bear SmIg. The SBA+ fraction responded well to concanavalin A and PHA, and only poorly to LPS, whereas the SBA- fraction responded well only to LPS, further demonstrating that the responses of hamster splenocytes to such T and B cell specific mitogens are indeed similar to those of other rodent species. The mitogenic response to concanavalin A of spleen cells from pregnant hamsters during mid or late gestation is 10%of that observed from age-matched virgin female animals; the normal magnitude of response returns within 10 days after parturition. Thus far, no other mammalian species has been described to exhibit such a level of depression in the response of lymphocytes to a mitogen during pregnancy. There are, however, conflicting reports on the depletion of circulatory T lymphocytes during human pregnancy. It was therefore of interest to examine the changes in T and B lymphocytes in pregnant hamsters (Weppner and Coggin, 1980). Such an examination was made possible b y the availability of the method for separation of hamster lymphocytes by SBA. Splenocyte preparations from both virgin female and mid-gestation pregnant hamsters were shown to contain normal proportions of T (65%)and B (30%)cells. However, the T lymphocyte suspensions isolated by SBA from the splenocytes of pregnant hamsters also displayed the same depressed reactivity to concanavalin A, substantiating the notion that the immunoregulatory phenomenon occurs at the level of the cell and is not a redistribution phenomenon. It is also apparent that only the T splenocytes become immunodeficient, since the level of blastogenesis induced by a B cell-specific mitogen, LPS, was unaltered in pregnancy.

c. CATTLE AND SHEEP The binding of five different lectins (concanavalin A, PNA, SBA, WGA, and UEA) to bovine peripheral blood lymphocytes was examined by fluorescence microscopy (Pearson et al., 1979). No lymphocytes that bound UEA were detected, whereas the other four lectins bound to various proportions of the cells. Double labeling experiments with a goat anti-bovine immunoglobulin reagent coupled with rhodamine and fluoresceinated lectins showed that both PNA and

284

NATHAN SHARON

SBA bound to lymphocyte subpopulations comprising, respectively, 45 and 17%of the total peripheral blood lymphocytes. The lectin binding cells were all essentially devoid of SmIg. Only about one-third of the SmIg- cells bound SBA, whereas over 70% of these cells bound PNA. Similar results were obtained regardless of the breed of cattle used as the source of peripheral blood lymphocytes, indicating that the receptor for PNA may be considered as a marker for bovine T lymphocytes, apparently the only marker available for these lymphocytes. The finding that PNA binds almost exclusively to bovine T lymphocytes has been confirmed by Fahey (1980), who further suggested that with the lectin it may now be possible to isolate T lymphocytes, particularly those from bovine lymph, and study their properties in detail. Using a fluorescent heterologous anti-bovine thymocyte antiserum and PNA, identical populations of bovine T lymphocytes were identified (Usinger and Splitter, 1981). Double fluorescent labeling and capping experiments showed that both reagents bound to approximately 62% of peripheral blood lymphocytes and virtually to all thymocytes. It was pointed out that PNA has distinct advantages over antisera for the identification and isolation of bovine T cells. Passage of bovine peripheral blood lymphocytes over a column of Sephadex G-10 and subsequent negative selection on plastic dishes which had been coated with F(ab’), anti-Ig or with PNA resulted in highly enriched populations of T cells bearing receptors for PNA (99% PNA+) and B cells (84% SmIg+, 10% PNA+, 6% null), respectively (Usinger et al., 1981). The level of monocytes remaining in each cell fraction was less than 0.1%. Examination of the mitogenic response of these isolated lymphocyte subpopulations demonstrated that bovine T cells can be strongly stimulated by concanavalin A, PHA, and PWM without apparent need for auxiliary B cells or monocytes. The binding of seven fluorescein-labeled lectins to sheep tissues and cells was examined (Fahey, 1980). Concanavalin A, Ricinus communis agglutinin, and WGA bound strongly to all sheep circulating cells, whereas DBA and UEA did not bind at all. PNA and SBA bound to thymus and lymph node cell suspensions, and to polymorphs and monocytes. After treatment of the peripheral blood lymphocytes by carbonyl-iron to remove the phagocytic cells, 40-50% of the lymphocytes and 65-70% of the popliteal lymph node cells were PNA+. Using an anti-sheep Ig reagent coupled with rhodamine, it was found that over 98% of the above PNA+ cells were SmIg-. Approximately 20-30% of the peripheral blood lymphocytes and 10-20% of the cells in lymph were PNA-SmIg-, and these were termed null cells. Lambs have substantial numbers of PNA+ cells in their circulation

LECTIN RECEPTORS

285

from at least 1 week of age (Fahey, 1980; Fahey et al., 1980), and no significant difference was detected in the percentage of PNA+ cells in peripheral blood lymphocytes from lambs and ewes. It was concluded that the PNA receptor is an excellent marker for T cells in sheep, as it is in cattle. Binding of HRP-PNA to frozen sections of thymic cortex of mesenteric lymph nodes of sheep was observed but the interaction of the lectin with other lymphoid organs of this animal was not examined (Rose and Malchiodi, 1981).

D. HORSE In preparations of isolated equine mononuclear peripheral blood leukocytes, two subpopulations were identified (Banks and Greenlee, 1981). One of these had surface characteristics identical to thymocytes, in that it readily bound PNA but lacked receptors for complement or Ig and did not have SmIg. This population could be isolated, for example, using nylon wool columns. The other class of lymphocytes had equine complement receptors, Ig receptors, and SmIg, but did not bind PNA.

