ADVANCES IN
Immunology EDITED BY FRANK J. DIXON Scripps Clinic and Research Foundation La Jolla, California ASSOCIATE EDITORS
K. FRANKAUSTEN LEROYE. HOOD JONATHAN W. UHR
VOLUME 45
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ADVANCES IN IMMUNOLOGY, VOL. 45
Cellular Interactions in the Humoral Immune Response ELLEN S. VITETTA, RAFAEL FERNANDEZ-BOIRAN, CHRISTOPHER D. MYERS, A N D VIRGINIA M. SANDERS Department of Mlcrobiology, Unlverslty of Texas Southwestern Medical Center. Dallas, Texas 752SS
I. Introduction
The immune system has evolved primarily to combat infection by pathogenic organisms. It is characterized by its virtually infinite repertoire of specificities, its highly specialized effector components, its complex regulatory mechanisms, and its mobility. In contrast to most other organ systems, the immune system is not confined to a single site in the body: immunocytes and their secreted molecules traffic within and among lymphoid organs and various body compartments. Hence, a highly complex system of communication has developed among the various cell types in the immune sytem. One important mechanism of communication is the requirement for interactions among cells for the activation and differentiation of resting B lymphocytes into antibody-secreting cells. These cellular interactions involve both cell/cell contact and the release of mediators (cytokines) that can act in either an autocrine or paracrine fashion on cells both within and outside the immune system. In the present review we will discuss the interactions between T and B cells and the role of accessory cells and cytokines in the generation of specific antibody responses. The first portion of the review is a historic perspective (Section I), followed by a summary of present-day concepts (Sections 11-V): in the final section we speculate on how the different components of the immune system might function in mvo (Section VI).
A. MODELS OF T CELL/BCELLINTERACTIONS In 1966, Claman and his co-workersprovided the first direct evidence that T and B cells interact in the generation of an antibody response to sheep red blood cells (SRBCs)* (1). Lethally irradiated mice were *Abbreviations used: ABC, antigen-binding cell; AIDS, acquired immune deficiency syndrome; APC, antigen-presenting cell; BCGF, B cell growth factor; BCDF-y, B cell differentiation factor for IgG,; BSF-1, B cell stimulatory factor-1; BSF-2, B cell stimulatory factor-2; C, constant region; CML, cell-mediated lysis; CSF, colonystimulating factor; DNP, dinitrophenyl; DTH, delayed-type hypersensitivity; EAF, 1 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
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injected with thymocytes or bone marrow cells from normal or immune syngeneic donors. The cell transfer was followed by challenge with antigen (SRBC) and the spleen cells from the recipient mice were assayed at various times for the secretion of hemolytic antibodies. Such experiments showed that neither thymus-derived nor bone marrow-derived cells could, on their own, elicit an anti-SRBC response. However, when the two types of cells were present in the same recipient, an antibody response was elicited. Studies by Davies et al. (2) and Mitchell and Miller (3-7)confirmed and extended these conclusions and established that the antibodyforming cell precursors were derived from the bone marrow population and that thymus cells could recognize and react specificallywith antigen, but did not, themselves, produce antibody. It was suggested that the thymocytes or their mature progeny played a role in helping the B cells to differentiate into antibody-producing cells. With the advent of in vitro tissue culture techniques developed by Mishell and Dutton (8, 9) and Marbrook (10, 11), it was possible to further elucidate the roles of different cell types under more controlled experimental conditions. Using in Vitro culture, Mosier and his colleagues (12-14) first demonstrated that when spleen cells were separated by virtue of their ability to adhere to plastic, neither the adherent nor nonadherent population of cells could, on their own, make an antibody response. The cells in the adherent population required for an antibody response were macrophages, while the cells in the nonadherent population were primarily T and B cells. Experiments carried out by many investigators confirmed that T and B cells were required for both primary and eosinophil activation factor; EBV, Epstein-Barr virus; EDF, eosinophil differentiation factor; FCS, fetal calf serum; FDC, follicular dendritic cell; F I X , fluorescein isothiocyanate; HGF, hybridoma growth factor: HIV, human immunodeficiency virus: HSA, human serum albumin; ICAM-1, intercellular adhesion molecule-1; IFN , interferon; Ig, immunoglobulin; IL, interleukin; Ir, immune response (gene); b,dissociation constant; KHF, killer helper factor; KLH, keyhole limpet hemocyanin; LAF, lymphocyteactivating factor; LAK, lymphokine-activated killer cell; LFA, lymphocyte function associated; LPS, lipopolysaccharide; LT, lymphotoxin; MABC, memory ABC; MAF, macrophage-activating factor; MHC, major histocompatibility complex: mAbs, monoclonal antibodies; MTOC, microtubule organizing center; NK, natural killer cell: OVA, ovalbumin; PC, phosphorylcholine; PCT-GF, plasmacytoma growth factor; PFC, plaque-forming cell; PGEp, prostaglandin E,; PKC, protein kinase C; PMA, phorbol myristate acetate; PNA, peanut agglutinin; PPD, purified protein derivative; r, recombinant; R, receptor; RAMIg, rabbit antimouse Ig; SDS, sodium dodecyl sulfate; sIg, surface immunoglobulin; SN, supernatant; SRBC, sheep red blood cell; Tc, cytotoxic T cell; TCGF, T cell growth factor; TcR, T cell receptor; TD, thymus dependent; Th, T helper cell; TI, thymus independent: TNF, tumor necrosis factor; TNP-ABCs, trinitrophenylantigen-binding cells; TNP-MABCs, memory TNP-ABCs; TRF, T cell replacing factor; B,T suppressor cell; TT, tetanus toxoid; V, variable region.
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secondary antibody responses and that macrophages served as accessory cells (7, 12, 15). B. INTERACTIONS BETWEEN T AND B CELLS SPECIFIC FOR DIFFERENT DETERMINANTS O N THE SAME ANTIGEN (HAPTEN-CARRIER EFFECT) An important but initially perplexing observation was the demonstration of the “hapten-carrier’’ effect. It had been known for many years that immunization with a small nonimmunogenic molecule or hapten was effective only when the hapten was coupled to a “carrier” molecule which was immunogenic. It was demonstrated that cooperative interactions between distinct lymphocytes specific for carrier and haptenic determinants were essential for the development of an antihapten antibody response. The first direct evidence for cooperative participation of two cells with distinct determinant specificitieswas obtained by Mitchison (16). Spleen cells obtained from syngeneic donor mice that had been immunized with a hapten-carrier conjugate secreted antihapten antibodies following challenge with a homologous haptencarrier conjugate, but not with a heterologous conjugate containing the correct hapten but another carrier. In contrast, when spleen cells from donors immunized with a hapten-carrier conjugate were mixed with spleen cells from donors immunized with another carrier, a good secondary response could be obtained using the hapten conjugated to the second carrier. Thus, cells specific for the second carrier helped the hapten-specificB cells to make an antihapten response. It was later shown by Raff and colleagues (17, 18) that the carrier-specificcooperating cells, or helper cells, were thymus derived, whereas the antihapten antibodyforming cells were bone marrow derived. These experiments were made possible by the identification of a marker on thymus-derivedcells called 8 (now Thy-1), and the development of an antibody against 8, which, in the presence of complement, could lyse the thymus-derived cells. By eliminating 8 cells from cell mixtures, it was demonstrated that they were responsible for carrier specificity and for cooperating with the B cells in the elaboration of an antibody response to the hapten. The phenomenon of cooperation between carrier-specificT cells and haptenspecific B cells was also demonstrated independently by Rajewsky et al. (19), who immunized rabbits with a hapten-carrier conjugate and observed that the animals made a significant antihapten antibody response if they received a supplemental intervening immunization with free, unconjugated carrier. It was further demonstrated that the intervening immunization with the carrier primed a second helper T cell population, which could then cooperate with the hapten-specific B cell +
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population in responding to the hapten-carrier conjugate. While subsequent in vilro studies went a long way in defining the cell types involved in the linked response to hapten and carrier, they also pointed to the vast array of artifacts involved in obtaining antibody responses in vitro as opposed to in vivo. Numerous papers focused on the roles played by the constituents of the tissue culture media, fetal calf serum (FCS), plastic ware, and the addition of reagents (such as 2-mercaptoethanol) in obtaining optimal antibody responses in m’tro (9, 20-24). During this time, immunologists were faced with the frustrating problem that it was often difficult to repeat experiments among different laboratories because of technical differences in the culture systems. Nevertheless, with time, it became evident that the basic tenet of T cell/B cell collaboration involving hapten-specific B cells and carrier-specificT cells was correct, albeit with some qualifications. For example, experiments carried out by Miikela and his associates (25, 26) and others (27-31) established that an increasing density of repeating antigenic epitopes correlated with decreasing dependency on T cells, and that epitope density affected not only the magnitude of the response, but the isotype of antibody secreted as well. Additional variables that influenced the in vilm antibody response included the physical nature of the carrier (particulate or soluble), virgin versus memory cells, and differences among mouse strains (32, 33). C. THEROLEOF T CELLSON THE QUALITY OF THE ANTIBODY RESPONSE As discussed above, experiments carried out in the 1960s demonstrated that T cells are required for B cells to respond to most antigens. It was further demonstrated that T cells were also required to induce the progeny of the B cells to switch from the secretion of IgM to IgG antibodies (34-42). Furthermore, other experiments showed that T lymphocytes were involved in affinity maturation of the antibody response and that the more “T independent” the response, the poorer the affinity maturation (39, 43-46). It was concluded that T cells play an essential role not only in the activation of resting B cells, but also in isotype switching and affinity maturation of this response. Thus, T cells can regulate the levels and affinity of serum antibodies of different isotypes. Different Ig isotypes are essential for providing the most effective means of eliminating a given type of pathogen, e.g., viral, parasitic, or bacterial. Finally, T cells play a major role in generating memory B cells which can respond more effectively to subsequent challenge with antigen (47).
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D. SUPPRESSION VERSUS ENHANCEMENT OF THE IMMUNE RESPONSE Gershon and Kondo (48, 49) were the first to develop the concept of suppressor T cells. In general, suppression appeared to be nonspecific (50, 51); however, there were claims of antigen-specific suppressor T (Ts) cells (52-55). Subsequently, additional mechanisms for down regulating immune responses were suggested, including antibody-mediated suppression generated by immune complexes (2, 56-68) or antiidiotypic antibodies (69-71). It is now apparent that at least some types of suppressor cells can be distinguished from helper T (Th) cells by virtue of the expression of different surface markers. More recent evidence suggests that suppression may be mediated, at least in certain cases, by soluble factors elaborated by the suppressor cells that can act on helper cells and, possibly, on other immunocytes as well (72). The mechanisms by which Ts cells manifested their effect were controversial and have still not been well defined. These observations led to experiments in which it was demonstrated that supernatants (SNs) from activated T cells could, in some instances, substitute for T cells in enhancing or suppressing the immune response (73). In retrospect, much of the confusion concerning suppression can be accounted for by two findings: (1) different subsets of T cells secrete a large array of different lymphokines which exert both helper and suppressor activities (74, 75); (2) soluble factors act primarily on cells which had been activated by a T cell/B cell cooperative interaction and which can induce the partially activated B cells to differentiate and, in some cases, can suppress this differentiation (50, 51). E. THEROLEOF THE MAJORHISTOCOMPATIBILITY COMPLEX IN T CELL/BCELLINTERACTION A large number of experiments in the 1970s established the relationship between immune response (Ir) genes, encoded by the major histocompatibility complex (MHC), and the ability of T and B cells to interact in the elaboration of an immune response (76-79). Molecules encoded by the MHC, in particular the I region of the MHC, influenced the ability of T cells to interact with macrophages and B cells (76, 78, 80-84). This implied that the products of Ir genes (now class I1 MHC genes) were involved in cellular interactions between T cells and B cells and indicated that in order for a T cell to recognize a B cell or an antigenpresenting cell (APC), recognition of class I1 molecules was necessary. T cell receptors (TcRs) involved in recognizing class I1 molecules were either the same or different from those recognizing antigen. In the last
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few years, the basis for genetic restriction has been elucidated and the concept has emerged that a single TcR can recognize both the antigenic moiety and the MHC molecule on the surface of an APC (85, 86). It has also been shown that accessory molecules on both T and B cells influence the interaction of the two cells. F. ANTIGEN-SPECIFIC RECEPTORS ON T AND B CELLS Perhaps no issue in immunology has had a more colorful and controversial history than the elucidation of the antigen-specific receptors on T and B cells. Studies in the early 1960s demonstrated that B cells express antibody molecules that served as antigen-specific receptors (87-97). It was initially thought that the antigen-specific receptors were the same as the major class of immunoglobulin (Ig) in the serum, e.g., IgG (97). However, studies in the early and mid-1970s established that the major antigen-specific receptors on the B cells are monomeric IgM (98) and IgD molecules (99), both of which share the same antigen specificity (90, 100, 101) and idiotype (102-104), and each of which contains a C-terminal transmembrane domain responsible for anchoring the receptor in the membrane of the B cell (105-109). It was later reported that B cells can express other classes of Ig on their surface depending upon their state of differentiation (110-112) and that these receptors can also express transmembrane segments (112). Furthermore, B cells often express more than one isotype of surface Ig (sIg) (110-112). Based on previous studies, it was assumed that the Ig receptors on B cells are responsible for recognizing the haptenic portion of a hapten-carrier conjugate, whereas, the T cells express a receptor of a different specificity that recognizes portions of the carrier. For a long time, there was heated controversy concerning the nature of the TcR which divided the immunologic community into two camps, i.e., those who were convinced that the TcR was Ig (113) and those who were not (114). In 1980, pioneering experiments by Hedrick et al. (115) and Yanagi et al. (116) demonstrated that the receptor expressed on the T cell was indeed different from that expressed on the B cell. The major TcR consists of disulfidebonded a and @ chains, each having a M , of 45,000-55,000. The a and @ chains express transmembrane and cytoplasmic domains. It was further shown that the two chains of the receptor can be divided into variable (V) and constant (C) region segments (117-119) which show structural similarities to V and C domains of Ig (116, 120-122). Indeed, the TcR is encoded by V, D, and J segments of DNA, in a manner analogous to the Ig receptor on the B cell (123). The a@ dimer of the TcR recognizes both antigen and portions of the MHC molecule (123-125). Finally, it was shown that other surface molecules on the T cell
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[such as cluster-designation (CD)3, CD4, CD8, lymphocyte functionassociated antigen (LFA)-I]interact with the crp complex or with molecules on the B cell (125, 126). More recently, 6 and y chains of the TcR have been identified but their role in antigen recognition has not been fully defined, even though T cells expressing 6y receptors have been identified (127-130). Most importantly, T cells, in contrast to B cells, do not recognize soluble native antigen, but recognize “processed’ antigens (131-136) or endogenously synthesized antigens (137) that are bound to the surface of APCs, e.g., macrophages, dendritic cells, Langerhans cells, B cells, or tumor target cells. In the case of exogenous antigen, the presented form of the antigen arises as a consequence of a series of intracellular processing events after the native antigen binds to and is internalized by the APC (138). It is thought that these processing events involve degradation and/or denaturation of the antigen, recycling of fragments of denatured antigen to the surface of the presenting cell, and association of the fragments with class I1 MHC-encoded molecules. Recent studies supporting this concept will be presented later in the chapter. G. THEANTIGEN-BRIDGING MODELOF T CELL/BCELL INTERACTION The original model postulated by Mitchison (16) to describe associative recognition of antigen by T and B cells suggested that receptors on B and T cells recognize the hapten and carrier portion of the native antigen, respectively, and, hence, antigen bridges the two cells. Following bridging by antigen, the T cell delivers activating signals to the B cell. This model has now been revised to include the current-day notion that the B cells bind the haptenic portion of the antigen, internalize the haptencarrier complex, degrade the carrier, and present peptide fragments of the carrier in association with a class I1 molecule to specific T h cells. Th cells also recognize peptides of the processed carrier (taken up nonspecifically)on the surface of a macrophage or other accessory cells (138). In fact, both Th and B cells may interact on the surface of a macrophage where the B cells recognize the native hapten and the T cells, the processed carrier. This becomes important in the physiological setting of a lymphoid organ where T cells and B cells are concentrated in different portions of the organ (139). In this system, it is attractive to postulate trafficking of cells through the lymph node to a common site where antigen is present in sufficient concentration to bind to the relevant cells and where these cells can interact. In the following sections of this review, we discuss the mechanisms underlying the hapten-carrier effect, T cell/B cell interaction, the role
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of Th cells in inducing affinity maturation and Ig class switching, genetic restriction, T cell suppression, and the role of accessory cells and soluble mediators in T cell/B cell interaction and activation. Furthermore, the role of the MHC will be discussed both in the context of processing and presentation of antigen to T cells by APCs and in the context of T cell/ B cell interaction. The focus of this review will be on the mechanisms involved in the interaction between T and B cells, and the functional outcome of these interactions. Ii. Antigen Processing and Presentation
A. REQUIREMENT FOR PROCESSING Between 1970 and 1980, experimental findings related to antigen processing and presentation established that (1) T cells are required for the generation of antibody by the differentiated progeny of activated B cells responding to TD antigens; (2) B and T cells do not recognize the same epitope on a TD antigen; (3) the antigenic epitopes recognized by T and B cells must be physically linked on the antigen in order for the antigen to elicit an antibody response; and (4) T cells cannot bind native, soluble antigen, but have receptors for “processed” antigen associated with class I1 molecules on APCs. These findings suggested that APCs must first bind native antigen and, at a later time, present some other form of this antigen to a T cell in an antigen-specific, MHCrestricted manner. As a result of the interaction of the T cell with an APC, the T cell becomes activated. The term “presentation” can be used to define the capacity of APCs to express altered forms of antigen to T cells. Antigen “processing” describes the steps by which an APC converts native antigen to a form which can be recognized by a T cell. Processing may involve proteolytic degradation, denaturation, or modification of the antigen. Processing also involves the association of antigenic fragments with class I1 molecules and the expression of these complexes of processed antigen and class I1 molecules on the surface of APCs. The earliest studies to correlate antigen catabolism with presentation were carried out by Ziegler and Unanue (140), who exposed macrophages to the bacterium Listeriu monocytogenes. Following extensive washing of these treated macrophages, Listenu-primed T cells were added to the cultures and were allowed to bind. Unbound T cells were decanted from the cultures and the depletion of antigen-specificcells was assessed. These experiments showed that (1) a processing period of approximately 1 h is required before the T cells can bind to the antigen-pulsedmacrophages;
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(2) antigen presentation is decreased after treatment of the APCs with lysosomotropic agents: (3) presentation is inhibited when the macrophages are fixed prior to exposure to antigens; and (4) after the APC has been exposed to antigen for at least 1 h , subsequent fixation or treatment with lysosomotropic agents no longer affects their ability to present antigen. These experiments also demonstrated that the binding of T cells to antigen-presenting macrophages is “restricted” by class I1 molecules and that the native antigen is ingested and catabolized by the macrophages.