E. CHICKEN The pattern of binding of PNA to chicken lymphocytes was markedly different from that of the other animals tested. Thus, in cryostat sections all the lymphocytes of chicken thymus, spleen, and cecal tonsil appeared to bind HRP-PNA (Rose and Malchiodi, 1981). In the bursa of Fabricius, the medulla but not the cortex was positive (Fig. 29). Interestingly, the basement membrane associated epithelium at the corticomedullary border was strongly positive, as was the basement membrane of the plical epithelium. Upon analysis in the FACS of cells from various organs of 10-weekold chicken, about 80% of PNA+ the cells found in bursa and thymus were PNA+; splenocytes and peripheral lymphocytes contained 50 and 74%PNA* cells, respectively (Schauenstein et al., 1982). These values are somewhat lower than what could be expected from the staining of cryostat sections described above, but are considerably higher (in spleen and peripheral blood) than what is normally found in mammals. For all four chicken organs tested, the distribution of PNA+ cells showed monophasic patterns in the FACS. The PNA+ and PNA- cells in various organs were separated by selective agglutination with PNA, and some of their functional properties were assayed. The PNA- fraction was unresponsive to the mitogenic action of concanavalin A, PHA, and PWM, whereas the

286

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FIG.29. Binding of HRP-PNA to bursa of Fabricius. C, Cortex; M, medulla; arrow points to CM, corticomedullaryjunction; arrow points to PE, plical epithelium. ~ 2 5 0 . (From Rose and Malchiodi, 1981.)

PNA+ cells responded equally well, or even slightly better, as compared with the unfractionated cells. Similar reactivity patterns of PNA+ and PNA- fractions were obtained in the MLR as well as in the in vitro antibody response against sheep red blood cells. The PNA+ cells were shown to suppress T cell functions (responses to concanavalin A and PHA), as well as the MLR, whereas the PNA- cells suppressed responses involving the B cell system, such as response to PWM and the antibody response to sheep red blood cells, leaving pure T cell functions unaffected. I t thus appears that PNA permits separation of chicken suppressor cells of different target specificity. The high proportions of PNA+ cells found in the peripheral lymphoid organs of the chicken indicates that the postulated sialylation of surface galactose residues during cell differentiation is far less pronounced in this species. This is in agreement with earlier biochemical data that demonstrated the presence of high concentrations of free glycoproteins with terminal galactose in the serum of chickens (Lunney and Ashwell, 1976). Eight other lectins were screened for their ability to agglutinate cells

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from different organs of 28-day-old chicken (Flower and Wilcox, 1981). Only with LTL was selective agglutination observed of cells from one organ: the lectin strongly agglutinated bursal cells, whereas those from spleen were only poorly agglutinated, and cells from thymus or peripheral blood were not agglutinated at all. Sixty percent of the bursal cells were stained with FITC-LTL or rhodamine-conjugated LTL: a slightly higher proportion of cells bound to latex beads to which the lectin had been attached. In spleen and peripheral blood, only a small percentage of the cells reacted with the lectin in the above tests (6-10%).The percentage of LTL+ cells varied with the age of the chicken. This variation was particularly pronounced in spleen and peripheral blood, where the levels of LTL+ cells increased, respectively, to 52 and 17%. It was suggested that since LTL does not agglutinate chicken T lymphocytes, and agglutinates only about two-thirds of the B (bursal) lymphocytes, the latter cells may represent a unique subpopulation of chicken B lymphocytes. VI. Concluding Remarks

From the foregoing discussion it is clear that lectin receptors are characteristic markers of distinct lymphocyte subpopulations in mouse, man, and several other animals. Just like antigenic surface markers, lectin receptors are extremely useful for the identification and separation of lymphocytes and therefore for studies on the functions and lineages of cells with a given phenotype. Some of them may even be considered as differentiation markers. Based on the data presented in Table VIII it is possible to assign lectin receptor phenotypes to certain lymphocytes. Thus, mouse cortical thymocytes are PNA+SBA+, medullary thymocytes are PNA-SBA+, spleen T cells are HPAs+PNA-SBA-WGA- (the S in HPAs denoting that the lectin receptor is expressed after treatment of the cells with sialidase), and spleen B cells are HPAs-PNA-SBA+WGA+. The PNA receptor is of special significance, since it has become a widely used standard marker for cortical (immature) thymocytes in the mouse, and to a more limited extent also in man. In addition, it is for the time being the only marker for bovine and sheep T lymphocytes. Cell separation by lectins-which is simple, inexpensive, and as a rule highly reproducible-has permitted access to previously inaccessible lymphocyte subpopulations, such as murine cortical thymocytes, human cortical and medullary thymocytes, as well as murine and human pluripotential stem cells. This has led to better

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TABLE VIII LYMPHOCYTES IDENTIFIED AND SEPARATED BY LECTINS

Animal

Organ

Lectin used

Lectinpositive cells

Lectin negative cells

~

Mouse

Human

Rat Hamster Cattle Sheep

Thymus Spleen Lymph node Bone marrow Thymus PBLb Cord blood Bone marrow Spleen Spleen PBL PBL

PNA HPA S BA WGA SBA PNA and SBA PNA HPA S BA PNA S BA HPA S BA HPA PNA PNA

Cortical Ta B B B Stem cells Cortical ,P

T helper Immature ,P

B ,P

T T

Medullary Ba T T T Medullary B' T suppressor Mature Stem cells B' T B' B B

Sialidase-treated cells. PBL, Peripheral blood lymphocytes.

characterization of some of these subpopulations. In particular, the availability of the PNA fractionation techniques has enabled the extensive characterization of murine thymocyte subpopulations in terms of surface markers, biochemical characteristics, and distribution of suppressor and helper cells. In addition it has made possible studies of the mechanisms of generation of cytotoxic cells and the action of thymic hormones on lymphocyte subpopulations. It has, however, not resolved the question whether maturation of thymocytes occurs via one pathway (i.e,, cortical to medullary) or two independent pathways. Lectin receptors characteristic of other cells have only been mentioned briefly in this article, but these too are attracting increasing attention. A recent example is the demonstration that stimulated murine macrophages express a new receptor which reacts with Griffonia (Bandeiraea)simplicifolia I-B4 isolectin (Maddox et al., 1982). Separation of the stimulated from the resident macrophages was achieved by affinity chromatography on the immobilized lectin. The question has often been raised concerning the biological significance and function of the lectin receptors on lymphocytes. Here

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again more information is available on the PNA receptor than on that for any other lectin. In the mouse, the original suggestion that the PNA receptor is a marker for immature lymphocytes appears still tenable, although the receptor is not present on certain immature cells, e.g., prothymocytes. It is also noteworthy that there are PNA receptors on murine embryonal carcinoma cells but not on the differentiated cells segregating during i n vitr5 differentiation (Muramatsu et al., 1979; Ogiso et al., 1982; Reisner et al., 1977). In the human lymphoid system, the distribution of the PNA receptor is somewhat similar to that of the mouse. There is also much evidence to support the suggestion that the disappearance of the PNA receptor in the course of lymphocyte differentiation and maturation is a result of its masking by sialic acid residues, presumably catalyzed by the action of suitable sialyltransferases. Under pathological conditions this masking may be defective, and PNA+ cells may appear in large numbers, as in many cases of human leukemia. However, as pointed out very recently by Rose (1982), the pattern of PNA binding to lymphocytes of animals other than mouse or man, and especially to nonlymphoid cells (also of mouse and man), makes it unlikely that the PNA receptor is a general marker of immaturity. Detailed studies of the interaction of PNA with sections of mouse organ tissues have revealed numerous morphologically distinct structures in the respiratory, intestinal, and uroepithelial tracts and elsewhere in the body, to which the lectin specifically binds (Stoward et al., 1980; Watanabe et al., 1981).Also, PNA binding is species-specific, being observed with most types of chicken lymphocytes but not with lymphocytes from rabbit, guinea pig, or hamster (Rose and Malchiodi, 1981). Rose (1982) has therefore suggested that the PNA binding properties of lymphocytes may reflect their tendency to recirculate or to be sessile. This suggestion is based on the well-known observation that lymphocytes which have been treated with sialidase to reveal galactose residues do not recirculate normally in vivo but become trapped in the liver, just like asialoglycoproteins are rapidly cleared from the circulatory system into the liver (Ashwell and Morell, 1974). Interestingly, murine thymocytes and PNA+ cells from the germinal centers of Peyer’s patches migrate to the liver even if they have not been treated with sialidase. In the chicken, the observation that nearly all the lymphocytes are PNA+ accords well with the fact that this species has 10 times as much circulating asialoglycoproteins (i.e., glycoproteins with terminal galactose and N-acetylgalactosamine) as mammals (Lunney and Ashwell, 1976; Neufeld and Ashwell, 1980). In a broader context, it is possible that the specific acquisition of

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components at the cell surface occurring during differentiation affects cell location by providing surface receptors for interaction with lectins in certain tissues (Hughes and Pena, 1981). We may thus postulate that the PNA+ cells are kept in the cortex by binding to a putative endogenous membrane-bound lectin with specificity similar to that of PNA (Fig. 30). Once the PNA receptor is masked, the cortical thymocytes are free to migrate to the medulla (or directly to the periphery). Alternatively, we may assume that lectin receptors on lymphocytes may be required for cell activation or cooperation in the immune system. A prerequisite for understanding the function of lectin receptors is their isolation and structural characterization, particularly of their carbohydrate moieties. The structure of the receptors is certainly much more complex than suggested by the specificity of lectins for mono- or disaccharides. Although in several cases lectin receptors have been isolated from lymphocyte membranes, they have only been poorly characterized. Such characterization is still greatly hampered by the difficulties in preparing large amounts of pure plasma membranes, and very often b y the lack of sensitive microtechniques for the isolation and quantitation of membrane components. Only rarely has it been possible to prepare sufficient material for the complete chemical characterization of carbohydrate units of lymphocyte membrane. An extreme example is a recent study in which 6.78 g of delipidated plasma membrane was used as starting material for the isolation and complete characterization of 18 asparagine-linked acidic sugar chains of the glycoproteins of calf thymocyte plasma membranes (Yoshima et al., 1981). A serious limitation to any approach that requires membrane solubilization is that membranes may contain many glycoconjugates that react with lectins, only some of which may

CORTEX

.,

MEDULLA

FIG.30. Hypothetical model showing the role of PNA receptors in thymus. 0, Rereceptor for SBA only; 0,sialic acid. ceptor for SBA and PNA;

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be expressed on the surface of the intact cells, and thus accessible to the Iectin in situ. Methods for receptor isolation, such as photoaffinity crosslinking (Jaffe et al., 1979, 1980) or plucking (Jakobovits et aZ., 1981), which do not require prior membrane disruption, should therefore be used in preference. Whatever the biological role of lectin receptors, there is no doubt that structural characterization of the receptors will provide insight for the understanding of the changes that occur on the surface of lymphocytes during differentiation and maturation, both under normal and pathological conditions. The results of such studies will also contribute greatly to clarify the role of cell surface sugars and of the membrane in general, in the complex mechanisms functioning in the immune system. ACKNOWLEDGMENTS I wish to thank Dr. Marlene Rose for providing me with Figs. 8,11,22, and 29. Special thanks are due to Mrs. Dvorah Ochert for her devoted and efficient help in the preparation of this manuscript for publication.