TYPES OF APCs B. DIFFERENT The macrophage was the first APC to be identified. Monocytes/ macrophages exist both in the circulation and in the tissues, and together with polymorphonuclear leukocytes (PMNs) form the first line of defense against foreign pathogens. Macrophages have the ability to engulf particulate and soluble antigens, to degrade them, and to present antigen fragments to T cells (138). More recent studies have demonstrated that in addition to macrophages, other cells can also act as effective APCs. In general, all cells capable of presenting antigen to T cells constitutively express class I1 antigens on their surface or can be induced to do so. These cells include dendritic cells (141-144), Kupffer cells (145), Langerhans cells (146-148), vascular endothelial cells (146), Schwann cells (149), astrocytes (150), thymic stromal cells (151), human dermal fibroblasts (152), B lymphocytes (153), and human class 11+ T cells (154). Although the role.played by these APC cells in mu0 is not completely understood, evidence from a number of laboratories suggests that macrophages, B lymphocytes, and dendritic cells are the major APCs present in lymphoid tissues. Steinman and co-workers (141-144) have demonstrated that dendritic cells are very efficient APCs. In fact, their studies suggest that the dendritic cell may be a more important cell than the macrophage for the initial activation of resting T cells in viuo. Another special type of APC is the follicular dendritic cell (FDC) (155, 156). This cell is unique in that it carries immune complexes of native antigen on its surface in a nonprocessed form. In vivo, the FDC may serve as a reservoir for native antigen which can be subsequently bound and processed by other APCs and, in particular, B cells. Since 1980, a number of groups have demonstrated that B cells can process and present antigen to T cells both in mlro and in mvo (153). This demonstration supports the earlier hypotheses of antigen bridging in T cell/B cell interactions. Once it became clear that B cells could bind native antigen, process it, and present processed fragments of
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antigen to T cells in an MHC-restricted manner, the mechanism of cognate interaction between B and T cells became clearer. In the remainder of this section we focus on antigen processing and presentation by B cells, a comparison of the ability of B cells versus other APCs to present antigen, and proposals concerning the roles of the different APCs in viuo. C. B CELLSAS APCs Benacerraf first hypothesized that B cells were major APCs (157). This was deduced from two findings: (1) B cells express high densities of class I1 molecules, which were thought to be involved in T cell/B cell communication, and (2) since the initiation of an antibody response requires the “bridging” of T and B cells, it would be logical to presume that B cells should function as APCs. The first report to clearly demonstrate that this was the case was that of Chesnut and Grey (158). Using rabbit antimouse Ig (RAMIg) as an antigen and rabbit Ig-specific T cells, they circumvented the problem of the low frequency of B cells which could specifically bind to a purified protein antigen, because RAMIg can bind to all sIg+ B cells while antigen binds to
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of irradiating the presenting B cells (which also proliferate as a consequence of T cell-mediated activation). Under these conditions, normal splenic B cells depleted of T cells and macrophages were unable to present KLH to T cells except at very high antigen or cell concentrations. In contrast, both the BAL and the macrophages were very efficient APCs and induced significant levels of IL-2 secretion from the T cells at 100-fold lower cell numbers. When normal primed T cells (instead of T hybridoma cells) were used as responding cells, the normal B cells were, again, very poor APCs for KLH. Although the BAL were better APCs than the B cells, they were 50 times less efficient than splenic adherent cells. Two issues were raised by the aforementioned experiments. (1) There were marked differences in the capacity of different B cells to present antigen to T cells and this appeared to be dependent upon their state of activation; i.e., resting B cells were less effective than activated or neoplastic B cells. (2) There was a dichotomy between the ability of B cells to present antigens which they could bind specifically via sIg, e.g., RAMIg, versus nonspecifically, e.g., KLH and ovalbumin (OVA). This latter point raised two further questions: (1) whether presentation of antigen by resting B cells was physiologically relevant under conditions where the B cells did not express sIg receptors specific for the antigen in question, and (2) whether resting B cells could process irrelevant antigen (as opposed to RAMIg) as effectively as macrophages. Between 1983 and 1985,a number of papers were published with conflicting views as to whether resting B cells could act as APCs. Cowing and co-workers(163,164)and Grey and co-workers(131,165-167)showed that resting B cells could not act as APCs, while Parker and associates (168,169) and Pierce and colleagues (170)demonstrated that they could. This controversy was partially resolved by Ashwell et al. (171)who showed that resting B cells could present antigen, but that this event was radiosensitive. This was subsequently confirmed in several reports (163,172, 173). In earlier experiments by some groups, the resting B cells had been irradiated prior to using them as APCs. Although studies have now confirmed the exquisite radiosensitivity of the presentation process in resting B cells, this may not be the entire explanation for the lack of antigen-presentingactivity reported previously. In 1985,Krieger and co-workers (167)irradiated B cells with 500 rads and subsequently examined them for their ability to act as APCs following Percoll density gradient fractionation into cells of low and high density. Only the low-density cells were capable of eliciting a significant T cell response. These results conflicted with those of Parker and co-workers (169, 174), who separated B cells on the basis of density using a
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countercurrent elutriation system and found that the dense cells could act as APCs. The difference in the two studies may be attributed not to irradiation of the B cells, but to differences in the antigens used. In the experiments of Krieger and co-workers (167), OVA was used as the antigen, while in the studies of Parker and colleagues (169, 174), RAMIg was used. Thus, the difference in efficiency of uptake of these two different antigens, which were (RAMIg) or were not (OVA) bound to the resting B cells via sIg, undoubtedly influenced the ability of the B cells to act as effective APCs. Hence, antigens bound to sIg on resting B cells may be more effectively internalized and/or routed to different intracellular compartments than are antigens which are taken up by fluid-phase pinocytosis. Furthermore, fluid-phase pinocytosis is probably a relatively inefficient process in resting B cells and would not result in uptake of large enough quantities of irrelevant antigen unless the concentrations of antigen in the medium were very high. In contrast to resting B cells, large or activated B cells may pinocytose antigen more efficiently.
D. ANTIGENPROCESSING A number of studies have addressed the issue of what intracellular events are involved in “antigen processing” and how these events compare in B cells and macrophages. The picture that has emerged, although not yet complete, suggests that antigen processing takes place intracellularly, is time- and temperature-dependent, and is inhibited by agents that interfere with lysosomal function. In addition, processing involves recycling of antigenic fragments to the surface of the APC. Finally, B cells are more effective at binding and presenting specific antigen because of their ability to capture antigen via sIg molecules. However, processing events in macrophages and B cells may be very similar, although it is possible that B cells process sIg-bound antigens in endosomes while macrophages process antigens in lysosomes. In addition, antigen taken up by fluid-phase pinocytosis in B cells may also be routed to lysosomes. 1. Intracellular Events
Different investigators have established that antigen processing is an intracellular event, since metabolic inhibitors, inhibitors of secretion, and inhibitors of lysosomal enzymes can prevent processing. Furthermore, cells which cannot internalize antigen, or cells from which bound antigen is removed by enzymes, cannot present antigen. In this regard, Metezeau et al. (175) demonstrated that anti-Ig antibodies bound to B cells are rapidly internalized, but that the endocytic process is blocked either by incubation at 4OC or by treatment with metabolic inhibitors
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such as azide and 2-deoxyglucose. Furthermore, Fab’-anti-Ig is routed primarily to endosomes while divalent anti-Ig is routed to lysosomes. Hence, the degree of cross-linking of sIg molecules on B cells may determine how antigen-sIg complexes of different sizes are routed intracellularly. The approach used by Unanue and others (176-178) has been adapted to analyze internalization of antigen by B cells (131). i.e., cells are treated with antigen at 4OC and then washed extensively. After warming the cells to 37OC, they are fixed to prevent further internalization. Cells are then treated with proteolytic enzymes and the amount of antigen released from the cell surface is determined. Any remaining cell-associated antigen is assumed to be intracellular. Results from such studies confirmed that specifically bound antigen is rapidly internalized while nonspecifically bound antigen remains on the cell surface (131). A similar approach has also been used to measure the intracellular transit time and reappearance of processed antigen on the cell surface of macrophages and B cells. The first studies of this type were carried out by Ziegler and Unanue (140) using macrophages pulsed with Listenu. This study established two important points: (1) light fixation of APCs (1% paraformaldehyde) blocks antigen processing, but does not inhibit antigen presentation after processing has ocurred; and (2) macrophages require 30-60 min after pulsing with antigen before they can present it to T cells. In this case, antigen presentation was measured by the direct binding of T cells to macrophages. More recently, Lanzavecchia and co-workers (179) have analyzed the kinetics of antigen processing and presentation by antigen-specificB cells by measuring Ca2+fluxes in the T cells; Ca2+fluxes occur very rapidly once a T cell binds to antigen on an APC. Using a tetanus toxoid (TT)specific B cell line and specific antigen, it was confirmed that a processing period of 60 min was required before the APCs would present specifically bound antigen to T cells. Taken together, the results of Ziegler and Unanue and Lanzavecchia demonstrate that a period of 30-60 min is required before antigen bound to either B cells or macrophages can be presented in a form which the T cell can recognize. Information concerning intracellular routing of antigen has been obtained by using inhibitors of proteolysis or agents which raise the pH of lysosomal compartments (e.g., NH&l or chloroquine) and inhibit the activity of lysosomal enzymes. These agents block antigen processing and/or presentation and imply that processing occurs in lysosomes and involves proteolysis. However, it should be noted that both chloroquine and N h C l have multiple effects on cells, such as interfering with recycling of intracellular vesicles and fusion of endosomes with lysosomes
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(180-182). In order to understand how these inhibitors effect processing, Ziegler and Unanue (183) treated macrophages with 0.01 M NH4C1 or 0.1 mM chloroquine and found that this treatment had a negligible effect on antigen binding and ingestion. Likewise, chloroquine or NH&l added after antigen processing had a minimal effect on the binding of T cells to presenting macrophages. However, when the macrophages were treated with lysosomotropic agents prior to treatment with antigen, both presentation and catabolism of antigen were reduced by greater than 50%. The results of these experiments suggest that NH4Cl and chloroquine do not effect T cell/macrophage interactions, but do effect antigen degradation and/or recycling of antigen to the cell surface. The effect of chloroquine on antigen presentation by B cells and macrophages was studied by Grey and co-workers(131, 165). Their results demonstrated that 40 p M chloroquine is sufficient to totally ablate antigen presentation by B lymphoma cells and macrophages from peritoneal exudates. Furthermore, they demonstrated that chloroquine only inhibits antigen processing and not presentation; once an antigen had been processed by B lymphoma cells, subsequent addition of chloroquine has no effect on the presentation of this antigen, even though a second antigen added following chloroquine treatment can no longer be presented. These and similar findings (184) using B cells must be interpreted with caution in light of recent observations that B cells are far more sensitive to the toxic effects of lysosomotropic amines than are macrophages. In this regard, C.D. Myers and E.S.Vitetta (unpublished data) have shown that concentrations of chloroquine as low as 0.1 mM cause irreversible damage to B cells in 3-4 h. Furthermore, Nowell and Quaranta (185) demonstrated that incubation of B cells with 1.0 pM chloroquine or 10 mM NH4Cl inhibits transport of newly synthesized class I1 molecules to the cell surface, resulting in the intracellular accumulation of nascent molecules. Considering the major role of class I1 molecules in antigen presentation, the notion that chloroquine effects only lysosomal catabolism of antigens should be reevaluated. It is important to note that monensin (131) and xyloside (186), two agents which block antigen presentation, prevent the transpot of newly synthesized proteins to the cell membrane.
2. Processing by Macrophages versus B Cells Although the studies mentioned above demonstrate that both B cells and macrophages are able to process antigens and present them to T cells, it was not clear whether B cells and macrophages handled antigen differently. In this regard, Grey and colleagues examined the fate of radiolabeled proteins bound to the surfaces of B lymphoma cells or
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15
macrophages (165). Conversion of the radiolabel from acid precipitable to acid soluble was used as a measurement of protein degradation. Using radiolabeled RAMIg and KLH as antigens, macrophages handled them in a similar fashion; over the course of 20 h, SO-SO% of the proteinassociated radioactivity initially bound to the cells was released into the supernatant (SN) in an acid-solubleform, and 20% in an acid-precipitable form. In contrast, in the B lymphoma cells, the processing of RAMIg and KLH was different. Radiolabeled RAMIg was processed with kinetics similar to those reported for macrophages; 50 % of the protein was released in 20 h in an acid-soluble form and 20% was released in an acidprecipitable form. Analysis of antigen fragments on sodium dodecyl sulfate (SDS)polyacrylamide gels showed that both macrophages and B lymphoma cells degraded intact RAMIg (M,= 150,000) into fragments of 50,000. However, when the B lymphoma cells were pulsed with low concentrations of radiolabeled KLH, almost 90% of the labeled protein remained cell bound and acid precipitable. These findings confirmed earlier results that B cells can degrade a specifically bound antigen, e.g., RAMIg, in a manner analogous to that observed for macrophages. However, antigen not bound to sIg is not readily internalized or degraded. Chesnut and co-workershad previously compared processing of antigen by B cells and macrophages (131) using the secretion of IL-2 by T hybridoma cells as an assay for antigen presentation by B lymphoma cells or macrophages. Their studies demonstrated that paraformaldehyde furation effectively blocks presentation of antigen by B cells within 45 min after its addition to the cells. Thus, some time-dependent event is required prior to presentation. Antigen which had bound to the cells at 4OC could be entirely removed by pronase treatment immediately after binding, but became pronase-resistant after the cells were warmed to 37OC, suggesting that antigen is rapidly internalized. Both macrophages and B cells are able to present high concentrationsof either native or denatured OVA to an OVA-specific T hybridoma cell and this presentation can be blocked by either lysosomotropic agents or carboxylic ionophores, e.g., chloroquine or monensin. Taken together, these findings provided further evidence that any antigen bound to a macrophage or B cell in high enough concentrations can be internalized and denatured, and that B cells do not have to take up antigen via sIg if the antigen is present in high enough concentrations to be internalized by fluid-phase pinocytosis. E. ANTIGEN PRESENTATION BY ENRICHED POPULATIONS OF ANTIGEN-SPECIFIC B CELLS Although nonspecific pinocytosis of antigens by resting B cells is generally inefficient as compared to the same events in macrophages, B cells have a significant advantage over macrophages in capturing
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ELLEN S. VITETTA ET A L
specific antigen via sIg. It could be argued that presentation by B cells of antigens which do not bind via sIg is an in vitro artifact resulting from the high concentrations of both antigen and cells used in culture. Thus, in a physiological setting, B cells might present only their specific antigen to T cells. As mentioned above, studies by Grey and co-workers (153) as well as Parker and co-workers (168, 169, 174) have examined presentation of antigen by B cells using the “polyclonal” antigen, RAMIg. In both cases, the specific antigen (RAMIg) was presented at 10,000-fold lower concentrations than was nonspecific antigen (normal rabbit Ig). The first studies to use an entirely antigen-specific system were those of Abbas and associates (172, 187-190) and Lanzavecchia (184, 191, 192). Abbas and colleagues purified trinitrophenyl-antigen-binding cells (TNP-ABCs) from the spleens of either primed or unprimed mice on TNP-gelatin (172, 187). When the gelatin was warmed and the bound cells were recovered, 20-40oJo could bind to TNP. These cells were analyzed for their capacity to present either TNP-derivatized or native poly(g1ycine-leucine-phenylalanine) (GL4) to GL$-specific, I-Edrestricted T hybridoma cells. As previously demonstrated using RAMIg as antigen, TNP-ABCs could present TNP-GL4 (as compared to GL4) to T cells at least 1000-fold more efficiently; presentation was I-Edrestricted. Likewise, the importance of antigen uptake by sIg on the B cell was demonstrated by blocking (TNP-GL4) uptake with anti-Ig or TNP-human serum albumin (HSA) (187, 188). Further studies by the same group demonstrated that TNP-ABCs from unprimed mice had properties similar to those of TNP-ABCs from primed mice (172). The hapten and carrier specificity of these responses were further demonstrated by using both OVA and GL4 as carrier molecules, TNPspecific and fluorescein isothiocyanate (F1TC)-specificB cells, and TNPor FITC-haptenated antigens (172). Finally, Abbas and co-workers(172, 187-190) have used TNP-ABCs to demonstrate that presentation of antigen by the TNP-ABCs is specific at low antigen concentrations (when antigen binds to sIg) and nonspecific at high concentrations (when antigen is not taken up by sIg molecules). As described previously, Lanzavecchia (184) utilized TTspecific human B cell clones which had been transformed by Epstein-Barr Virus (EBV) to present antigen to TT-specific T cell clones. The TT-specific B cells were able to present their antigen at 104-foldlower concentrations than were cells from EBV-transformed B cell lines not specific for TT. Likewise, when a second irrelevant antigen was used, purified protein derivative (PPD), the TT-specific B cells presented antigen only at high concentrations. Lanzavecchia (192) further demonstrated that TT-specificB cells are capable of presenting antigen at concentrations lower than the
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17
dissociation constant (Kd)of the sIg on the B cells, suggesting that only a very small fraction of the sIg molecules is necessary to take up the amounts of antigen necessary for subsequent presentation. These findings led to the proposal of the “vacuum cleaner” model of antigen presentation in which B cells “concentrated” small amounts of antigen from their environment, processed it, and presented fragments (at a high enough concentration) on their surface to initiate interaction with specific T cells (192). BY ENRICHED POPULATIONS OF F. ANTIGENPROCESSING ANTIGEN-SPECIFIC B CELLS
The same approaches described above for the study of presentation by antigen-specific B cells have been used to analyze processing. Lanzavecchia (192) has shown that presentation of antigen by TT-specific human B cell clones follows many of the same patterns described for normal splenic B cells and RAMIg. Hence, B cells present specific antigen at very low concentrations and presentation requires a period of time for processing. Processing can be inhibited by fixation or by treatment with 10 p M chloroquine. In these experiments, the B cells were EBV transformed and were therefore radioresistant. Unfortunately, no direct comparison of presentation of antigen and anti-Ig has been reported in this system. One direct approach to the analysis of intracellular events occurring in antigen processing is the measurement of protein degradation by the B cell. C.D. Myers and E.S. Vitetta (unpublished results and Ref. 193) have used TNP-ABCs purified by rosetting spleen cells from normal mice with TNP-horse erythrocytes (194, 195). From 60 to 80% of the TNP-ABCs bind specific antigen, and some of the properties of these cells are summarized in Table I (196). Although TNP-ABCs can degrade both anti-Ig and TNP-proteins, the kinetics of degradation are different. TNP-ABCs treated with anti-Ig degrade proteins in a manner similar to that previously reported for both unpurified B cells and macrophages. TABLE I GENERAL CHARACrERISTlCS OF ANTIGEN-SPECIFIC B CELLS 1. 2. 3. 4. 5.
From 60 to 90% of the cells bind TNP (194, 195, 369) From 80 to 90% of the cells are sIg+ (194, 369) Greater than 95% of the cells are in Go (369) Greater than 92% are small B cells (as determined by cell volume) (369) Of the cells, 1/2 grow in response to dextran sulfate plus LPS (194) and 1/15 secrete IgM in response to T h cells and TD antigen (369, 453)
18
ELLEN S. VITETTA ET A L .
However, when specific antigen is used, degradation is more rapid and reaches a plateau at about 4 h. The percentage of antigen released into the culture medium as acid-solublefragments is only 30% (of the antigen bound) as compared to 60% when anti-Ig is used. It is important to note that the kinetics of release of acid-solublefragments of antigen into the medium parallel the kinetics of antigen presentation as determined by the formation of specific T cell/B cell conjugates (197) (to be discussed later). OF ANTIGEN FRAGMENTS WITH CLASS I1 G. INTERACTION MOLECULES The experimental approaches thus far described suggest that protein degradation and denaturation are important steps in antigen processing. In addition, the requirement for expression of class I1 molecules by APCs suggests that there is a relationship between processed antigen fragments, class I1 molecules, and antigen presentation. An approach used by many groups to demonstrate that APCs present fragments of antigen has been to fix cells, to incubate them with either native or partially degraded proteins, and to evaluate their ability to present proteins versus protein fragments to T cells. Allen (198) has reviewed data based on similar approaches and described three different types of antigen: (1) those which require proteolytic degradation or cyanogen bromide fragmentation before they can be presented by fixed cells; (2) those which require only reduction of disulfide groups to allow presentation by fixed cells; and (3) those which can be presented as native antigen by fixed cells. Other groups have described protein structures which require different degrees of degradation prior to presentation by fixed cells (133, 199-202). Using fmed cells or artifical membrane systems such as those described by Watts and McConnell(203), a number of groups have analyzed the structure of “minimal peptides” required for antigen presentation by APCs (84, 132, 136, 204-211). Schwartz et al. (82, 212-219) analyzed synthetic peptides of cytochrome c for their ability to stimulate T cells (when presented on APCs). By using a series of synthetic peptides which differed by one amino acid, and analyzing both the antigen and cell concentrations required for efficient antigen presentation, it was proposed that specific portions of the antigen fragment interact with either class I1 molecules on the APCs and/or the TcRs. Minimal peptide structures required for antigen presentation and binding of antigen fragments to class I1 molecules have been analyzed by Berzofsky and co-workers (220-224), who have demonstrated a common structural theme for many of these peptides, i.e., the majority of antigenic fragments recognized
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19
by T cells form an a-helical structure with an amphipathic nature. This structure, which was initially described for a fragment of sperm whale myoglobin, has been postulated to be important in immunodominance, i.e., the selection of T cell epitopes. This could be achieved by one of two mechanisms: (1) the amphipathic nature of the peptide may be specifically required for the binding of an antigen fragment to a class I1 molecule; or (2) since amphipathic fragments have a hydrophobic face, they may associate with the cell membrane, effectively producing a reservoir of fragments for class I1 molecules to bind to, either independently of the TcR or in its presence. The second possibility is supported by the data of Falo et al. (225, 226), who demonstrated that APCs treated with phospholipase (following antigen processing) lose their ability to present antigen but that the expression of class I1 molecules on the cell surface is unaffected. These data suggest that phospholipase treatment removes the antigen fragments from the surface of the APCs because of their weak binding to the cell surface. Alternatively, data from the same group (225) suggest that in vim processing includes the addition of a lipid moiety to antigen fragments, resulting in a covalent, phospholipase-sensitive attachment to the membrane. In an attempt to demonstrate that antigenic fragments can bind directly to MHC class I1 molecules, Babbitt et al. (135, 227) purified class I1 molecules from cells and performed equilibrium dialysis in the presence of synthetic peptides of antigen. In this and similar studies by Grey and colleagues (134, 136, 206, 228, 229) it was demonstrated that peptides had a measurable affinity for class I1 molecules and that binding was MHC-restricted with regard to the class I1 antigen used. However, since the K d of peptides and class I1 molecule is relatively low ( M), questions concerning the physiological relevance of the specific binding of antigenic peptides to class I1 molecules were raised. Hence, it is likely that the concentration of specific antigen fragments on the surface of APCs is too low to allow effective binding to class I1 molecules. Nonetheless, class I1 molecules could bind to peptides in a specific intracellular compartment (e.g., endosomes), where high concentrations of both peptides and class I1 molecules would be present. In this regard, experiments by Cresswell (230) demonstrated that endosomes (in which transferrin receptors are recycling) fuse with other endosomes carrying nascent class I1 molecules in transit between the Golgi apparatus and the cell surface. If antigen fragments are generated in similar endosomes recycling to and from the cell surface as suggested by several studies [(183, 193, 231, 232) and C.D.Myers and E.S.Vitetta, unpublished observation], it is possible that class I1 molecules and antigen fragments may associate and recycle to the surface in the same vesicles.
20
ELLEN S. VITETTA ET A L
This would provide the high local concentration of antigenic fragments necessary for binding to class I1 molecules. As mentioned above, this hypothesis is supported by the finding that chloroquine inhibits antigen presentation at concentrations which have no effect on antigen degradation by B cells, but which inhibit the transport of class I1 molecules to the cell surface [(185) and C.D. Myers and E.S.Vitetta, unpublished observation]. In any case, it is possible that random, transient, low-affinity interactions between class I1 molecules and peptides could be stabilized by simultaneous binding to TcRs. OF CLASS I AND CLASSI1 MOLECULES AND H. A COMPARISON THEIR ROLEIN ANTIGENPRESENTATION Bjorkman et al. (233, 234) have produced an X-ray crystallographic model of an MHC class I molecule and, from this, have hypothesized its three-dimensional structure. The class I molecule has a large cleft on its upper surface which might be the antigen-binding site. After the assignment of all the amino acids in the sequence of the class I molecule, there remained electron-dense material, apparently residing in the proposed antigen-binding cleft. This material may represent bound antigen peptides, although this has not been directly demonstrated. Nevertheless, if this material does represent antigen, it would suggest that most, if not all, class I molecules on the cell surface already have antigen fragments bound to them with sufficient affinity so as not to be removed during purification and crystallization. In any case, these findings have been extrapolated to class I1 molecules, for which hypothetical model structures have now been produced (235). In this regard, it is important to mention certain differences between antigen presentation by MHC class I and class I1 molecules, since the data described here have been extrapolated to antigen presentation in the context of class I1 molecules on APCs. Class II-restricted antigen presentation invariably involves CD4+ (human) or L3T4+ (mouse) cells, which usually function as T h cells (236), although there have also been reports of class II-restricted, CD4+/LST4+ T cytotoxic (Tc) cells (237). CD8 (human) Lyt-2+ (mouse) cells are invariably class I restricted and usually function as Tc or Ts cells (236), although there are also reports of CD8+/Lyt-2+ cells that can provide help for B cells (238). Furthermore, CD4+, class I-restricted cells have also been described (239). In general, there is a difference in the nature of the antigen presented in the context of class I or class I1 molecules on APCs. As described above, class II-restricted cells are primarily involved in the recognition of antigen which has been endocytosed by the cell, processed in endosomes or lysosomes, and then recycled to the surface (240, 241). Mechanisms by +
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21
which these antigenic fragments could become associated with class I1 molecules have been described in previous sections. In contrast, many class I-restricted T cells recognize antigen that has been synthesized by the cell (e.g., viral proteins or minor histocompatibility antigens) (240, 241). The interaction between newly synthesized antigens and class I molecules could occur during synthesis and assembly of both molecules. This possibility is supported by the finding of material in the proposed antigen-binding cleft of the crystallized class I molecules analyzed by Bjorkman and colleagues (233, 234, 242). However, a direct demonstration of the binding of purified class I molecules to viral or other antigens has not yet been carried out. OF ANTIGEN PRESENTATION in Vitro I. A SUMMARY
The results of studies carried out over the last decade have shown that B cells can serve as APCs and that they are most efficient at presenting antigens which they bind via sIg. A period of time for processing is required before antigen is presented to T cells. During this time, the antigen is denatured and/or proteolytically cleaved into small peptides, some of which are presented, but most of which are released into the culture medium. Very little is known about the intracellular mechanism(s) by which this proteolytic cleavage occurs or in which intracellular sites it takes place, although both endosomes and lysosomes have been implicated. However, fragments are in some manner recycled to the surface and become weakly associated with class I1 molecules. This information is summarized in the model depicted in Fig. 1. Receptors on T cells bind to the complex of antigen and class I1 molecules and this binding results in T cell activation. The activated T cells then activate the antigen-presenting B cells with which they specifically interact. J. THEB CELLAS
APC in Vivo Although it has been demonstrated that B cells can act as APCs in vitro, their role in vivo remains less well defined. Several lines of evidence indicate that B cells play a major role in antigen presentation, leading to T cell activation in mvo. Inaba and colleagues (141, 142) and Lassila et al. (243) have demonstrated that dendritic cells are critical for antibody responses to TD antigens in both mouse and man. Although it has not been definitively demonstrated that dendritic cells process antigen, it is possible that they bind fragments of antigen released by macrophages. Whatever the case, dendritic cells are the only APCs capable of eliciting a response when unprimed, resting T cells are used in m*tro(141, 142, 243). These results suggest that dendritic cells may be the first APCs responsible for activating T cells from unprimed AN
22
ELLEN S. VITETTA ET A L .
d
k L l *
FIG. 1. Antigen processing and presentation by B cells. (a) Antigen: in B cells, antigen uptake is mediated by sIg. At very high antigen concentrations, antigen uptake may occur by pinocytosis without specific binding to sIg; this leads to polyclonal activation of the antigen-presenting B cells following their interaction with T cells. The source of antigen could be soluble protein or immune complexes. These could be in solution, on the surface of FDCs or icoccomes, or on macrophages. (b) sIg: sIg on B cells plays a role in the specific binding of antigen and increases the efficiency of antigen capture. sIg (or fragments of it) may remain bound to antigen during processing and affect the peptides presented by protecting certain epitopes or making other epitopes more accessible to proteases in intracellular compartments. Cross-linkingof sIg on the B cell results in the induction of early activation events. Increased levels of class I1 molecules could also increase the efficiency of presentation. Finally, changes in the metabolism of the cell may result in the activation of enzymes involved in antigen processing, recycling, and presentation. (c) Intracellular routing: antigen is internalized into vesicles and is either processed in the endosomes and/or lysosomes. ( d ) Extent of antigen degradation: it is unclear how much antigen degradation is required for antigen presentation. In some situations, peptides of 8-10 amino acids are presented, while in others large fragments of several hundred amino acids may be presented without further processing. It is possible that a lipid or peptide may be attached to the antigen fragment to facilitate binding either to class I1 molecules or to the cell membrane. (e) Interactions between class 11 molecules and processed antigens-source of class I1 molecules: class I1 molecules may be derived from the cell surface or may be newly synthesized. In this instance, nascent class I1 molecules trafficking from the Colgi apparatus may interact with antigen fragments generated in the endosomes. Some fragments of antigen may have an affinity for the plasma membrane, either because of their amphipathic nature or because of the attachment of a lipid. These fragments would then recycle to the cell surface where they would be present at a high enough concentration to interact with class I1 molecules. The interaction of peptides and class I1 molecules may be further stabilized by the binding of TcRs to the class Wantigen complex.
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23
animals and that B cells (or macrophages) might present antigen only to activated, partially activated, or memory T cells. The general approach that has been used to analyze the importance of B cells as APCs in mvo has been to deplete mice of B cells (or B cells of certain specificities) and then to analyze the capacity of these mice to generate activated T cells. Using anti-p-treated mice which are deficient in B cells, Janeway et al. (244) demonstrated a requirement for B cells in the generation of T h cell activity in lymph nodes. These results were confirmed and extended by Ron et al. (245), who treated mice from birth with anti-p to deplete B cells and subsequently primed these mice with antigen plus adjuvant. Popliteal lymph nodes were then removed and the capacity of the T cells to proliferate in response to antigen on APCs was analyzed. T cells obtained from anti-p-treated mice were incapable of proliferating in response to antigen, even though they maintained their mitogen responsiveness. In addition, the APC activity of cells from the anti-p-treated mice was normal, as demonstrated by their ability to stimulate T cells from immunized mice. In contrast, the reciprocal experiment using T cells from anti-p-treated mice and normal mouse APCs was negative, demonstrating that the deficiency was due to a lack of T cell priming. These results were confirmed and extended in a second series of experiments which were carefully controlled for the presence of contaminating cells and clearly demonstrated a requirement for B cells in the priming of T cells in mvo (246). One alternative interpretation of these experiments is that a defect exists in the handling of antigen by macrophages or dendritic cells in anti-p-treated mice. Unless this is the case, results of the above experiment seem to contradict the conclusions of Steinman and associates (141, 142) that dendritic cells are the only cells which can activate a naive T cell. An alternative hypothesis which is supported by the work of Ron and Sprent (247) is that dendritic cells initiate the activation of naive T cells, but that B cells are necessary to further stimulate or clonally expand these T cells. Abbas and co-workers(248) have also analyzed cells from anti-p-treated mice in an antigen-specific system. In their experiments, TNP-ABCs were adoptively transferred into the treated mice in order to prime T cells in vivo. This approach permits a more direct assessment of the role of B cells in priming T cells, since it is possible to compare specific versus polyclonal T cell responses. A high level of antigen-specific reconstitution was found. Nevertheless, in such experiments there was a small but reproducible T cell response to an unrelated antigen which was attributed to nonspecific binding of antigen to B cells of irrelevant specificity (Lea,not sIg mediated) and subsequent presentation to T cells. However, these data could also support the notion of an initial round of
24
ELLEN S. VITETTA ET A L .