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Index A

Acute phase, response, 141-145 Acute phase proteins, induction and control of synthesis of biosynthetic mechanisms in liver, 147-148 general considerations, 145-147 interleukin I, 148-150 prostaglandins, 150-151 Amyloidosis, serum amyloid P component and, 188-189 Antibodies, monoclonal, specific for Tpre, Tthy, Tind and Tsu preparation of, 4-9 Antiproliferative activities, of interferons, 110-111 Anti-Tsu serum, conventional, preparation of, 3-4 Antitumor effects, of interferons in animal systems, 130-132 Antiviral activities, of interferons, 110111 Antiviral state, interferons and, 101-102 B

B cells, and products, exclusion of Tpre, Tthy, Tind and Tsu from, 22-24 C Cattle, lymphocytes of, 283-285 Cell(s) bearing Tsu and Tind, in uiuo studies on function of, 32-33 expressing Tpre, T h y , Tind and Tsu, in uitro functional role of, 27-32 identification and separation of, 221223 lectin-binding, detection and enumeration of, 223-225 proliferation, interferons and, 106-107

purity of pure preparations, 229-230 techniques for separation of, 225-229 Cell cycle, interferons and, 107-109 Cellular parameters, other, interferon and, 109-110 Chicken, lymphocytes of, 285-287 Clinical studies, with human interferons, 132-133 C-reactive protein biological properties, 162- 168 definition and nomenclature, 151-156 functions, 168-175 measurement in clinical practice, 175183 synthesis and turnover, 160-162 structure and ligand specificity, 156159 Cytolysis, nonspecific, interferons and, 122-127 G

Gene products encoded in Dd region antigenic heterogeneity of, 41-46 chemical heterogeneity of, 46-50 quantitative comparisons of, 50-52 of H-2Ld, functional studies of, 52-54 Genomic clones, of H-2D region loci characterization of genes, 58-60 characterization of gene products, 60-63 H

H-2D region, evolutionary models and future approaches, 64-66 H-2Ld allelic products, searches for in other haplotypes, 54-58 H-2Ld gene products, functional studies of, 52-54 Horse, lymphocytes of, 285 299

INDEX

Human Ia gene structure and products in, 7180 interferons, clinical studies with, 132133 Ir gene function in, 80-92 I

Ir gene, structure and products in mice and humans, 71-80 IgT-C linkage group, genetic characterization of, 9-14 IgT-C region, unique T cell differentiation pathway and, 14-22 Immune responses, cellular, interferon effects on, 114-116 Immune system, humoral, interferon effects on, 111-114 Immunity cell-mediated, interferons and, 119122 interferon and other mechanisms related to, 129-130 Immunochemical characterization, preliminary, of Tsu and Tind, 33-34 Immunology, lectins and, 218-221 Inflammation, interferon and other mechanisms related to, 129-130 Interferon(s) actions of, antiviral and antiproliferative activities, 110-111 antiviral state and, 101-102 cell cycle and, 107-109 cell-mediated immunity and, 119122 cellular immune responses, and, 114-1 16 humoral immune system and, 111114 inhibition of cell proliferation and, 106,107 inhibition of virus replication, 102106 modulation of macrophage action, 116-1 19 nonspecific cytolysis and, 122-127 other cellular parameters and, 109110

pathogenesis of LCVM disease and, 127-128 antitumor effects in animal systems, 130-132 defense against viral infections and, 128-129 human clinical studies with, 132-133 other mechanisms related to immunity and inflammation and, 129-130 production of, 99-101 Interleukin 1, acute phase proteins and, 148-150 Ir gene, function in humans, 80-92 L

Lectins brief survey of, 215-218 human lymphocytes and, 277-281 immunology and, 218-221 murine lymphocytes and, 260-265 Liver, acute phase proteins and, 147148 Lymphocytes, of other animals cattle and sheep, 283-285 chicken, 285-287 horse, 285 rat, 281-282 Syrian hamster, 282-283 Lymphocyte subpopulations human receptors for other lectins and, 27728 1 receptors for peanut and soybean agglutinins and, 265-277 murine receptors for other lectins, 260-265 receptors for peanut and soybean agglutinins, 30-260 Lymphocytic choriomeningitis virus disease, pathogenesis, interferons and, 127-128 M

Macrophage(s), interferon-induced modulation of, 116-119 Mice, Ir gene structure and products in, 71-80 Monoclonal antibodies, specific for Tpre,

30 1

INDEX

Tthy, Tind and Tsu, preparation of, 4-9 P

Peanut agglutinin receptors human lymphocytes and, application to bone marrow transplantation, 276-277 other lymphoid organs, 269 peripheral and umbiIical cord blood, 269-275 thymus, 265-269 murine lymphocytes and, 230-235 application to bone marrow transplantation, 259-260 biochemical characteristics, 248-251 distribution, 235-245 functional properties, 251-258 surface markers, 245-248 Pentaxins, definition and nomenclature, 151-156 Prostaglandins, acute phase proteins and, 150-151 R

Rat, lymphocytes of, 281-282

polymorphism, 192 synthesis, 192-193 Serum amyloid P component definition and nomenclature, 151-156 ligand binding, 184-187 SAP and amyloidosis, 188-189 SAP-related material in normal human tissues, 189-190 serum levels, 187-188 structure, 183-184 Sheep, lymphocytes of, 283-285 Soybean agglutinin receptors human lymphocytes and, application to bone marrow transplantation, 275-277 other lymphoid organs, 269 peripheral and umbilical cord blood, 269-275 thymus, 265-269 murine lymphocytes and, application to bone marrow transplantation, 259-260 biochemical characteristics, 248-251 distribution, 235-245 functional properties, 251-258 surface markers, 245-248 Syrian hamster, lymphocytes of, 282-283