presentation of antigen by dendritic cells followed by clonal expansion of those T cells which can interact with, and activate, antigen-presenting B cells. The antigen-presenting capacity of germinal center B cells has also been examined by Tew and co-workers (249), who studied the fate of injected antigen in immune mice and demonstrated that the antigen was very rapidly bound to circulating antibody to form immune complexes which were transported to the draining lymph nodes. In the nodes, complexes were found on the surface of FDCs. These complexes were not processed, and with time the tips of the dendritic processes pinched off and formed small immune complex-coatedbodies (icoccomes). These icoccomes were endocytosed by both B cells and macrophages, which subsequently appeared to act as APCs in the germinal center. In a very recent study, germinal center B cells were primed in muo, highly purified, and used as APCs in Vitro. It was found that 1-8 days following secondary inoculation of mice with OVA, germinal center B cells were still capable of presenting antigen to T cells in the absence of additional antigen pulsing. Therefore, germinal centers may retain antigen for long periods of time using FDCs or icoccomes as antigen reservoirs. It remains unclear whether this process occurs in a primary response where immune complexes are limiting, and what role this pathway plays in the activation of T cells in mvo. 111. Helper T Cells
In the preceding section we discussed how B cells, macrophages, and other APCs process and present antigen, in association with class I1 molecules, to T cells, leading to their activation. Activated T cells play a fundamental role in the regulation of humoral and cellular immune responses through direct cell/cell contact and/or release of lymphokines. Three types of functionally distinct T cells have been described: Th cells, Ts cells, and Tc cells. T h cells mediate a variety of immunological functions and generally regulate the immune response. Th cells mediate delayed-typehypersensitivity (DTH) and help for B cellmediated antibody responses, and are involved in the activation of Tc cells. Ts cells generally down regulate the immune response and Tc cells are responsible for killing virus-infected cells, tumor cells, and other cells expressing foreign antigens on their surface. Over the past 15 years, T cell subsets which mediate specific functions have been identified and their interactions have been studied. The picture that has emerged is complex and incomplete, but supports the contention that (1) T cells mediate their responses by both cell/cell contact and by secreted
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25
lymphokines; (2) activation of T cells is related to the type of antigen and its route of administration; and (3) the regulation of T cell activation and proliferation is controlled in part via lymphokine feedback circuits. The delineation of subset-specific T cell surface markers by Cantor and Boyse (250) contributed three important concepts to immunology: (1) functional heterogeneity exists among T cells; (2) functionally different T cells can be distinguished based on their expression of cell surface markers ( T cells that help antibody responses are of the Ly-1+2-3phenotype in mice and CD4+ in humans, while T cells that mediate cytotoxicity or suppression are Ly-1-2 3 or L3T4- in mice and CD8+ in humans); and (3) diversification of T cell function takes place before, and independently of, antigenic exposure. +
+
A. T h CELLHETEROGENEITY Studies by Marrack and Kappler (251) and Janeway (252) suggested that additional heterogeneity existed among Th cells that provided help to B cells for antibody production. A number of studies (244, 253-257) later confirmed that T h cells were functionally heterogeneous and that two main types could be distinguished based on the type of help provided to B cells (251, 253, 254, 257). In general, one type of T h cells was MHC-restricted, carrier-specific,and augmented antibody responses via cognate interactions with B cells. The other type of T cell cooperated with B cells through the generation of “nonspecific soluble factors” and could generally activate multiple clones of B cells, irrespective of their antigen specificity. Both types of T h cells acted synergistically.A number of characteristics distinguished these two types of T h cells. Tada et al. (253) reported that T h cells of the first type were nonadherent to nylon wool and did not react with antisera raised against molecules encoded or controlled by the I region (class I1 antigens) of the MHC. The other type of T h cell was nylon-wool adherent and reactive with such antibodies. Imperiale et al. (257) reported that T cells (generated from the lymph nodes of KLH-primed mice) cultured at low density corresponded to the first type, whereas the second type was preferentially generated in cultures of higher density. In the last few years, with the development of techniques to clone and propagate functionally active and antigen-specific Th cells in vitro (258), their heterogeneity has become even more apparent. Kim et al. (259) studied the ability of a series of antigen-specific I-A-restricted T h cell clones to help B cells that secreted T-15 idiotype-positive or T-15 idiotypenegative antibodies. They reported the existence of four types of clones: the first two induced B cells to secrete antiphosphorylcholine (PC)
26
ELLEN S. VITETTA ET A L .
antibody, although the resultant antibodies induced by the two clones varied in the proportion of T-15 idiotype-positive antibodies; the third type of clone could induce B cell proliferation but not antibody secretion and actually inhibited the Ig secretion induced by other types of clones. The fourth type of clone was autoreactive, inducing B cell proliferation and antibody secretion in the absence of antigen. It has recently been reported that the pattern of lymphokines secreted by clones of established T h cells can delineate two types of functionally distinct cells (Table 11). Mosmann et al. (75) studied a large panel of clones of LST4+ MHC-restricted T h cells raised against a variety of antigens in a number of mouse strains. Virtually all clones fell into one of two groups, depending on the pattern of lymphokines secreted after mitogenic or antigenic stimulation and on the type of “help” they provided to B cells. Cells from one type of clone, designated as Thl, secreted IL-2, interferon-y (IFN-y), and IL-3, but not IL-4, and some Thl clones (but not others) could help B cells. The other type of clone, Th2, secreted IL-4, IL-5, and IL-3, but not IL-2 or IFN-y, and provided excellent help to B cells to induce the secretion of IgGl and IgE. This study not only demonstrated that functional subsets of T h cells could be distinguished based on their pattern of secreted lymphokines, but supported the notion that the differential secretion of lymphokines was at least partially responsible for the functional differences between the two types of Th clones. Subsequently, the list of lymphokines produced by each type of T h cell has been extended (260) (see Table 11). An alternative classification, proposed by Bottomly and colleagues (259, 261, 262), divides T h cells into “helper” and “inflammatory” subtypes. These TABLE I1 LYMPHOKINES SECRETED BY DIFFERENT MURINEThl AND Th2 CLONESa Lymphokine IL-2 IFN-./ LT IL-4 IL-5 IL-3 GM-CSF TNF LT
Thl
Th2
+ + +
-
-
-(?)b
+ + + +
+ + + +
f ( < Thl) -
OFrorn Cherwinski et al. (260). + (263).
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27
two subtypes are for the most part equivalent to the Th2 and T h l subtypes, respectively, described by Mosmann et al. (75, 263). Finally, it should be noted that rare clones have been described which secrete lymphokines usually produced by only one or the other type of clone (263a; R. Fernandez-Botran, unpublished observations). Thus, it remains possible that either the delineation of all clones into T h l or Th2 types is generally, but not always, the rule, and/or that T h l and Th2 clones represent T h cells “frozen” at different stages of differentiation, i.e., that normal T h cells of one type can give rise to T h cells of the other type. Hence, these rare clones may represent “transitional” cells. B. FUNCTIONAL DIFFERENCES BETWEEN T h l AND Th2 CELLS Evidence has accumulated that clones representing the T h l and Th2 types of T h cells defined by Mosmann et al. (75) are functionally different. Cher and Mosmann (264) reported that only Thl cells can mediate antigen-specific DTH reactions. Other studies have analyzed the relationship between the nature of B cell help and the type of lymphokines secreted by T h cell clones (75, 260, 261, 264). As mentioned above, in the original paper describing the classification of T h cells into Thl and Th2, both subtypes were able to provide help to B cells for antibody responses, although Th2 clones were better helper cells than Thl clones. In this regard, Killar et al. (261) studied three functional subtypes of T h clones based on their ability to induce an in vitro antihapten antibody response to hapten-carrier conjugates. They reported that clones that failed to produce IL-4 could not provide help to antigen-specific B cells. If the IL-4-secreting T cells correspond to the Th2 cell type (helper), then the T h cells that failed to provide help would be of the Thl type (inflammatory), based on their failure to produce IL-4 (and their ability to secrete IFN-y). In contrast to these findings, Stevens et al. (265) reported that some clones of Thl cells can induce TNP-ABCs to secrete IgM, IgGS, and IgGz, antibody. Thus, there is a discrepancy with regard to the role of Thl cells in providing help to B cells that will subsequently secrete antibodies. Two possible reasons for this discrepancy are as follows: (1) IFN-y (secreted by activated Thl cells) often provides negative signals to B cells (266-271); it is unclear why IFN-y is sometimes, but not always, inhibitory. It is possible that different clones of Thl cells secrete different amounts of IFN-y and that the high secretors prevent B cell help; (2) B cells which respond to Thl clones may represent distinct subsets (e.g., memory versus virgin B cells) or cells at different stages of activation. In light of the last point, Killar et al. (261) used small splenic B cells, Stevens et al. (265) used TNP-ABCs prepared by rosetting, and Mosmann et al. (75) used LPS-activated B cells. A number
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of studies have now suggested that the ability of Th cells to help B cells is, in fact, dependent on the activation state of the B cells. Bottomly and colleagues (259, 261, 262) have reported that both types of clones (ThMdammatory and Th2/helper) are capable of mediating polyclonal B cell responses, whereas only Th2/helper clones are able to mediate antigen-specific responses. Moreover, they have reported that antigenspecific responses are elicited from small B cells, whereas polyclonal responses are elicited from large B cells. Taken together, the above studies suggest that clones of Th2 cells, possibly because of their ability to secrete IL-4 and IL-5 (260, 261), are probably responsible for the major portion of the helper activity mediated by T h cells. The reason for the conflicting evidence concerning the helper activity of Thl clones remains to be clarified.
MARKERSOF THE DIFFERENT T h CELLSUBTYPES C. SURFACE Although the characteristic pattern of lymphokine secretion can be used as a functional “marker” to distinguish murine T h l and Th2 cells (75, 260), no cell surface marker has been described that is present exclusively on one type (Thl or Th2) of murine T h cell. However, antibodies have been reported to distinguish between functionally different T h cells in several species. Arthur and Mason (272) reported that the antibody MRC-OX22 could separate rat Th cells into those responsible for the majority of IL-2 production (OX22+) and those responsible for B cell help (OX22-). MRC-OX22 antibodies recognize an epitope on the common leukocyte antigen or T-200 molecule (CD45R) (273). Based on recent reports, antibodies recognizing epitopes on human (274) and mouse (263, 275) CD45R molecules also appear to distinguish between functionally different T h cells. Birkeland et al. (275) have used two monoclonal anti-CD45R antibodies recognizing epitopes on the T-200 molecule to stain normal T cells and clones of T h l and Th2 cells in an indirect immunofluorescence assay. The different subtypes of clones stain to different degrees, i.e., Th2 clones stain with greater intensity than do Thl clones. In addition, another anti-CD45R antibody has been described which distinguishes functionally different murine T h cells based on their density of the reactive T-200 epitopes. “CD45R-low”and “CD45R-high’T cells produce IL-4 or IL-2 and IFN-y and correspond to Th2 and Thl cells, respectively (263). Recently, Clement et al. (276) have studied the relationship between two human CD4 + T h cell subsets defined by reactivity with monoclonal antibodies (mAbs) directed against the CD45R (T-200)molecule. They have reported that the CD45R+ and CD45R- subsets, corresponding to “suppressor-inducer” and T h cells, respectively, are not composed of fully mature and functionally stable
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T cells, but that they represent cells at different stages of maturation in a postthymic activation-dependent differentiation pathway. Although the ontogenetic relationship between CD45R+ and CD45R- T h cells is not known, these findings are not inconsistent with the notion that antigen or other factors may cause the conversion of one T h cell type into another. If this were the case, there would be a predictable sequence of lymphokines secreted during an immune response and these lymphokines would regulate the order of Ig isotypes secreted and the types of effector cells activated.
D. THEPROLIFERATIVE RESPONSE OF CLONES OF T h l AND Th2 CELLS T h cell clones do not constitutively secrete lymphokines but are dependent upon antigenic or mitogenic stimulation to do so (75, 260, 277). Another important consequence of T h cell activation after antigen presentation is the acquisition of a state of “competence,” which is characterized by the responsiveness of the T h cells to the proliferative effects of some of the secreted lymphokines, particularly IL-2 (277-279) and IL-4 (277, 280). T h cells are stimulated by the binding of either anti-TcR antibodies or antigedclass I1 (on an APC) to their receptors (138, 281). This results in the induction of lymphokine secretion and the expression of cell surface receptors (R) for IL-2 (IL-2R) and IL-4 (IL-4R) (282, 283). The secreted lymphokines act via autocrine and paracrine mechanisms to induce the proliferation and expansion of the activated T h cells as well as proliferation and differentiation of the B cells which they help (277, 280, 284-287).
E. REGULATION OF THE ACTIVATION OF T h l AND Th2 CELLS The different types of T h cells appear to differ in their sensitivity to the stimulatory or inhibitory effects of a variety of lymphokines (IL-4, IL-1, and IFN-y) (280, 287, 288), raising the possibility that lymphokinemediated circuits regulate the activation, expansion, and eventual down regulation of each particular type of T h cell. In this regard, IL-2 was originally defined as a lymphokine which mediated the proliferation of activated T cells (289), and IL-4 was defined as its counterpart for activated B cells (290). Following the generation of a mAb against IL-4 [llBll (291)], and the cloning and expression of IL-4 cDNA (292, 293), a number of laboratories reported that IL-4 could also mediate the proliferation of some T cell lines (293-297) or resting T cells [in combination with phorbol myristate acetate (PMA)] (298). Moreover, it was demonstrated that proliferation was not mediated through the production and utilization of IL-2, since neither anti-IL-2 nor anti-IL-2R
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antibodies were able to block the IL-4-mediated proliferation (277, 294, 296). The delineation of the different Th cell subsets by Mosmann et al. (75, 260) and the characterization of their lymphokines prompted investigations of how antigen-induced proliferation was mediated in Th2 cells, which were reported to lack the ability to secrete IL-2. A number of laboratories (277, 285, 286) reported that when stimulated in vitro by antigen on APCs, Th2 cells could proliferate in response to IL-4 in an autocrine fashion. In Thl cells, IL-2-mediated proliferation was also an autocrine event (285). Fernandez-Botran et al. (277, 280) reported that activated Th2 cells could also utilize IL-2 as a paracrine growth factor and that their responsiveness to both IL-2 and IL-4 increased after antigenic stimulation. These results suggest that the proliferation of one T h cell type can be modulated by lymphokines secreted by the other T h cell type. Kurt-Jones et al. (288) and Greenbaum et al. (287) have studied IL-2and IL-4-mediated proliferation of “activated’ T h clones. They reported that both clones respond to IL-2, but that Th2 cells were more responsive to IL-4 than were Thl cells. Thl cells did not respond to IL-4 except in the presence of suboptimal concentrations of IL-2 (287) and Th2 cells required the presence of IL-1 in order to respond to IL-4 (287, 288). IL-1 had no effect on Thl cells. In a recent report, Fernandez-Botran et a2. (280) reinvestigated the responsiveness of both types of T h cells to IL-2 and IL-4 as a function of the time elapsed after antigenic stimulation. Both Thl and Th2 cells respond to IL-4 and IL-2 shortly after antigenic stimulation and responsiveness to both lymphokines decreases with time after activation. Thl clones lose their responsiveness to IL-4 more rapidly and to IL-2 more slowly than do Th2 clones. In agreement with previous reports (287, 288), Th2 cells are more responsive to IL-4 than are Thl cells. When both IL-2 and I L 4 are added to cultures of Thl or Th2 cells, the proliferative response of both cell types is synergistic, suggesting that the presence of both lymphokines might be required for an optimal response. No requirement for IL-1 on the proliferation of Th2 cells in response to either IL-2 or IL-4 was identified, suggesting that there might be heterogeneity among Th2 cells with regard to their requirement for IL-1. Thl-derived IFN-y also has a regulatory effect on the proliferative response of Th2 cells. IFN-7 is generally inhibitory for IL-4-mediated activities on B cells (266-271). IFN-y also inhibits the IL-2- and IL-4-mediated proliferation of Th2, but not T h l cells (280, 299). Table I11 summarizes the responsiveness of the two T h subsets to different lymphokines. In summary, a number of mechanisms might exist in vivo whereby the initial stimulation of one or both types of Th cells could be amplified
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TABLE 111
RESPONSIVENESS OF Th SUBSETS TO DIFFERENT LYMPHOKINES' Lymphokine
Thl
Th2
IL-1 IL-2 IL-4 IFN--/
++ +
-
+ ++ ++ +
-
Assay (Costimulation with IL-2 and IL-4) (Proliferation) (Proliferation) (Inhibition of IL-2- and IL-4-mediated proliferation)
"From Fernandez-Botran e t al. (280).
and subsequently down regulated. T h cells would become stimulated after antigen presentation by APCs, leading to lymphokine secretion and the expression of receptors for these lymphokines. The secreted lymphokines would then act via autocrine or paracrine mechanisms to induce the expansion of clones of activated T cells and proliferation and differentiation of activated B cells. Eventually, when antigen concentrations decrease, T cell activation would become self-limiting by down regulation of surface IL-2Rs and IL-4Rs and by the cessation of lymphokine secretion. In addition, it has become clear that lymphokines produced by one T h cell type (i.e., IFN-7) can down regulate the growth of the other T h cell type (280, 299). F. T h l AND Th2 CELLSin Vim The characterization of the two types of T h cells has been carried out in vi'tro using established clones of cells (75, 260, 261, 264). It is therefore possible that variables in the techniques used for isolating, growing, and cloning T h cells might select clones that are not representative of their in mvo counterparts. On the other hand, since the phenotype of these T h clones is stable and since most clones fall into one of the two categories described by Mosmann et al. (75), it is likely that they are representative of their normal counterpart in vivo (263, 300, 301). Indeed, studies correlating the phenotype of normal T cells (CD45R-low and CD45R-high) with their pattern of lymphokine secretion and other functional characteristics provide strong evidence that analogs of T h l and Th2 cells exist in vivo (263, 275). However, as mentioned previously, it is still not clear whether Thl and Th2 cells represent two different lineages which differentiate from a common precursor, or whether they represent different stages of the T h cell in the same differentiation pathway, although there is evidence to support the latter possibility (276). Powers et al. (302) have used limiting dilution cultures to analyze the frequency of IL-2- and IL-4-secretingprecursors in naive
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and antigen-primed mice and have found that most freshly isolated Th cells produced IL-2 following primary stimulation. IL-4 production was detected only after further in Vitro stimulation. Similarly, Swain et al. (301) studied the generation of Thl and Th2 cells in vitro from freshly isolated T cells and reported that, in contrast to T h l cells, Th2 cells were dependent on priming before they secreted detectable levels of IL-4 or IL-5. They have suggested that precursors of Th2 cells require a maturation event before they can be stimulated to secrete IL-4 or IL-5. The delayed development of IL-4-producing cells (Th2) after repeated antigenic exposure suggests a potential role for Th2 cells in the generation of immunologic memory and/or in the immune response against antigens that are persistent or frequently encountered (302).