S

T

Serum amyloid A protein apoSAA, 191-192 functions, 193-195 introduction, 190 measurement in clinical practice, 195198

T cells alloantigens, cross-reactive determinants shared in linkage group and T cell products, 24-27 unique differentiation pathway, IgT-C region and, 14-22

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CONTENTS OF PREVIOUS VOLUMES Cellular Genetics of immune Responses

Volume 1

G. J. V. NOSSAL

Transplantation Immunity and Tolerance Antibody Production by Transferred Cells

M. HASEK,A. LENGEROVA, AND T. HRABA

CHARLES G. COCHRANE AND FRANK J. DIXON

Immunological Tolerance of Nonliving Antigens

Phagocytosis

DERRICKROWLEY

RICHARDT. SMITH

Antigen-Antibody Reactions in Helminth Infections

Functions of the Complement System

ABRAHAMG. OSLER

E. J. L. SOULSBY In Vitro Studies of the Antibody Response Embryological Development of Antigens

ABRAMB. STAVITSKY

REED A. FLICKINGER Duration of Immunity in Virus Diseases

J. H. HALE

AUTHORINDEX-SUBJECTINDEX

Fate and Biological Action of AntigenAntibody Complexes

WILLIAM0. WEIGLE Volume 3 Delayed Hypersensitivity to Simple Protein Antigens

In Vitro Studies of the Mechanism of

P. G . H. CELLAND B. BENACERRAF Anaphylaxis K. FRANK AUSTENAND JOHN H. The Antigenic Structure of Tumors HUMPHREY P. A. GORER AUTHORINDEX-SUBJECT INDEX

The Role of Humoral Antibody in the Homograft Reaction

CHANDLER A. STETSON Volume 2

immune Adherence

D. S. NELSON immunologic Specificity and Molecular Structure

Reaginic Antibodies

FREDKARusn Heterogeneity of y-Globulins JOHN

L. FAHEY

D. R. STANWORTH Nature of Retained Antigen and i t s Role in Immune Mechanisms

DAN H. CAMPBELL AND JUSTINE S. GARVEY

The Immunological Significance of the Thymus

J. F. A. P. MILLER, A. H. E. MARSHALL,AND R. G. WHITE

Blood Groups in Animals Other Than Man

W. H. STONEAND M. R. IRWIN

303

304

CONTENTS OF PREVIOUS VOLUMES

Heterophile Antigens and Their Significance in the Host-Parasite Relationship

C. R. JENKIN AUTHORINDEX-SUBJECTINDEX

The Immunology of Insulin

c. G . POPE

Tissue-Specific Antigens

D. C. DUMONDE AUTHORINDEX-SUBJECTINDEX

Volume 4 Ontogeny a n d Phylogeny of Adaptive lmmunity

ROBERTA. GOODAND BEN W. PAPERMASTER Cellular Reactions in Infection EMANUEL SUTER AND HANSRUEDY

RAMSEIER Ultrastructure of Immunologic Processes

JOSEPH D. FELDMAN Cell W a l l Antigens of Gram-Positive Bacteria

MACLYNMCCARTYAND STEPHEN I. MORSE Structure a n d Biological Activity of Immunoglobulins

SIDNEYCOHENAND RODNEYR. PORTER Autoantibodies and Disease

H. G. KUNKELAND E. M. TAN Effect of Bacteria a n d Bacterial Products on Antibody Response

J. MUNOZ AUTHORINDEX-SUBJECTINDEX

Volume 6 Experimental Glomerulonephritis: Immunological Events and Pathogenetic Mechanisms

EMILR. UNANUEAND FRANK J. DIXON Chemical Suppression of Adaptive Immunity

ANN E. GABRIELSONAND ROBERTA. GOOD Nucleic Acids as Antigens

OTTO J. PLESCIAAND WERNER BRAUN In Vifm Studies of Immunological Responses of lymphoid Cells

RICHARDW. DUTTON Developmental Aspects of Immunity

JAROSLAV STERZL AND ARTHURM. SILVERSTEIN Anti-anti bodies PHILIP

G . H.GELLAND ANDREWs.

KELUS Conglutinin a n d lmmunoconglutinins

P. J. LACHMANN Volume

5

Natural Antibodies a n d the Immune Response

AUTHORINDEX-SUBJECTINDEX Volume 7

STEPHENV. BOYDEN Immunological Studies with Synthetic PoIy p eptide s

MICHAEL SELA Experimental Allergic Encephalomyelitis a n d Autoimmune Disease

PHILIP Y. PATERSON

Structure a n d Biological Properties of Immunoglobulins

SYDNEYCOHENAND CESAR MILSTEIN Genetics of lmmunolobulins in the Mouse

MICHAELPOTTER LIEBERMAN

AND

ROSE

CONTENTS OF PREVIOUS VOLUMES Mimetic Relationships between Group A Streptococci a n d Mammalian Tissues

JOHNB. ZARRISKIE lymphocytes a n d Transplantation Immunity

DARCYB. WILSONAND R. E. BILLINGHAM

305

Volume 10 Cell Selection b y Antigen in the Immune Response

GREGORYW. SISKINDAND BARUJ BENACERRAF Phylogeny of Immunoglobulins

Human Tissue Transplantation JOHN

P. MERRILL

AUTHOR INDEX-SUBJECT INDEX

HOWARDM. GREY Slow Reacting Substance of Anaphylaxis

ROBERT P. ORANGE AND K. FRANK AUSTEN

Volume 8 Chemistry a n d Reaction Mechanisms of Complement

HANSJ. MULLER-EBERHARD Regulatory Effect of Antibody on the Immune Response JONATHAN U H R AND GORAN

w.