G. T CELL-MEDIATED REGULATION OF ISOTYPE SWITCHING IN B CELLS - MOLECULAR AND CELLULAR CONSIDERATIONS Since the early 1970s, it has been recognized that T cells influence the class of Ig secreted by B cells in response to TD antigens (34, 303-310). IgM and IgG3 responses are relatively thymus independent, whereas IgGl, IgGz, IgA, and IgE responses require T cells and their secreted lymphokines. These conclusions were deduced from experiments in which animals that were either carrier primed or lacked a thymus were injected with antigen. Carrier-primed mice made significant IgG responses whereas athymic n u h u mice did not (35, 311, 312), implying that carrierprimed T cells were necessary for switching. It was subsequently reported that an IgG response could also be induced in the presence of an “allogeneic” effect (74, 313-315). In contemporary terms, this finding suggests that T cells and their lymphokines play an important regulatory role both in the secretion of IgG antibodies and in the generation of immunological memory. During the past decade, numerous experiments have elucidated the organization and expression of the Ig constant region (CH) genes on the chromosomes of mouse and man (316). Not only has the 5 ’ -3’ order of the CH genes’been assigned in both species, but some of the molecular events underlying isotype switching have been identified. Thus, B cells can switch, at the DNA level, from the production of a functional transcript encoding contiguous V, D, J, and CM segments (exons) to VDJ juxtaposed to other downstream CH genes (317), which in the mouse consist of y3, yl, y2b, y2a, E , and a (5’43’). In most cases, switching involves deletion of intervening intronic segments of DNA as well as intervening exons encoding other CH genes. Hence, by switch recombination and deletion, a new constant region gene is juxtaposed to VDJ. Initial reports suggested that proliferating B cells switched
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33
sequentially and stochastically from the expression of 5’ CH genes to other 3’ CH genes. However, Isakson et al. (318) first demonstrated that lymphokines strongly influenced the class of Ig secreted by LPSstimulated B cells, implying that switching was not entirely stochastic with regard to CH gene organization (i.e., 5’-3‘). More recent experiments have implicated T cell-derived lymphokines as mediators of directed switching to other isotypes of Ig as well (75, 268, 284, 318-322). To date, most experiments have used anti-Ig or mitogenstimulated B cells to study lymphokine-mediated switching. As described in a previous section, during the interaction of antigenspecific T and B cells, lymphokines are released by the activated T cells and these lymphokines can act not only on the cells involved in the interaction, but also on other cells as well. As noted earlier, Thl cells secrete IFN-y and IL-2, and Th2 cells secrete IL-4 and IL-5. With regard to these four lymphokines, a number of laboratories have demonstrated that B cells cultured with these lymphokines and with the mitogens LPS or purified protein from Salmonella typhimurium (STM) can selectively secrete certain classes of Ig (75, 268, 284, 318-323). For example, STM or LPS and IL-4 induce B cells to secrete IgGI (318, 323) and, at higher doses of IL-4, IgE (268, 322). IFN-y induces LPS-stimulated B cells to secrete IgGza(321), and IL-5 induces LPS-stimulated B cells to secrete IgA (300, 324). Therefore, at least with respect to IgGl, IgGea, IgA, and IgE in the mouse, the aforementioned lymphokines clearly play a major role in inducing B cells to secrete the isotypes indicated. A recent study by Stevens et al. (265) has placed some of these observations into the context of T cell/B cell interactions involving subsets of Thl versus Th2 cells which secrete lymphokines involved in class switching (265, 325). Using two KLH-specific T cell clones of the Thl or Th2 types, it was shown that the interaction between a TNP-ABC and a Th2 cell (that secretes IL-4 and IL-5) resulted in the production of IgGl, whereas interaction between a TNP-ABC and a Thl cell (that secretes IL-2 and IFN-y) resulted in the production of IgGza. During the interaction of TNP-ABC with Thl cells (which normally induce production of IgGz,), the addition of exogenous IL-4 resulted in the production of IgGl. Conversely, if B lymphocytes were incubated with Th2 cells (which normally induce the production of IgGl), the addition of an exogenous IFN-y failed to induce the production of IgGza but inhibited the IgGl-inducing effects of the Th2 cells. Since an individual TNP-ABC is capable of interacting with either a Thl or a Th2 cell (326), it can be postulated that the interaction between a B cell and a T cell (Thl or Th2) induces a state of responsiveness to many, but perhaps not all, lymphokines. Taken together with the results obtained using
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mitogen plus lymphokines, these results suggest that the class of Ig secreted by a B cell is dependent upon the type of T cell with which it interacts and the lymphokines which the activated T cell subsequently secretes. Although the relationships between T cells and their lymphokines and the molecular events involved in isotype switching have not yet been related in a cause-and-effectmanner, some insights have emerged from studies utilizing the Th2 cell-derived lymphokine, IL-4. IL-4 can induce mitogen-activated murine sIgG - B cells to secrete increased levels of IgGl and IgE and decreased levels of IgGs and IgGzb (268, 318). The enhanced secretion of IgGl is accompanied by an IL-4-mediated increase in the transcription of y l genes (327), and an increase in the precursor frequency of IgG1-secreting cells (328), suggesting that IL-4 induces sIgM+ B cells to undergo a heavy-chain class switch from IgM to IgGl. Although both mitogen and IL-4 are necessary for the switch to IgGl secretion, IL-4 is required very early in the culture, several days before switch recombination can be detected (318, 329). The mechanism by which IL-4 regulates isotype switching is unknown, but recent studies on the molecular basis of isotype switching may provide some clues. Analyses of murine hybridomas and normal murine B cells have provided strong support for the hypothesis that switch recombination is a directed event which occurs within the tandemly repeated elements of the same switch region on both the productive and nonproductive alleles (330-333). This directed switch might be due to the induction or activation of switch recombinases specific for each switch region. Recent work suggests that isotype switching is regulated at the level of accessibility of the switch regions to a common switch recombinase (334-337). Stavnezer et al. (338, 338a) have demonstrated in the IgM+ 1.29 B cell lymphoma line that the specificity of class switching correlates with hypomethylation and transcriptional activation of the corresponding germ line CH gene prior to switching. Recently, IL-4 has been shown to induce transcription of germ line Cy RNA in normal B cells before switch recombination access (338b). This induction is dosedependent and markedly uninhibited by IFN-7 (338b). Yancopoulos et al. (339) have also shown that Abelson-transformed pre-B cell lines, which switch spontaneously in Vitro to IgG2b, produce small germ line (“sterile”)y2b transcripts prior to switch recombination. Most recently, it has been shown that LPS induces the synthesis of germ line y2b transcripts in Abelson-transformedpre-B cell lines and in normal splenic B cells prior to y2b class switching (340). IL-4 inhibits the induction of these transcripts in splenic B cells (341). This correlates with the
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previous observations that IL-4 inhibits the secretion of IgGzb by LPSstimulated B cells (342). These studies have suggested an accessibility model for the regulation of isotype switching (342a), i.e., that the initial events in the process involve the opening up of the chromatin in the switch region, followed by transcriptional activation and eventual recognition of DNA segments by switch recombinases. Whether transcriptional activation actually directs specific recombination or is a manifestation of a more accessible chromatin structure remains to be determined. Nevertheless, the accessibility of switch regions is probably affected by DNA-binding proteins (343) that recognize elements in the switch regions with partial or perhaps complete isotype specificity. It will be important to identify and characterize such regulatory proteins and their corresponding sequence elements and to understand how they might be regulated by external stimuli such as lymphokines and mitogens. It is also possible that some lymphokines (e.g., IL-5) might have no effect on the switching machinery, but might instead induce secretion of Ig in activated postswitch B cells (those that already express sIgG or sIgA). Future studies will be aimed at relating T cells and their lymphokines to the molecular events leading to early changes in CH-encoded DNA and to switch recombination. Eventually, these concepts must be placed in a physiological setting to take into account how the Thl and Th2 cells that secrete different lymphokines are activated in vivo and how activation is related to the nature of the antigenic stimulus (e.g., parasite, virus, or bacterium). Finally, the anatomical site in which the T h cells reside must be considered. IV. Physical Interaction between T and 6 Cells
A. CELLULAR INTERACTIONS IN THE IMMUNE RESPONSE A large body of work spanning nearly two decades has established the basic requirements for, and consequences of, the interactions between immunologically relevant cells such as T h cells with macrophages and Tc cells with their target cells. The major findings are summarized as follows: 1. Resting T cells from unprimed mice express clonally distributed receptors which bind to processed antigen in association with class I or class I1 molecules (344). The class I or II/antigen complex must be presented on the surface of an APC.
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2. The antigen-specific, MHC-restricted binding of resting T cells to APCs leads to their activation, the release of lymphokines, and proliferation and expansion of the T cell clones (289). 3. Although any cell expressing the correct MHC-restricting elements and antigen can act as an APC, the major APCs in the immune system are dendritic cells (345, 346), macrophages (347, 348), epidermal Langerhans cells (349, 350), and B cells (162, 171, 351). 4. The binding of T cells to APCs is cation and temperature dependent (352,353) and may involve not only TcRs, but also other cell surface adhesion molecules on both cells, strengthening the binding affinity between the APC and the T cell. 5. The APCs and T cells (in the presence of antigen and MHCrestricting elements) form tight interactions (352,354-357) which, at least in the case of the Tc cell, facilitate the secretion of pore-forming proteins. 6. The activated T cells detach from the APC after a period of time and are able to initiate effector function, e.g., activated T h cells support antibody responses by resting B cells (358). 7. After their detachment from APCs, activated T cells can be maintained in a proliferating state by endogenously produced or exogenously added lymphokines (277, 280, 289). 8. Eventually, the activated T cells return to a resting state, probably as a consequence of both the depletion of lymphokines necessary for growth and the discontinued stimulation by APCs (278, 280). However, these resting T cells can be activated once again with antigenpulsed APCs (258). The requirements for the interaction between T and B cells are, in general, similar to those described above. Recent studies on T cell/B cell interaction are described in detail below. We will not address the issue of interactions among B cells or the activation of Ly-1 B cells and their possible involvement in autoantibody production. For discussions on these topics, the reader is referred to recent reviews (359-362). +
B. MODELSYSTEMS TO STUDY THE PHYSICAL INTERACTIONBETWEEN T AND B CELLS Both T and B cells are required for an immune response to a thymusdependent antigen (1, 2, 5, 16, 19). This observation led to the suggestion that the T and B cells physically interact, perhaps by antigen bridging. The first description of physical interactions between antigen-specific T and B lymphocytes was reported by Marrack et al. (363). Radiolabeled antigen-specific, I-region-restrictedT cell hybridomas were cultured with
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37
adherent antigen-pulsed B cell lymphomas or hybridomas. Nonadherent cells were removed and the percentage of T cells attached to the adherent monolayers was estimated by measuring radiolabel remaining in the culture wells. Adherence of T cells to B cells was both antigen specific and MHC restricted and occurred within 15 min. Two additional experimental models have made it possible to quantify the number of T and B lymphocytes which adhere to each other and have facilitated the elucidation of factors required for conjugate formation. Experiments by Kupfer et al. (364) showed that the formation of conjugates between B hybridoma cells (LK35.2) and antigenand class II-specific cloned T h cells (DlO.M.1) required antigen presentation by the B hybridoma cells and MHC-restrictedinteraction between the two cells. Cells of the D10 clone were specific for conalbumin and IaKdeterminants. The LK35.2 cells expressed both IaKand Iad determinants but were not conalbumin specific. In the presence of high concentrations of conalbumin, the LK35.2 cells were able to nonspecifically bind the antigen and present it to the D10 cells. When LK35.2 cells and D10 cells were mixed, centrifuged, and resuspended, tight physical interactions or conjugates were formed; the number of cells capable of interacting was quantitated microscopically. The formation of antigenspecific, MHC-restricted conjugates induced reorientation of the microtubule organizing center (MTOC) inside the helper T cell to face the site of T cell/B cell contact. When conjugates formed in an antigennonspecific, MHC-nonrestricted manner, random reorientation of the MTOC was observed. Sanders et al. (197) employed a fully antigen-specific system, utilizing a highly enriched population of B cells specific for TNP (TNPABCs) and T cells specific for the carrier portion (KLH or OVA) of the TNP-carrier. The T hybridoma cells recognized processed antigen in the context of I-Ad molecules on the antigen-presenting TNP-ABCs. Because of the differences in size between the T hybridoma cells and the normal B cells, conjugates could be studied at the light and electron microscopic levels. In this way, two types of interactions were described. One type involved the interdigitation of one or more microvilli from each cell type (i.e., loose interaction). Loose interactions occurred in the absence of antigen and, hence, were not immunologically specific. Antigen-nonspecific, MHC-nonrestricted interactions between T cells and macrophages have also been reported (354, 356, 358, 365-367); the rate of dissociation of cells in these conjugates is rapid (354, 365, 368). The second type of interaction between the T and B cells involved an extensive area of close contact between the two cells (i,e., tight interaction). Tight interactions occurred in an antigen-specific, MHC-restricted
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manner and the number of conjugates and the degree of close contact between the two cell types increased as a function of time. OUTCOME OF T CELL/BCELLINTERACTION C. THEFUNCTIONAL As reported by Sanders et al. (197), the functional importance of physical interactions between TNP-ABCs and T hybridoma cells is supported by two findings: (1) in the presence of the appropriate TD antigen (197), the T hybridoma cells induced the differentiation of TNP-ABCs into antibody-secretingcells; and (2) the number of TNP-ABCs forming tight interactions with T cells (197) was similar to, or greater than, the number of TNP-ABCs differentiating into antibody-secreting cells (369). These observations suggest that those B cells that form tight conjugates with T cells differentiate into antibody-secreting cells. Definitive proof of this hypothesis will require isolating conjugated versus unconjugated B cells to compare their ability to secrete antibody. FOR T CELL/BCELL D. BIOCHEMICAL EVENTS REQUIRED INTERACTION
1. Antigen Processing
As discussed in the previous section, the physical interaction between T and B cells is dependent upon the processing of native antigen by the B cells and presentation of processed, antigenic fragments or denatured antigen in association with class I1 molecules to TcRs. Following exposure of the B cells to antigen, a period of several hours is required before the B cells are able to conjugate to T cells. This suggests that antigen processing plays a rate-limiting role in conjugate formation. Supporting this hypothesis are the following data: (1) chloroquine, a lysosomotropic agent, inhibits the conjugation of TNP-ABC and T cells by 40% (197); (2) chloroquine inhibits MTOC reorientation in 50% of T cells that form conjugates (370); and (3) the rate of release of soluble radiolabeled antigen fragments from antigen-pulsed TNP-ABCs (C.D. Myers and E.S. Vitetta, unpublished observations) follows kinetics similar to those of conjugate formation between TNP-ABCs and T hybridoma cells (197). 2. Time and Temperature Conjugation between T and B cells is temperature dependent; a maximal number of conjugates forms at 37OC, fewer conjugates form at room temperature, and no conjugates form at 4OC (197). This implies that an energy-dependent event is involved. Once the B cells have processed antigen, their physical interaction with T cells occurs rapidly;
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39
a maximal number of T cell/B cell conjugates forms within 5 min (197). Rapid adherence of antigen-specific T hybridoma cells to monolayers of adherent B cell lymphomas and hybridomas has also been reported (358). In contrast to the rapid rate of adherence, the rate of dissociation of specifically conjugated T and B cells is slow, i.e., the half-life is 4 h (V. M. Sanders, J. W. Uhr, and E. S. Vitetta, unpublished observations). E. INTRACELLULAR EVENTS WHICHOCCURDURING T CELL/BCELL INTERACTION As discussed above, an important event that follows specific T cell/ B cell interactions is reorientation of the MTOC in the T cell to face the site of contact with the B cell (364, 370). This event is reminiscent of the reorientation of the MTOC and the Golgi apparatus in Tc cells interacting with target cells (371). As shown for the Tc cell/target cell interaction (371), reorientations direct secretory vesicles from the Golgi apparatus toward the area of cell/cell contact. It has been hypothesized that the reorientation of these intracellular components in the T h cells that conjugate to B cells may serve a similar function (364, 370). These changes may direct the release of lymphokines into the intracellular space between the conjugating cells such that small amounts of lymphokines can effectively stimulate the interacting B cells because of their close proximity. Hence, local transfer of lymphokines might not effect neighboring cells. A concomitant localization of the membrane-associated cytoskeletal protein, talin, occurs under the membrane of the T h cell participating in specific conjugation (370). A rearrangement of talin is associated with cell interactions that involve the membrane-associated proteins of the integrin family (372), e.g., LFA-1. The integrin family includes molecules which act as receptors for extracellular matrix molecules involved in cell/cell adherence (372); these molecules have a common /3 subunit (95 kDa) and a variable a subunit (372). F. MONOGAMOUS NATURE OF T CELL/BCELLINTERACTION
The tight physical interactions that occur between normal T and B cells invariably involve one B cell and one T cell (326). In contrast, a large T hybridoma cell sometimes binds to two different B cells. The monogamous nature of B cells was reported in recent experiments using TNP-ABCs which could bind to either Thl or Th2 cells (326). In competition studies, the number of cells of one type of T h cell (e.g., Th2) was kept constant in the presence of increasing numbers of the other type of T h cell (e.g., Thl). Although the same antigen-specific B cell could bind to either a Thl or a Th2 cell, during the course of these
40
ELLEN S. VITETTA ET AL.