MOLLER The Mechanism of Immunological Paralysis

Some Relationships among Hemostasis, Fibrinolytic Phenomena, Immunity, a n d the Inflammatory Response

OSCARD. RATNOFF Antigens of Virus-Induced Tumors

KARL HABEL Genetic and Antigenetic Aspects of Human Histocompatibility Systems

D. BERNARDAMOS

D. W. DRESSERAND N. A. MITCHISON AUTHORINDEX-SUBJECT INDEX In Vitm Studies o f Human Reaginic Allergy

ABRAHAM G. OSLER,LAWRENCE M. LICHTENSTEIN, AND DAVIDA. LEVY

AUTHOR INDEX-SUBJECT INDEX

Volume 11

Volume 9

Electron Microscopy o f the Immunoglobulins

Secretory Immunoglobulins THOMASB. TOMASI, JR., AND JOHN

BIENENSTOCK Immunologic Tissue Injury Mediated by Neutrophilic leukocytes

CHARLESG. COCHRANE The Structure and Function of Monocytes a n d Macrophages

ZANVILA. COHN The Immunology a n d Pathology o f NZB Mice

N. MICHAELGREEN Genetic Control of Specific Immune Responses

HUGH0. MCDEVITTAND BARUJ BENACERRAF The lesions i n Cell Membranes Caused by Complement

JOHNH. HUMPHREY AND ROBERT R. DOURMASHKIN Cytotoxic Effects of lymphoid Cells in Vitro

PETER PERLMANN AND GORAN HOLM

J. B. HOWIEAND B. J. HELYER Transfer Factor

AUTHORINDEX-SUBJECT INDEX

H. S. LAWRENCE

306

CONTENTS OF PREVIOUS VOLUMES

Immunological Aspects o f Malaria Infection

In Vifro Approaches to the Mechanism of Cell-Mediated Immune Reactions

BARRYR. BLOOM

IVOR N. BROWN AUTHORINDEX-SUBJECTINDEX

immunological Phenomena in Leprosy and Related Diseases

J. L. TURKAND A. D. M. BRYCESON

Volume 12 The Search for Antibodies with Molecular Uniformity

RICHARDM. KRAUSE Structure and Function o f yM Macroglobulins

HENRYMETZGER

Nature a n d Classification of ImmediateType Allergic Reactions

ELMERL. BECKER INDEX AUTHORINDEXSUBJECT Volume 14

Transplantation Antigens

lmmunobiology of Mammalian Reproduction

The Role of Bone Marrow in the Immune Response

Thyroid Antigens and Autoimmunity

R. A. REISFELT AND B. D . KAHAN

NABIH I. ABDOU AND MAXWELL RICHTER Cell Interaction in Antibody Synthesis

D. W. TALMAGE, J. RADOVICH, AND H. HEMMINGSEN

ALAN E.BEER AND R. E. BILLINGHAM SIDNEYSHULMAN

Immunological Aspects of Burkitt's Lymphoma

GEORGEKLEIN Genetic Aspects of the Complement System

The Role of Lysosomes in immune Responses

GERALDWEISSMANN AND PETER DUKOR Molecular Size and Conformation of Immunoglobulins

KEITH J. DORRINGTON AND CHARLES TANFORD

AUTHOR INDEX-SUBJECTINDEX

A. &PER

AND

FREDs.

The Immune System: A Model far Differentiation in Higher Organisms

L. HOOD AND J.

PRAHL

AUTHORINDEX-SUBJECTINDEX

Volume 15 The Regulatory Influence o f Activated T Cells on B Cell Responses to Antigen

Volume 13 Structure a n d Function of Human Immunoglobulin E

HANSBENNICHAND

CHESTER

ROSEN

s. GUNNAR0.

JOHANSSON

Individual Antigenic Specificity of Immunoglobulins

JOHN E. HOPPERAND ALFRED NISONOFF

DAVID H. KATZ AND BARUJ BENACERRAF The Regulatory Role of Macrophages in Antigenic Stimulation

E. R. UNANUE Immunological Enhancement: A Study o f Blocking Antibodies

JOSEPH D. FELDMAN

307

CONTENTS OF PREVIOUS VOLUMES Genetics a n d Immunology of Sex-Linked Antigens

DAVIDL.

GASSER AND WILLYS

K.

VINCENT

P.

BUTLER,

Jn. AND SAM M.

BEISER

SILVERS

Current Concepts of Amyloid EDWARD

Antibodies to Small Molecules: Biological a n d Clinical Applications

c. FRANKLIN AND

DOROTHEA ZUCKER-FRANKLIN AUTHOR INDEX-SUBJECT

INDEX

AUTHOR INDEX-SUBJECT INDEX

Volume 18 Genetic Determinants of Immunological Responsiveness

DAVIDL. GASSERAND WILLYS K. SILVERS

Volume 16 Human Immunoglobulins: Classes, Subclasses, Genetic Variants, a n d ldiotypes

J. B. NATVICAND H. G. KUNKEL

Cell-Mediared Cytotoxicity, Allograft Rejection, and Tumor Immunity JEAN-CHARLES CEROTTINI AND T H E O D O R E BRUNNER

K.