studies it was observed that if one T h cell had bound to a B cell, another Th cell (of the same or different T h cell type) was unable to do so. One possible explanation for the monagamy of B cells is that membrane-associated class I1 molecules are clustered at the interaction site between the T and B cells and would not be available for interactions with other T h cells. However, two experiments have excluded this possibility: (1) immunofluorescence staining of T cell/B cell conjugates has demonstrated that class I1 molecules remain relatively uniformly distributed on the B cell surface (V. M. Sanders, J. W. Uhr, and E. S . Vitetta, unpublished observations); and (2) when TNP-ABCs were conjugated to antigen-specific T h cells, they could not conjugate at the same time to alloreactive T h cells (which recognize only class I1 molecules) (326). This indicates that clustering of class I1 molecules at the interaction site does not occur to any great degree and is not responsible for the monogamy of B cells. It is possible, however, that other accessory molecules on B cells do cluster at the interaction site (e.g., LFA-1) and are not available to facilitate interactions with other T h cells. In any case, this does not rule out the possibility that, after the dissociation of the TNP-ABC from the T h cell, the TNP-ABC can subsequently bind to another Th cell [which is the case for T h cells and macrophages (368, 373) and Tc cells and target cells (357)l. G. CELLSURFACE Ig AS AN ACTIVATING MOLECULE As noted earlier in this review, because B cells express monoclonal sIg molecules, antigen-specific B cells are more efficient than macrophages in capturing specific antigen and presenting it in a processed form to T cells (169, 184, 187). However, the role of sIg per se in activating B cells has remained controversial. Anti-Ig stimulation of splenic B cells or antigen stimulation of TNP-ABCs induces a change in phospholipid metabolism (374-377), a decrease in membrane potential (378, 379), CaZ+ fluxes (380), and an elevation in the levels of mRNA for the c-myc protooncogene (381). These findings imply that B cells can be stimulated via sIg in the absence of T cells. However, stimulation of resting splenic B cells (382) with antigen alone is insufficient to induce their proliferation or differentiation into antibody-forming cells. The differentiation of resting TNP-ABCs into antibody-secreting cells requires the presence of T h cells in addition to antigen (383). In this regard, Julius et al. (384) cultured resting B cells with T h cells specific for B cell membrane molecules other than sIg [i.e., the male antigen or H-2 (class I) molecules]. In such experiments, antibody secretion did not occur unless anti-Ig antibodies were added to the cultures. These results showed that two signals were required for the activation of resting B cells. One signal was derived from the interaction between T h cells
CELLULAR INTERACTIONS
IN THE HUMORAL IMMUNE RESPONSE
41
and B cells and the other was provided by anti-Ig antibodies. In a study by LoCascio et al. (385), SRBC-specific “resting” B lymphoma cells, CH12, were cultured with T h cells which were specific for H-2 (class I1 molecules) on the CH12 cells. The B cells cultured with Th cells secreted antibody in the presence, but not in the absence, of specific antigen, supporting the conclusion that both sIg and cell/cell contact are involved in the induction of an antibody response. H. CELLSURFACE MOLECULES INVOLVED IN T CELL/BCELL INTERACTION Cell surface molecules involved in the formation of conjugates between T and B cells include L3T4, LFA-la, Thy-1, and TcRs on the T cell, and class I1 molecules and LFA-la on the B cell. This conclusion has been deduced from the observation that mAbs directed against these molecules inhibit the formation of conjugates in a concentrationdependent manner (197, 386). Furthermore, immunofluorescence staining shows localization of L3T4 and TcRs on the T cell at the site of specific conjugation with the B cell (387). Finally, antibodies directed against these molecules modulate both in vi2ro and in vivo functional responses of the B cells. 1. Class 11 Molecules
As discussed earlier, it has been postulated that antigenic fragments processed by APCs associate with class I1 molecules (235) and are recognized by MHC-restricted, antigen-specificT h cells (76, 78, 80-84). Hence, the production of antibody in response to a TD antigen is restricted by class I1 molecules on B cells (76-79, 388). In this regard, the experiments of LoCascio et al. (385) also demonstrated the necessity for class I1 compatibility between B cells and T cells activated by TD antigens and, in addition, implicated class I1 molecules as transmembrane signaling molecules. However, the state of activation of the B cell can determine its requirements for a class 11-restrictedT cell/B cell interaction in the induction of an antibody response (389-391). Resting B lymphocytes require a class 11-restrictedinteraction whereas activated B cells require only the lymphokines produced by T cells (previously activated by another APC in a class 11-restrictedmanner). In addition, recent observations have challenged the absolute requirement for the involvement of class I1 molecules in the activation of resting B cells (392-395); these findings will be discussed in more detail in a later section. A number of approaches have been used to show that class I1 molecules are involved in the events associated with the initial physical interaction
42
ELLEN S. VITETTA ET A L .
between antigen-specificT and B cells. Kupfer et al. (364, 370) have shown that specific reorientation of the MTOC occurs when class 11-compatible, as opposed to class 11-incompatible,B cells conjugate with T cells. Likewise, Sanders et al. (197, 386) reported that anti-class I1 antibodies inhibit the formation of antigen-specific tight interactions between T and B cells. Modulation of the level of expression of class I1 molecules on B cells has been accomplished and these cells have been evaluated for their ability to conjugate to T cells (396). The results of such experiments extended earlier observations that the density of class I1 antigens on B cells is directly related to the frequency of successful T cell/B cell interactions, leading to the activation of B cells (397-399). Hence, pretreatment of TNP-ABCs with IL-4 increased by twofold both the density of class I1 molecules on the TNP-ABC and the number of antigen-pulsed TNP-ABCs capable of conjugating to T cells (396). The same twofold increase in the number of IL-4-pretreatedTNP-ABCs differentiatinginto antibody-secretingcells was observed (282, 396). Thus, both the presence and the density of class I1 molecules on the B cell surface are important in determining the number of successful T cell/B cell interactions that occur. There is controversy as to whether the level of glycosylation of class I1 molecules on a resting B cell is a relevant factor in the association of T and B lymphocytes (163, 400). In one case, Frohman and Cowing (163) reported that class I1 molecules on resting B cells versus adherent APCs were glycosylated to different degrees and that this level of glycosylation explained the ability of adherent APCs, as opposed to resting B cells, to present antigen more effectively. By altering the level of glycosylation of surface molecules on resting B cells by neuraminidase treatment (which removes terminal N-acetylneuraminic acid residues), resting B cells became more effective APCs even though they remained in a resting state. The authors concluded that changes in the level of glycosylation of class I1 molecules on resting B cells are responsible for enhanced interactions between T and B cells. However, they did not prove that class I1 molecules, rather than other cell surface glycoproteins, mediated the observed effect. In this regard, and in contrast to the above conclusions, Krieger et al. (400) showed that planar membranes, into which class I1 molecules obtained from resting (“highly glycosylated’) and activated (“poorly glycosylated”) B cells had been inserted, could be pulsed with antigenic fragments and used as artificial APCs. Levels of glycosylation were indirectly evaluated by the ability of neuraminidase to enhance the ability of the intact B cells to present antigen. Results showed that both types of class 11-containingplanar membranes were equivalent in their antigenpresenting capacity. These studies suggest that glycosylation of cell surface
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43
molecules other than class I1 molecules may be important in T cell/B cell interactions and the subsequent antigen-presenting capacity of B cells. The role of class I1 molecules as signal-traducing molecules in B cells has remained controversial (401). The major approach that has been used to address this issue has been to utilize anti-class I1 antibodies to stimulate B cells. Although some monoclonal anti-class I1 antibodies can stimulate resting B cells in a T-independent manner, treatment of mitogen-stimulated B cells can result in either inhibition (402-404) or stimulation of antibody secretion (405). Anti-class I1 antibodies also inhibit both anti-Ig-induced B cell proliferation (406) and differentiation induced by antigen and T cells (407, 408). Cambier and colleagues (409,410) have shown that anti-classI1 antibodies can stimulate the translocation of protein kinase C (PKC) from the cytoplasmic pool to a nuclear membrane-associatedpool, suggesting that class I1 molecules have a signaling function. This observation may also explain the inhibitory effects of anti-class I1 antibodies on mitogen-induced B cell responses, since a class 11-inducedtranslocation of PKC to the nuclear membrane would deplete cytoplasmic stores of PKC, which are usually translocated to the plasma membrane pool after mitogen stimulation (411). In summary, class I1 molecules on B cells are involved in the MHCrestricted interactions between antigen-specific T and B cells and may be involved in the transduction of activating signals to the B cell. Modulation of the density of class I1 molecules on the B cell influences both the number of B cells capable of conjugating to T cells and those B cells differentiating into antibody-secreting cells. 2 . LFA-1
Involvement of LFA-1 in cell/cell adhesion has been demonstrated primarily in the interaction between Tc cells and target cells (412, 413). Recently, LFA-1 has been implicated in interactions between T and B cells (197). LFA-1 is a heterodimer consisting of an a! subunit of 180 kDa noncovalently associated with a subunit of 95 kDa (414, 415). LFA-1 is expressed on resting and activated T and B cells (416). Springer and colleagues (417, 418) have reported that the intercellular adhesion molecule-1 (ICAM-1) may be the receptor for the LFA-1 molecule. An antibody directed against the ICAM-1 molecule inhibits LFA-1-dependent adhesion events and identifies ICAM-1 on many cells of hematopoietic and nonhematopoietic origin. T D antibody responses can be inhibited by anti-LFA-1antibodies (419-422) and it has been postulated that this is due to an effect of LFA-1 on intercellular interactions. The relative importance of LFA-1 as an adhesion molecule may be related to the avidity of the interaction between the antigedclass I1 molecule and the
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ELLEN S. VITETTA ET AL.
TcR. Golde et al. (423) demonstrated that LFA-1 is strongly involved in the interaction between T cells and APCs only if the avidity of the TcR for processed antigedclass I1 molecules on the APC is low. These conclusions were deduced from experiments in which cells from a panel of T hybridomas (which released IL-2 in response to antigedclass 11) were treated with anti-LFA-1 antibodies. The ability of anti-LFA-1 antibodies to inhibit IL-2 release was inversely related to the binding avidity of the TcR to antigedclass I1 molecules on the APCs. Along the same lines, when TNP-ABCs from unprimed mice were used in the T cell/B cell conjugate assay, pretreatment of either the T cells or TNPABCs with anti-LFA-la antibody inhibited the formation of conjugates (197). In contrast, when TNP-ABCs from primed mice were used, pretreatment of the T cells, but not the TNP-ABCs, with anti-LFA-la antibody inhibited the formation of conjugates (386). A discussion of these results is presented in a later section of this review. Anti-LFA-1 also inhibits cluster formation between autoreactive T cells and neuraminidase-treated resting B cells (424), an observation which is consistent with the idea that autoreactive interactions of this type may be of low affinity and hence may require accessory adhesion molecules. A possible signaling function for LFA-1 has been reported by Mishra et al. (425). In this study, a mAb recognizing LFA-la (G-48) mimicked many of the actions of IL-4 on B cells. G-48 induced increased levels of expression of class I1 antigen on resting B cells, augmented the proliferation of anti-&stimulated B cells, and in insolubilized form, induced IgGl secretion in LPS-activated B cells. Of several anti-LFA-la antibodies tested, G-48 was superior in its stimulatory functions (W. Lee and E. S. Vitetta, unpublished observations). In this regard, this particular antibody also enhances, rather than inhibits, conjugate formation (V.M. Sanders and E. S. Vitetta, unpublished observations). A similar enhancement of conjugate formation was observed if TNP-ABCs were treated with IL-4 in the presence of antigen prior to conjugation with T cells (396). These data suggest a possible signaling function associated with antibodies directed against this particular epitope of LFA-la. In summary, LFA-1 participates in T cell/B cell interactions. The degree of participation of LFA-1 in these interactions may be influenced by not only the avidity of the TcR for antigedclass 11, but also by the ability of B cells to process antigen and express high densities of class I1 molecules. LFA-1 may also be involved in the transduction of activating signals to the B cell. 3. L3T4 (CD4) L3T4, the murine equivalent of human CD4, is a T cell-associated surface molecule which may be involved in the recognition of class I1
CELLULAR INTERACTIONS IN THE HUMORAL IMMUNE RESPONSE
45
molecules on the surface of APCs (426-429), and, as such, plays an important role in strengthening interactions between T cells and other cells (430). In vitro, L3T4 plays an essential role in both T h cell activation and T cell-mediated activation of B cell proliferation (426-428). In an attempt to elucidate the role of L3T4 in functional responses of T cells, it was shown that anti-L3T4 antibodies inhibit conjugate formation between T and B cells (197, 386). In addition, when conjugates between T and B cells form, L3T4 localizes to the site of interaction, as does the TcR (387). Marrack et al. (430) have shown that the requirement for L3T4, like LFA-1 (423), is inversely proportional to the avidity of the TcR for the antigedclass I1 complex on the APC. Thus, L3T4 functions primarily as an adhesion molecule: its natural ligand is probably the class I1 molecule on APCs (426-428). Recent data suggest that L3T4 may also have a signaling function. It has been postulated that L3T4 plays a direct role in the inactivation of T cells. A number of workers have reported that antibodies directed against L3T4 molecules inhibit T cell activation by conconavalin A or anti-TcR antibodies in the absence of TcR recognition of antigen in association with class I1 molecules (431-433). Thus, it has been suggested that L3T4 may function as a negative signaling molecule when the T cells interact with cells lacking the appropriate antigenic fragment in association with class I1 molecules. During such a nonspecific interaction between a T and B lymphocyte, the binding of L3T4 (on the T cell) to its putative ligand (class I1 molecules) on the B cell in the absence of an interaction between the TcR and antigedclass I1 may cause the T cell to dissociate from the B cell so that it can “sample” other B cells that might express the relevant antigedclass I1 complex. In summary, L3T4 participates in T cell/B cell interactions by serving primarily as an adhesion molecule. L3T4 may also mediate signals to T cells and be involved in mediating dissociation between APCs and T cells. 4 . The T Cell Receptor
The majority of T cell receptors are composed of disulfide-bonded a and 0chains, each of which has a molecular weight of 45,000-55,000 and both of which have transmembrane and cytoplasmic domains. Other T cell receptors are composed of y and 6 chains, but it is not yet clear how these receptors function. The a0 chains of the TcR recognize processed antigen and class I1 molecules on the surface of APCs (132, 134, 136, 206, 227). This is probably the key interaction which initiates specific conjugation between a T cell and a B cell. Kupfer et al. (387) have shown that TcRs localize at the site of contact between T and B cells. Crosslinking of TcRs with antireceptor antibodies in metroactivate T cells and
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ELLEN S. VITETTA ET A t
result in the directed release of lymphokines to the site of clustered TcRs (434). In addition, Taplits et al. (435) have shown that the secretion of enzyme-containing granules from T h cells exposed to anti-TcR antibodies occurs in an MHC-restricted, antigen-dependent manner and correlates with T cell proliferation. Taken together, these findings suggest that during the close interaction between T and B cells, clustering of TcRs results in T cell activation followed by the directed release of lymphokines and/or enzymes into the interaction site. 5. Thy-1
Thy-1 is expressed primarily on T cells although there is a report that activated B cells can also express Thy-1 (C.M. Snapper and W.E. Paul, personal communication). Involvement of the Thy-1 antigen in T cell/ B cell interaction was suggested by the finding that anti-Thy-1antibodies inhibit conjugate formation (197). In contrast to their ability to block conjugate formation, anti-Thy-1 antibodies activate T lymphocytes directly (436-439). In addition, Kroczek et al. (440)reported that antiThy-1 antibodies increased the concentration of intracellular Ca2 in T cells. Hence, anti-Thy-1 antibodies can activate T cells, but prevent T cell/B cell interaction. It would be informative to add anti-Thy-1 antibodies to cultures of T and B cells to determine whether specific antibody production can be inhibited. For example, it could be postulated that anti-Thy-1would inhibit the activation of resting B cells by T D antigens (by inhibiting T cell/B cell conjugation). At the same time, anti-Thy-1 might enhance the secretion of antibodies by lymphokine-responsiveactivated B cells (which had already undergone an interaction with T cells), since the T cells which had been activated by the anti-Thy1 antibody would be secreting lymphokines. +
I. CELLCONTACT VERSUS LYMPHOKINES IN T CELL/BCELL ACTIVATION The role of cell/cell contact versus the role of T cell-derived lymphokines in the activation of B cells by TD antigens has been addressed in several ways. In Vitro studies by Corbel and Melchers (441) have demonstrated that LPS-activated B cells can be induced to secrete antibody by the addition of soluble mediators. LeClerq et al. (442, 443) have shown that resting B lymphocytes can be stimulated to proliferate and produce antibody in the presence of T cell-derived lymphokines alone. In an attempt to distinguish between the requirement for cell/cell contact and the requirement for soluble mediators in the stimulation and differentiation of resting B cells into antibody-secreting cells, a
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47
number of model systems have been developed. Melchers and colleagues (389, 444, 445) were among the first to study the early events involved in B cell activation by TD antigens. Blast transformation of small, resting B cells required antigen-specific, MHC-restricted interactions between B and T h cells. Differentiation of B cell blasts into antibody-secreting cells required antigen-nonspecific,MHC-unrestrictedsoluble factors produced by T h cells which had interacted specifically with MHCcompatible adherent cells in the presence of specific antigen. In this regard, Singer and Hodes (446, 447) reported that B cells expressing the cell surface antigen, Lyb-5, responded to T cell-derivedlymphokines alone. In contrast, Lyb-5-negativeB cells required MHC-restricted T cell interactions. These results indicate that some cell contact-mediated event was required to induce responsiveness of the Lyb-5-negativecells to T cellderived lymphokines. However, the relationship between Lyb-5-positive and Lyb-5-negative cells remains unclear. In addition, activation of Lyb-5-positive cells is MHC restricted in other experimental systems (398, 448, 449). Noelle et al. (383) cultured TNP-ABCs with antigen-specific, MHCrestricted T lymphocytes and antigen for 24 h. The T cells were then eliminated with anti-Thy-1antibody and C’ and the remaining B cells were cultured with lymphokines. Only the B cells which had interacted with the T cells became responsive to T cell-derived lymphokines and differentiated into antibody-secreting cells. These results indicate that resting TNP-ABCs must be activated by T cells and antigen via cell/cell contact prior to becoming responsive to lymphokines. However, lymphokines released at the contact site during the interaction could be solely responsible for the initial activation event. This possiblity was addressed by Krusemeier and Snow (450), who cultured TNP-ABCs with antigenspecific T cells that had been treated (prior to interaction) with cyclosporin A. This pharmacologic agent inhibits the release of lymphokines from activated T lymphocytes (451, 452). It was demonstrated that although cyclosporin A-treated T lymphocytes were unable to release lymphokines, they could physically conjugate to TNP-ABCs. TNP-ABCs which had interacted with cyclosporin A-treated T cells were unable to differentiate into antibody-secreting cells in the absence of exogenously added T cell-derived lymphokines, but were able to differentiate when specific lymphokines were added to the cultures. Thus, cell contact could be distinguished from lymphokine utilization. The development of lymphokine responsivenessby B cells cultured with cyclosporin A-treated T cells was partially blocked by the addition of anti-class I1 antibodies, suggesting that MHC-restricted interactions of TNP-ABCs with T cells render the TNP-ABCs responsive to lymphokines.
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Rasmussen et al. (262) compared the requirements for specific versus polyclonal B cell activation by Th2 (IL-4- and IL-5-producing) cells. Hapten-specific antibody responses were observed only when the small B cells had physically interacted with T h cells. Polyclonal responses were observed when large B cells were cultured with lymphokines in the absence of T cell/B cell contact. The requirement for physical contact was determined by culturing cells in chambers separated by a nucleopore membrane. Thus, T cells could be physically separated from B cells without interfering with the diffusion of lymphokines and antigen. AntiIL-5 antibodies completely abrogated both the specific and polyclonal antibody response. This result suggests that IL-5 is essential for differentiation of resting or activated B cells and that T cell/B cell contact is critical for the induction of a state of IL-5 responsiveness in small, resting B cells. The characteristics of antigen-specific, MHC-restricted interactions between T and B cells are summarized in Table IV and a model is presented in Fig. 2.
CHARACTERISTICS
OF
TABLE IV ANTIGEN-SPECIFIC, MHC-RESTRICTEDFORMATION OF T CELL/BCELLCONJUGATES
Characteristic ~~
~~
Reference ~
1. Frequency: B cells forming conjugates with T cells (1/15) Frequency: B cells secreting antibody (1/15) 2. Antigen specific, MHC restricted 3. Antigen processing by B cell required 4. Time and temperature dependent 5 . T cell surface molecules involved: L3T4 LFA-la Thy-1 TcR B cell surface molecules involved: I -A LFA-la 6.
7.
8. 9. 10.
sk
T cell-associated L3T4, TcR, and talin cluster at the T cell/B cell contact site Class I1 molecules and sIg do not cluster at the contact site Reorientation of MTOC in the T cell Monogamous interaction of B cells A single B cell can conjugate with either a Thl or a Th2 cell
(197) (369) (197, 364) (197, 370, 386) (197, 370) (197, 386, 387, 424) (197, 386, 424) (197, 386) (387) (197, 364, 386, 396) (197) (424) (370, 387) (197, 326) (364, 370) (326) (326)
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ICAM-1
B Talin antigenklass 11 complex
FIG. 2. Physical interactions between T and B cells. (Top) T and B cells involved in a “loose” interaction which occurs in the absence of antigen. Cell surface molecules do not cluster at the interaction site and no reorientation of the MTOC or talin occurs in the T cell. This type of interaction may involve cell surface adhesion molecules on one cell and their respective receptors on the other. (Bottom) A “tight” interaction which is antigen specific and MHC restricted. Class I1 molecules and LFA-1 on the B cell, and L3T4, LFA-la, TcR, and Thy-1 on the T cell, are involved in this interaction. T cellassociated LST4 and TcR cluster at the contact site and a reorientation of the MTOC and talin in the T cell occurs to face the interaction site with the B cell. These types of interactions are monogamous for the B cell since a single B cell conjugates to only one T cell at a time.
1. The Conjugation of Antigen-Specqic Memory B Cells to T Cells A large number of studies (47) have demonstrated that the differentiated progeny of memory B cells, as compared to virgin B cells, secrete large amounts of non-IgM antibody and that memory B cells themselves are more readily activated by smaller quantities of antigen and by fewer T cells. Furthermore, following antigenic stimulation, the latency period
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ELLEN S. VITETTA ET A L .
for antibody secretion is shorter and the avidity of the antibody which the activated memory cells secrete is higher. These observations have raised questions concerning the mechanisms by which memory cells versus virgin cells interact with, and are activated by, T cells and antigen. Two technical advances have facilitated the characterization of the initial interaction between T cells and either antigen-specific virgin (TNP-ABC) or memory (TNP-MABC) B cells. The fmt technique involves the enrichment of TNP-ABCs and TNP-MABCs by virtue of differences in the avidity of their sIg molecules for specific antigen (194, 369, 453). The second technique involves the characterization and quantitation of the number of enriched antigen-specific B cells which physically conjugate to antigen-specific T hybridoma cells (197). Together, these two techniques have established that different B cells (e.g., virgin versus memory) differ in their requirements for physical conjugation to antigen-specific T cells. Physical and functional characteristics of both TNP-specific B cell populations are summarized in Tables I1 and V. Differences in conjugate formation between antigen-specific T cells and TNP-ABCs or TNP-MABCs are summarized in Table VI. When the number of B cells forming conjugates was correlated to the concentration of antigen to which the B cells were exposed prior to conjugate formation, TNP-ABCs, as compared to TNP-MABCs, required 10-fold higher antigen concentrations for half-maximal conjugate formation. This finding is not surprising, since TNP-MABCs express sIg receptors of a higher affinity for antigen than do TNP-ABCs (369). Therefore, these TNP-MABCs are better able to capture antigen and present it to T cells. In addition, the higher levels of expression of class I1 molecules on TNP-MABCs (369) probably contribute to the overall efficiency of antigen presentation at low antigen concentrations.
TABLE V CHARACTERISTICS OF TNP-MABCs
AND
TNP-ABCsa
1. TNP-MABCs can be prepared by lowering the TNP density fivefold on the red
blood cells used for rosetting the TNP-ABCs 2 . TNP-MABCs, but not TNP-ABCs, can be activated following adoptive transfer into irradiated, carrier-primed mice 3. TNP-MABCs, as compared to TNP-ABCs, express sIg receptors of a higher affinity for antigen 4. TNP-MABCs express higher levels of class I1 antigens than do TNP-ABCs 5. TNP-MABCs secrete high-affinity IgG after activation by a T D antigen and T h cells in Vitm, whereas TNP-ABCs secrete primarily low-affinity IgM “From Yefenof et al. (369).
CELLULAR INTERACTIONS IN THE HUMORAL IMMUNE RESPONSE
TABLE VI DIFFERENCES IN CONJUGATE FORMATION BETWEEN T CELLS TNP-ABCS OR TNP-MABCS‘
51
AND
1. TNP-ABCs require a higher antigen concentration for conjugate formation than do TNP-MABCs 2. TNP-ABCs require more time to process and present antigen than do TNP-MABCS 3. LFA-1 molecules on TNP-MABCs, as opposed to those on TNP-ABCs, are not involved in conjugate formation ~
“From Sanders et al. (386).
TNP-ABCs require more time than TNP-MABCs to process and present antigen to T cells (386). When the number of B cells forming conjugates with T cells is correlated to the time during which B cells are exposed to antigen prior to their conjugation with T cells, at least 4-6 h is required for the TNP-ABCs to form a maximal number of conjugates. Presumably, this period of time is required for maximal processing and reexpression of antigen on the surface of the TNP-ABCs. In contrast, TNP-MABCs require only 2 h of exposure to antigen for maximal conjugate formation with T cells. The decreased time required for the TNP-MABCs to acquire the ability to conjugate to T cells may again reflect the increased affinity of their sIg receptors for antigen. Thus, TNP-MABCs may bind and take up antigen more efficiently than TNPABCs. Preliminary studies with the lysosomotropic drug, chloroquine, suggest that antigen processing may differ between virgin and memory B cells (386). Nanomolar concentrations of chloroquine added during the time of antigen processing by TNP-MABCs can effectively inhibit the ability of these cells to conjugate with T cells. In contrast, the same concentrations of chloroquine only partially inhibit the ability of TNP-ABCs to conjugate to T cells. These results also suggest that different intracellular compartments are involved in the processing of antigen in virgin versus memory B cells. As determined by the ability of antibodies against LFA-1 molecules to inhibit conjugate formation, studies by Sanders et al. (197) demonstrated that LFA-1 molecules on T cells, as well as those on TNP-ABCs, are involved in conjugate formation. In contrast, pretreatment of TNPMABCs, as compared to pretreatment of T cells with anti-LFA-la antibodies, did not inhibit conjugate formation. This finding suggests that LFA-1 molecules on TNP-MABCs are not involved in conjugate formation, even though TNP-MABCs express the same levels of LFA-1 on their surface as TNP-ABCs (V. M. Sanders and E. S. Vitetta, unpublished
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ELLEN S. VITETTA ET A L .
observations). A more likely explanation is that the interaction between memory B cells and T cells is of a much higher avidity and, consequently, the requirement for LFA-1 molecules on the B cells is not as stringent as for the interaction between virgin B cells and T cells. Similar results have been described for primary versus secondary Tc cells which interact with target cells (454). These data suggest that the differences between the activation requirements of virgin and memory B cells may be explained, in part, by differences in the efficiency with which the B cells specifically interact with T cells. 2 . MHC- Unrestricted Th Cell-Dependent Activation of Resting B Cells
As presented in the preceding section, T h cell-dependent activation of resting B cells involves a sequence of events which includes the sIgmediated uptake and processing of antigen by the B cell, and the subsequent presentation of antigenic fragments in association with class I1 molecules to Th cells during direct T cell/B cell interaction. Antigen plus class I1 molecules bind to the TcR, resulting in both cell/cell contact and aggregation of TcRs (387). This results in the activation of the T cell, which then releases lymphokines toward the contact site with the B cell (434). Thus, the main role of antigedclass I1 complexes on B cells is to bind to and aggregate the TcRs. As mentioned earlier, it is not clear whether class I1 molecules per se are involved in signaling B cells, although many workers have concluded that the activation of resting B cells requires both stimulation of B cell surface determinants (e.g., class I1 molecules) during contact with the T cell and exposure to lymphokines, which mediate growth and differentiationof both the T and B cells. In contrast to MHC-restricted, antigen-specific activation of B cells, resting B cells can be induced to differentiate into antibody-secreting cells in a polyclonal fashion in the presence of histoincompatible T cells and high concentrations of antigen (455). These findings question the absolute requirement for the binding of TcRs to class I1 molecules on B cells in order to activate them and suggest that the activation of T cells by aggregation of TcRs results in the release of T cell-derived lymphokines. Subsequently, interactions between activated lymphokine-secreting T cells and resting B cells lead to B cell activation. Three recent findings have supported the latter hypothesis: (1) Hirohata et al. (456) cocultured human T cells (stimulated with immobilized antiCD3 antibodies) and highly purified peripheral blood B cells under conditions which allowed cell/cell contact. The immobilized anti-CD3 would aggregate the TcRs. When the T cells were activated and cocultured
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with the B cells, polyclonal antibody secretion was induced. These results demonstrate that activated T cells provide all the necessary signals to induce B cell differentiation in the absence of antigen by a mechanism requiring direct cell/cell contact. (2) In a report by Owens (394), an anti-TcR antibody, F23.1, which recognizes the Vg8 region of the TcR, was attached to wells of microtiter plates. When T cells were added to the wells in the presence of histoincompatible splenic B cells, a polyclonal antibody response was elicited. The requirement for T cell/B cell contact was supported by two findings-(a) SNs from F23.1-activated T cells alone were unable to support B cell differentiation, and (b) B cells separated by a membrane from F23.1-activated T cells could not be activated. (3) Julius and colleagues (392, 393) used two different approaches to show that activation of resting B cells by T cells can be achieved by prior aggregation of TcRs. In the first (392), resting B cells were cultured with histoincompatible resting T h cells in the absence or presence of the F23.1 antibody. The Th cells were either F23.1-positive or F23.1-negative. The F23.1-positiveT cells, but not the F23.1-negative T cells, were able to support B cell differentiation in a manner comparable to that of LPS. In addition, an antibody response of similar magnitude was obtained if the Th cells were first cultured with irradiated histocompatible APCs and antigen and then added to cultures containing histoincompatible B cells. These results further support the notion that the activated T h cells can activate B cells in the absence of a class 11mediated signal. Any cell contact involved would therefore be dependent upon cell surface molecules other than class I1 molecules (e.g., LFA-1, L3T4). In the second approach, TNP-ABCs obtained from mice transgenic for Sp-6p.x genes (393) could be bridged to histoincompatible T cells with TNP-conjugated F23.1 antibodies. In such experiments, the TNP-ABCs were induced to differentiate into antibodysecreting cells in the absence of MHC-restricted interactions. When T h cells and B cells were cocultured with unconjugated divalent F23.1 antibody, but not univalent F23.1 antibody, an equivalent antibody response was obtained. This again suggests that aggregation of TcRs is the critical requirement for T cell activation and the subsequent induction of B cell differentiation. To explore this observation further, histoincompatible B and T cells were cultured in the presence of TNP conjugated to univalent fragments of the F23.1 antibody. Since this situation promoted B cell differentiation, whereas the addition of unconjugated (no TNP) univalent fragments of F23.1 antibodies did not, it was concluded that sIg (on the B cell) could effectively aggregate the TcR during antigen bridging and that recognition of class I1 molecules on the B cell is not necessary for induction of B cell differentiation.
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ELLEN S. VITETTA ET A L .