Immunological Unresponsiveness WILLubi

0. WEICLE

Antigenic Competition: A Review o f Nonspecific Antigen-Induced Suppression

Participation o f Lymphocytes in Viral Infections

E. FREDERICK WHEELOCK STEPHENT.TOY

c. G. COCHRANE AND D. K O F F L E R

The lmmunopathology of Joint Inflammation i n Rheumatoid Arthritis ZVAIFLER

AUTHOR INDEX-SUBJECT INDEX

Volume 17 Anti Iym phocyte Serum LANCE, P. B. MEDAWAR, AND ROBERT N. TAUB

EUGENE M.

In Vitro Studies o f Immunologically Induced Secretion o f Mediators from Cells a n d Related Phenomena ELVER

AND

DAVID

AND

Immune Complex Diseases in Experimental Animals and M a n

NATHAN J.

HUGHF. PROSS EIDLNCER

L.BECKERAND P E T E R M.

Effect of Antigen Binding on the Properties o f Antibody

HENRYM E T Z G E R Lymphocyte-Mediated Cytotoxicity a n d Blocking Serum Activity to Tumor Antigens KARL ERIKHELLSTROM AND INGEGERD HELLSTROM

AUTHOR INDEX-SURJECT INDEX

Volume 19 Molecular Biology of Cellular Membranes with Applications t o Immunology

S. J. SINGER Membrane Immunoglobulins a n d Antigen Receptors on B a n d T Lymphocytes

NOEL L.

WARNER

HENSON Antibody Response to Viral Antigens

KEITH M. COWAN

Receptors for Immune Complexes on Lymphocytes VICTOR

NUSSENZWEIG

308

CONTENTS OF PREVIOUS VOLUMES

Biological Activities of Immunoglobulins o f Different Classes and Subclasses

HANSL. SPIEGELBERG SUBJECTINDEX

Volume 22 The Role of Antibodies in the Rejection a n d Enhancement of Organ Allografts

CHARLES B. CARPENTER, ANTHONY J. F. D’APICE, AND ABUL K. ABBAS

Volume 20

Biosynthesis of Complement

Hypervariable Regions, Idiotypy, and Antibody-Combining Site

Graft-versus-Host Reactions: A Review

J. DONALDCAPRA AND J. MICHAEL KEHOE Structure and Function o f the J Chain

MARIANELLIOTTKOSHLAND Amino Acid Substitution and the Antigenicity of Globular Proteins

M o m s REICHLIN The H-2 Major Histocompatibility Complex and the I Immune Response Region: Genetic Variation, Function, a n d Organization

HARVEYR. COLTEN STEPHEN c. GREBE AND J. WAYNE STREILEIN

Cellular Aspects of Immunoglobulin A

MICHAEL E. LAMM Secretory Anti-Influenza Immunity

YA. s. SHVARTSMAN AND M. P. Z Y K O V

SUBJECTINDEX

Volume 23

DONALD C. SHREFFLERAND CHELLA Cellular Events in the IgE Antibody ReS. DAVID sponse KIMISHIGEISHIZAKA Delayed Hypersensitivity in the Mouse

ALFRED J. CROWLE SUBJECT INDEX

Volume 21 X-Ray Diffraction Studies of Immunoglobulins

ROBERTOJ. POLJAK Rabbit Immunoglobulin Allotypes: Structure, Immunology, a n d Genetics

THOMAS J. KINDT Cyclical Production of Antibody a s a Regulatory Mechanism i n the Immune Response

Chemical a n d Biological Properties of Some Atopic Allergens

T.P. KING Human Mixed-Lymphocyte Culture Reaction: Genetics, Specificity, a n d Biological Imp Iicat ions BO DUPONT,JOHN A. HANSEN,AND

EDMONDJ. YUNIS lmmunochemical Properties of Glycolipids a n d Phospholipids

DONALD M. MARCUSAND GERALD A. SCHWARTING SUBJECTINDEX

WILLIAM 0. WIECLE Thymus-Independent B-Cell Induction a n d Pa ralysis

ANTONIO COUTINHO AND GORAN MOLLER SUBJECT INDEX

Volume 24 The Alternative Pathway of Complement Activation

0. GOTZE AND H. J. MULLER-EBERHARD

309

CONTENTS OF PREVIOUS VOLUMES Membrane a n d Cytoplasmic Changes i n B Lymphocytes Induced by Ligand-Surface Immunoglobulin Interaction

R. UNANUE GEORGE

SCHREINER AND E h f I L

The B-Cell Clonotype Repertoire

HOWARDB. DICKLER Ionizing Radiation a n d the Immune Response AND

NOEL L.

WERNER

Immunologically Privileged Sites

R. E.

Major Histocompatibility Complex Restricted Cell-Mediated Immunity

GENEM. SHEARER AND ANNE-MANE SCHMITT-VEHHULST Current Status o f Rat lmmunogenetics

DAVIDL. GASSER Antigen-Binding Myeloma Proteins of Mice

MICHAEL POTTER Human Lymphocyte Subpopulations

L. CHESSAND S. F. SCHLOSSMAN SUBJECTINDEX

Volume 26 Anaphylatoxins: C3a a n d C5a

TONYE. HUCLIAND HANSJ. M ULLER-EBERHARD

H-2 Mutations: Their Genetics a n d Effect on Immune Functions

KLEIN

The Protein Products o f the Murine 17th Chromosome: Genetics a n d Structure

ELLENs. VITETTA AND J. DONALD

CAPHA

SIGAL AND

NORMAN R.

SUBJECTINDEX

Volume 27

JON

Volume 25

JAN

NOLAN H. KLINMAN

Autoimmune Response to Acetylcholine Receptors in Myasthenia Gravis a n d Its Animal Model

SUBJECTINDEX

C L Y D E F. BAKERAND BILLINGHAhl

K. EICH~IANN

R.

lymphocyte Receptors for Immunoglobulin

ROBERT E. ANDERSON

Expression and Function of ldiotypes on Lymphocytes

LINDSTROM

MHC-Restricted Cytotoxic T Cells: Studies on the Biological Role o f Polymorphic Major Transplantation Antigens Determini n g T-cell Restriction-Specificity, Function, a n d Responsiveness

ROLF M. ZINKEHNACEL C. DOHERTY

AND P E T E R

Murine Lymphocyte Surface Antigens

IANF. C. MCKENZIE AND TERRY POTTER The Regulatory a n d Effector Roles of Eosinophils

PETERF. WELLERAND EDWARD J. GOETZL SUBJECTINDEX

Volume 28 The Role of Antigen-Specific T Cell Factors in the Immune Response TOM10

TADAAND

K O OKUMURA

The Biology a n d Detection of Immune Complexes

ARGYRIOSN. THEOFILOPOULOS AND

FRANK J. DIXON The Human la System

R. J . WINCHESTERAND H. G . KUNKEL

310

CONTENTS OF PREVIOUS VOLUMES

Bacterial Endotoxins a n d Host Immune Responses

DAVIDc. MORRISONAND JOHN L. RYAN

Responses to Infection with Metazoan a n d Protozoan Parasites in Mice

GRAHAMF. MITCHELL SUBJECT INDEX

Molecular Biology a n d Chemistry o f the Alternative Pathway of Complement

HANSJ. M~LLER-EBERHARDAND ROBERT D. SCHREIBER Mediators o f Immunity: Lymphokines and Monokines

Ross E.ROCKLIN, KLAUSBENDTZEN, DIRKGREINEDER

Adaptive Differentiation o f lymphocytes: Theoretical Implications for Mechanisms of Cell-Cell Recognition a n d Regulation o f Immune Responses

DAVIDH.

HENRYN. CLAMAN, STEPHEN D. MILLER, PAUL J. CONLON, AND JOHN W. MOORHEAD Analysis o f Autoimmunity through Experimental Models of Thyroiditis a n d Allergic Encephalomyelitis

WILLIAM 0. WEICLE The Virology and lmmunobiology o f lymphocytic Choriomeningitis Virus Infection

Volume 29

AND

Control o f Experimental Contact Sensitivity

UT Z

Antibody-Mediated Destruction of VirusInfected Cells

J. G. PATRICK SISSONS AND MICHAEL B. A. OLDSTONE Aleutian Disease of Mink

DAVIDD. PORTER,AUSTIN E. LARSEN,AND HELENG . PORTER Age Influence on the Immune System

TAKASHI MAKINODANAND MARGUERITEM. B. KAY

M. J. BUCHMEIER,R. M. WELSH, F. J. DUTKO.AND M. B. A. OLDSTONE

INDEX

Volume 31 The Regulatory Role o f Macrophages in Antigenic Stimulation Part Two: Symbiotic Relationship between Lymphocytes and Macrophages

EMILR. UNANUE T-cell Growth Factor a n d the Culture of Cloned Functional T Cells KENDALL

A. SMITH AND FRANCIS w.

RUSCETTI Formation of B lymphocytes i n Fetal a n d Adult l i f e

PAULW. KINCADE Structural Aspects and Heterogeneity o f Immunoglobulin Fc Receptors

JAYC. UNKELESS,HOWARD FLEIT, AND IRA s. MELLMAN

SUBJECTINDEX The Autologous Mixed-lymphocyte Reaction

Volume 30 Plasma Membrane a n d Cell Cortex Interactions in lymphocyte Functions

FRANCIS LOOR

MARC E. WEKSLER,CHARLES E. MOODY, JR., AND ROBERT w. KOZAK INDEX

CONTENTS OF PREVIOUS VOLUMES

311

Volume 32

Volume 33

Polyclonal B-Cell Activators in the Study of the Regulation of Immunoglobulin Synthesis in the Human System

The CBA/N Mouse Strain: An Experimental Model Illustrating the Influence of the X-Chromosome on Immunity

THOMAS A . WALDMANN AND SAMUEL BRODER Typing for Human Alloantigens with the Prime Lymphocyte Typing Technique N . MORLING, B . K. JAKOBSEN, P. PLATL, L. P. RYDER, A . SVEJGAARD, AND

M. THOMSEN

Protein A of Staphylococcus aureus and Related lmmunoglobulin Receptors Produced by Streptococci a n d Pneumonococci JOHN

J. LANGONE

Regulation of Immunity to the Azobenzenea rsonate H a pten

MARK I. GREENE, MITCHELL J. NELLES, MAN-SUN S Y , AND

IRWIN SCHER

The Biology of Monoclonal Lymphokines Secreted by T Cell Lines a n d Hybridomas

AMNONALTMANAND DAVIDH. KATZ

Autoantibodies to Nuclear Antigens (ANA): Their lmmunobiology a n d Medicine

ENGM. TAN The Biochemistry a n d Pathophysiology o f the Contact System of Plasma CHARLES G . COCHRANE AND JOHN

ALFRED

NISONOFF Immunologic Regulation of Lymphoid Tumor Cells: Model Systems for lymphocyte Function

Binding of Bacteria t o Lymphocyte Subpopulations

MARIUS TEODORESCU AND EUGENE P. MAYER

AWL K. ABRAS

INDEX

H.

GRIFFIN

INDEX

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