Taken together, the above studies demonstrate that it is not obligatory for the TcR to deliver an activating signal to the B cell via recognition of class I1 molecules. However, these studies have not ruled out the possible requirement for other B c e l l - d a t e d molecules (e.g., LFA-1)in cell contact or B cell activation. One explanation for these results is that weak interactions between B cells and activated T cells can be mediated by LFA-1 or other adhesion molecules. In the presence of high concentrations of lymphokines (secreted by the activated T cells), these weak interactions might bypass signals normally provided by interactions with class I1 molecules. In support of this hypothesis, and as discussed in a previous section of this review, LFA-1 has been implicated in B cell signaling in other models (425). This hypothesis could be further explored by characterizing the molecules involved in the formation of conjugates between activated T h cells and histoincompatible B cells. For example, if the histoincompatible B cells could not conjugate to the preactivated T h cells in the presence of anti-LFA-1,etc., this would implicate these molecules as mediators of cell contact and perhaps signaling. In support of this hypothesis, interactions between T and B cells were artificially induced in the absence of hapten-carrier complexes by using TNP-modified, antigen-specific T h cells and TNP-ABCs (457). Proliferation was induced in both T and B cells and differentiation was induced in most B cells. B cell activation was not MHC-restricted, but could be blocked with antibodies directed against class 11, L3T4, and LFA-1 molecules. Although not yet tested, it is possible that the lymphokine-containingSN from the activated T cells in the presence of antibodies which bind to LFA-la [e.g., G48 (425)] might activate the B cells. It is now evident that signaling of resting B cells via class I1 molecules can be bypassed when activated T h cells are present and T cell/B cell contact can occur. This may account for “bystander effects” frequently observed when T cells and B cells are cultured under conditions of noncognate help (79, 389, 390, 458). An analogous situation may occur in the local environment of an activated lymph node and explain how “natural” antibodies appear in the circulation, i.e., resting B cells not specific for the antigen in question would be activated by contacting Th cells activated by other antigens. However, in this case, activated Th cells would arise as a consequence of class II/antigen-TcR interactions: the B cell bystander effect would be MHC-unrestricted, class II-independent, and lymphokine-mediated. V. lnterleukinr
Our knowledge of the physiochemical and biological properties of interleukins (cytokines) has grown exponentially since the initial reports that soluble products from activated T cells (lymphokines) could induce
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B cells to secrete Ig after antigenic stimulation (74, 459). This has been due to the availability of mAbs, new cell culture techniques, and the molecular cloning of interleukins. Nonetheless, attempts to understand how interleukins regulate cellular activity, especially in vivo, have been complicated by the fact that their biological activities are usually extremely complex and pleiotropic (460-462). This complexity is exemplified by reports that (1) some lymphokines can act at different stages of development on a given cell type inducing different effects, some of which are inhibitory and some of which are stimulatory; (2) most lymphokines can act on cells from different lineages, including nonhematopoietic cells; (3) one lymphokine given in combination with another lymphokine can have both synergistic and antagonistic effects on different target cells; (4) in vi'tro activities of lymphokines from one species cannot be easily extrapolated to in vzvo models in the same species or even to in m'tro models in another species; and ( 5 ) with a few exceptions, the receptors for different lymphokines have not been well characterized. It is therefore possible that receptors for different lymphokines interact, share subunits, or bind other lymphokines with low affinity (460, 463-465). If so, this would call into question the physiological relevance of pleiotropism, particularly in Vitro, where large amounts of lymphokines are often added to cell cultures. In vivo, because of anatomical compartmentalization of different cells and the low concentrations of circulating lymphokines, some of the effects observed in vitro may not be physiologically relevant. The purpose of this section is to review our current knowledge of the biological properties of interleukins/cytokines that play a role in T cell/ B cell collaboration and activation. The discussion will focus primarily on in vitro systems in the mouse. Table VII lists the biological properties of some of the cytokines, the majority of which are secreted by activated Thl or Th2 cells, with special reference to their effects on T cells, B cells, and accessory cells. As discussed in the previous section, one of the consequences of T h cell stimulation by antigen, presented in the context of class I1 molecules on an APC, is the release of lymphokines (75, 260, 277). Such lymphokines have effects not only on cells immediately involved in cellular interactions, but also on other lymphoid and nonlymphoid cells as well (460, 461). In a physiological context, these pleiotropic effects presume that the activated T cell (or its lymphokines) can traffic to other areas of the body and that other target cells (not involved in the initial collaborative interaction between T cells and APC) express lymphokine receptors and perhaps class I1 antigens. This has not yet been proved to be the case in vivo. In addition, cells which are not directly involved in the immune response are able to secrete additional lymphokines that
TABLE VII MULTIPLEEFFECE OF CYTOKINE~ ON MURINET CELLS, B CELLS, AND ACCESSORY CELLS Lymphokine"
Sources
Biological activities
IGl -Two distinct but structurally related molecules have been cloned, IL-la! and IL-lfl (576, 577); [endogenous pyrogen (573), leukocyte endogenous mediator (574). lymphocyteactivating factor (LAF) (575)l
Monocytes, macrophages, B cell lines, T cell lines, fibroblasts, keratinocytes, Langerhans cells, astrocytes, endothelial cells, NK cells (reviewed in Ref. 571)
Effects on lymphocytic/myeloid cell precursors Maturation of pre-B cells (578, 579) Synergy with CSFs on immature precursors (571, 609) Increased synthesis of CSFs (571) Costimulatory activity on thymocytes (575) (reviewed in Refs. 570 and 572) Effects on B and T cells B cells Increased proliferation of B cells stimulated with anti-Ig + IL-4 (580) Increased proliferation of B cells stimulated with anti-Ig + dextran sulfate (581) Increased proliferation of antigen-stimulated B cells (484, 582) Increased PFC formation with IL-2 (527) T cells Costimulatory activity for growth of activated T cells (reviewed in Refs.570, 572, 609, 287, 288, and 583-585) Lymphokine release from activated T cells (reviewed in Refs. 570, 572, and 610-612) Costimulatory activity for IL-4 and IL-2-induced proliferation on Th2 but not Thl cells (280, 286-288) Effects on accessory cells (macrophages) (reviewed in Refs. 570 and 572) Increased synthesis of PGEz (613) Induction of cytotoxicity
IG2- [T cell growth factor, (TCGF) (reviewed in Refs. 289 and 522)]
Activated Thl cells (75, 260); Tc cells (614, 615)
IGS - [Mast cell growth factor, P-cell stimulating factor, multicolony stimulating factor, panstimulating hemopoietin (reviewed in Ref. 586)]
Activated T cells (Thl and Th2) (75, 260, 586), WEHI - 3 B (myelomonocytic cell line) (586, 587)
Increased migration Increased synthesis of IL-1, CSFs, and other cytokines Induction of release of collagenase and acute-phase proteins Effect on lymphocyte/myeloid precursors Growth and differentiation of thymocytes (523, 616, 617) Effects on B and T cells B cells Proliferation of B cells stimulated with anti-Ig + LPS (533) Proliferation and differentiation of antigenstimulated B cells (527-530) Anti-SRBC PFCs with IL-1 and IFN-y (527) Induction of IgM secretion (514, 530, 618) Differentiation with anti-Ig + IL-4 plus a second T cell-derived factor (516) T cells Induction of proliferation of resting T lymphocytes (high doses) (549, 550) Growth of activated T cells and thymocytes (both Thl and Th2) (reviewed in Reti. 289, 522, 523, 277, 280, and 288) Cytotoxic T lymphocyte generation (524-526) Effects on accessory cells Increased monocyte cytotoxicity (human) (554) Effects on lymphocytic/myeloid precursors Growth and differentiation of multipotential stem cells and myeloid progenitor cells (granulocytes, monocytic, eosinophilic, erythroid, megakaryocytic, and mast cells) (reviewed in Refs. 586 and 588-592) Growth of pro-B, pre-B, and pro-T cell clones (594, 595) Other Growth of mast cell lines (496, 591, 592) (continued)
TABLE VII (Continued) MULTIPLE EFFECTS OF CYToKINEs ON MURINET CELLS, B CELLS, Lymp hokine"
IL--[B cell growth factor-1 (BCGF I), B cell stimulatory factor-1 (BSF-l), T cell growth factor I1 (TCGF 11) (293, 619)]
AND
ACCESSORY CELLS
Sources
Biological activities
Activated Th2 cells (75, 260), mast cell lines (620)
Effect on lymphoid/myeloid precursors Growth factor for fetal thymocytes (496-498) Growth of thymocytes in the presence of IL-2 (499) Enhanced erythroid, granulocyte, monocyte, and mast cell colony formation in combination with erythropoietin, G-CSF, M-CSF, and IL-3, respectively (481, 482) Induction of cytotoxic precursors from thymocytes (496) Effect on B and T cells B cells Induction of class I1 MHC molecules on resting B cells (469, 470) Increase in size of resting B cells (472, 621) Induction of FccR on resting B cells (476, 477) Preparation of resting B cells to proliferate to antiIg (471, 472) Increased ability to conjugate with T cells (396) Synergy with Type I1 TI antigens (484) Increased secretion of IgGl and IgE by LPSactivated B cells or B cells stimulated with T cells and antigen (268, 284, 318, 319, 321, 473, 474, 485) Decreased IgGs, IgGs,, and IgGebsecretion (284, 318, 321, 474, 485) Increased IgA production with IL-5 (300, 324) T cells Proliferation of resting T cells with PMA (298)
IG5- [B cell growth factor I1 (BCGF 11) (501, 502), T cell-replacing factor (TRF) (500), killer helper factor (KHF) (508). eosinophilactivating factor (EAF) (506, 623)]
Activated Th2 cells (260, 300, 301)
Increased IL-2R expression in the presence of PMA (622) Growth factor for activated T cells (Thl and Th2) (277, 280, 285-28a, 294-296) Cytotoxic T lymphocyte activity (493-495) Effects on accessory cells Increased class I1 expression on macrophages (478-480) Increased tumoricidal activity (478-480) Growth factor for mast cells with I L 3 (293, 294, 475) Effects on lymphoid/myeloid precursors Eosinophil colony formation from bone marrow cells (proliferation and differentiation of eosinophils) (503, 506, 507) Differentiation of thymocytes into Tc cells (with IL-2) (508) Effects on B and T cells B cells Proliferation of normal activated B cells and B lymphoma cells (BCLI) (301, 500-502, 504, 514, 515) Increased IL-2R expression on activated B cells or B cell lines (509-511) Increased IgM and IgG secretion by activated B cells (300, 301, 500-502, 504, 510, 512, 514, 515, 517) Enhanced IgGl and IgE secretion with IL-4 (300, 517) Increased IgA secretion by LPS-activated B cells and lymphoma cells (with IL-4) (300, 320, 624) (continued)
MULTIPLE EFFF.CE
TABLE VII (Continued) MURINET CELLS. CELLS,
OF CYTOKINES ON
Lymphokine"
Sources
IG6- [Interleukin HPl (596, 597), plasmacytoma growth factor (PCT-GF) (598), B cell stimulatory factor-2 (BSF-2) (human) (60S), IFN-82 (human) (601, 602), 26-kDa protein (human) (600, 601)]
Activated T cells, macrophages (P388D1) and other cells (596, 598, 599)
AND
ACCESSORY CELLS Biological activities
Effects on lymphoidhyeloid precursors Support of colony formation by normal hematopoietic precursors in the presence of other factors (CSFs) (599, 625) Costimulatory activity on thymocytes (similar to IL-1) (599)
Effects on B and T cells B cells Proliferation and Ig secretion by activated normal B cells and lymphoblastoid lines (596-598, 603-604)
Hybridoma/plasmacytoma growth factor (596-600) T cells T cell activating factor (599, 606, 607) Cytotoxic T lymphocyte activity with IL-2 and IFN-7 (605) or in primary MLC (606) Cofactor for the expression of IL-2Rs (human T cells) (608)
IFN-y - [Macrophageactivating factor (MAF) W8)l
Activated Thl cells (75, 260) and Tc cells (615)
"Synonymous terms are given in brackets.
Effects on B and T cells B cells Differentiation factor for B cells (527, 565, 566) Induction of IgG2, production in the presence of LPS (265, 321, 568, 569) Decreased IL-4-induced activities, e.g., Ia expression (267, 269, 270); size increase (267); anti-Ig proliferation (266); FceR expression (476); IgG, and IgE secretion (268, 271, 300, 321) T cells Inhibition of IL-2- or IL-4-induced proliferation of Th2 but not Thl cells (280, 299) Effects on accessory cells Increased Ia expression (559-562) Increased FcyR expression (562) Induction of antimicrobial and tumoricidal activity (563) Enhancement of TNF and LT functions (564)
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might also affect the function of B and T cells. In general, although the release of lymphokines is a direct consequence of antigenic stimulation, the effects of such lymphokines are not antigen specific. It should be noted, however, that stimulation of certain T cells has been reported to result in the release of soluble antigen-specific helper factors (466, 467). The role played by these factors in T cell/B cell responses is highly controversial, and will not be discussed in this review. A. Th CELLSUBSETS AND B CELLACTIVATION As described in the previous section, a number of studies (75, 261, 263, 300, 462) suggest that the Th2 subset is more effective in providing help to B cells because of the secretion of IL-4 and IL-5. In contrast, the ability of Thl cells to provide help to B cells is more controversial (75, 261-263, 300, 462). Although some clones of Thl cells can provide help to B cells (75, 265, SOO), it is generally accepted that they are not as effective as Th2 cells, probably due in part to their production of IFN-y, a known inhibitor of several B cell functions (266-271), and their failure to secrete IL-4 and IL-5. Sanders et al. (326) have recently demonstrated that virtually all antigen-specificB cells that can physically interact with T cells in an antigen-specific, MHC-restrictedfashion can do so with either type of T h cells. Thus, if B cell activation and differentiation can be induced by different Th subsets, secreting different lymphokines, then at least two different pathways of B cell activation should exist. B. B CELLACHVATIONBY Th2 CELLS-THEROLES OF IL-4 AND IL-5 1. IL-4
“Resting”Th2 cells do not secrete IL-4 or IL-5 (75, 260, 277). In 2-4 h following antigenic stimulation, IL-4- or IL-5-specific mRNA can be detected in Th2 cells (260). Lymphokine activities can be detected in culture SNs within 4-6 h, reaching a peak after 24 h (277). No differences have been observed in the pattern of IL-4 secretion by T-286 cells (a Th2 cell line) when they are stimulated with antigen on different APCs (e.g., splenic adherent cells, peritoneal macrophages, or TNP-ABCs) (277). However, as noted in an earlier section, TNP-ABCs are much more effective at capturing antigen at lower concentrations and, hence, make outstanding APCs when low doses of antigen are used in vitro (158, 169, 172, 184, 187, 468). a. Effects of IL-4 on B Cells. IL-4 was first described by Howard et al. (290) as a lymphokine that was different from IL-2 and that could synergize with anti-p antibodies to induce the proliferation of resting
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B cells. It later became evident that IL-4 had a variety of effects which could be manifested on either resting or stimulated B cells. For example, IL-4 induced the increased expression of class I1 molecules on small, resting B cells (469, 470), and it prepared these cells to proliferate in response to anti-Ig (471, 472). These results suggested that IL-4 was a “progression”factor rather than a “growth” factor for resting B cells. IL-4 can also induce the increased secretion of IgGl after stimulation of resting B cells with LPS (284, 318, 473, 474) and is identical to the B cell differentiation factor for IgGl (BCDFyl) described by Isakson et al. (318, 473). It was later found that high concentrations of IL-4 also induced the secretion of IgE in B cells cultured with LPS (268). After the cloning and expression of IL-4 cDNA by two separate groups (292,293), it became evident that the activities of IL-4 were even more complex. IL-4 was reported to induce the proliferation of T cells (277, 280, 285-288, 294-296,298) and mast cell lines (293, 294,475), to induce the expression of FCEreceptors (CD23) on resting B cells (476) or B cell hybridomas (477), and to act on several other hematopoietic lineage cells, including mature macrophages (478-480) and precursors of macrophages, granulocytes, megakaryocytes, and erythrocytes (481, 482). From this brief description, it is clear that IL-4 can modulate the B cell response at many points in its differentiatiodactivation pathway. Its ability to do so may be programmed into the B cell when it is still in Go. How does IL-4 work during T cell/B cell collaboration? After cognate interaction between a Th2 and a B cell has occurred, secreted IL-4 could act in combination with putative signals provided by the physical interaction itself to activate the B cell and induce its proliferation. In fact, it has been reported by DuBois et al. (483) that a cloned T cell line which does not secrete IL-4 can induce the proliferation of B cells only if exogenous IL-4 is provided. IL-4 could also act on resting B cells that have not yet undergone cognate interactions with T cells to induce increased expression of class I1 molecules and prepare them to interact with T cells. In this way, IL-4 may enhance the efficiency of T cell/B cell collaboration. For this to happen, one would presume that the T cell with which the B cell interacts has already been activated by another APC and is secreting IL-4 before it interacts with a B cell. In this regard, Sanders et al. (396) have reported that treatment of small TNP-ABCs with IL-4 enhances the ability of such cells to physically interact with Th cells, an effect probably mediated by increased expression of class I1 molecules. The enhanced ability of IL-4-treated B cells to form conjugates with T h cells, correlated with a higher plaque-forming cell (PFC) response in treated versus untreated cells. Stein et al. (484) reported that IL-4 can act as a costimulant in
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the proliferative response of B cells to type I1 thymus-independent (TI) antigens. Thus, IL-4 also acts on B cells by preparing them to proliferate after interaction with anti-Ig (471, 472), antigen (TI/type 11) (484), or antigen and T h cells (TD antigens) (483). IL-4 acts on resting B cells (subsequently cultured with LPS) to induce the secretion of IgGl and IgE and to decrease the secretion of IgGs (268, 284, 318, 319, 321, 473, 474, 485), indicating that IL-4 is an “early-acting” factor that can program “later events.” Although it is not yet clear whether B cells stimulated during cognate T cell/B cell interaction behave similarly to LPS-activated B cells, it is plausible that if IL-4 is secreted during cognate T cell/B cell interaction it should “prime” the involved B cell and perhaps other B cells to subsequently secrete IgGl. In fact, both Snapper and Paul (329) and Bergstedt-Lindqvist et al. (486) have reported that IL-4 can program uncommitted B lymphocytes to switch to IgGl and IgE production. The decisive requirement for IL-4 in TD antigen-induced IgGI production in Vitro has been confirmed by Stevens et al. (265); Th2, but not Thl, cells (which do not secrete IL-4) induce IgGl secretion in TNP-ABCs; IgGl secretion can also be induced if Thl-activated B cells are cultured with exogenously added IL-4. Th2, but not Thl, cells induce IgE responses (75, 260, 300). This activity can be inhibited by anti-IL-4 antibodies, demonstrating that IL-4 is required in IgE production. IL-4 is also involved in IgE production in vivo in mice infected with the helminth Nippostrongylus bmsiliensis (487). Although IL-4 programs the secretion of both IgGl and IgE, Snapper et al. (322) have suggested that their production is differently regulated, based on the requirement for higher IL-4 concentrations to induce a polyclonal IgE response. 6. Effects of IL-4 on T Cells. A number of laboratories have reported that in addition to its effects on B cells, IL-4 also stimulates the proliferation of several T cell lines (277, 280, 285-288, 294-296) or resting T cells in combination with PMA (488). IL-4 also functions as the autocrine growth factor for antigen-stimulated Th2 cells (277, 285, 286). Although it has been reported that resting T cells express receptors for IL-4 (489-492), Fernandez-Botran et al. (277, 280) have observed that “resting” Th2 cells do not proliferate in response to IL-4, but become responsive to the growth-promotingeffects of IL-4 after antigenic stimulation. These results suggest that IL-4 induces proliferation only in cells that have been previously stimulated by antigen or mitogen. It is not clear whether IL-4 functions as an “activation” factor to prepare resting T cells to proliferate in response to antigen and APCs (in the same manner that it prepares resting B cells to proliferate in response to antiIg), or whether it acts as a “true” growth factor for T cells.
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In the context of T cell/B cell collaboration, it would be expected that following the activation of Th2 cells and subsequent lymphokine secretion, IL-4 would drive the activated Th2 cells to proliferate. Expansion of the T h cell pool would occur for 24-48 h, during which time the IL-4 would be secreted (277, 283). The responsiveness of the Th2 cells to IL-4 would slowly decrease in the absence of additional stimulation by antigen (280). IL-4 has additional effects on T cells. For example, IL-4 acts as a helper factor for the generation of Tc cells in unprimed and primed mice (493-495). In addition, IL-4 has been reported to act on thymocytes, stimulating their proliferation and differentiation (496). IL-4 synergizes with PMA in the generation of Tc cells from resting T cells (495) and has growth-promotingactivity on activated murine thymocytes (496-499). c. Effects of IL-4 on Other Cells. IL-4 induces increased expression of class I1 molecules on macrophages and enhances their cytotoxic activity (478-480). This effect is reminiscent of the effect of IL-4 on the expression of class I1 antigens on B cells. IL-4-treated macrophages also present antigen more effectively to T cells (480). Another important activity of IL-4 is its growth-promoting activity on mast cell lines in the presence of IL-3 (293, 294, 475). Mast cells mediate immediate hypersensitivity reactions by releasing histamine and other vasoactive substances. Thus, IL-4 not only acts by enhancing the humoral side of the hypersensitivity responses (increased IgE production), but enhances the effector phase (mast cell activation) as well. Finally, IL-4 has been reported to have effects on hematopoietic cell precursors (481, 482). Peschell et al. (481) and others observed that although IL-4 is not a colony-stimulatingfactor (CSF), it synergized with other CSFs such as granulocyte (G)-CSF, erythropoietin, monocyte (M)-CSF, a megakaryocyte-promoting activity, and IL-3 to stimulate colonies of several lineages, such as granulocytes, erythrocytes, monocytes, megakaryocytes, and mast cells, respectively. 2. IL-I
In the first reports that SNs of activated T lymphocytes could replace many of the functions of the T cells themselves, particularly the induction of Ig secretion by B cells, these activities were termed “T cell replacing factors,” or TRFs (74, 459, 500). Takatsu et al. (500) first established a T cell hybridoma, B151K12, which secreted a factor which induced B cell differentiation and was different from other known lymphokines. Swain and Dutton independently described a factor, originally called (DL) B cell growth factor (BCGF) (501) and later BCGF I1 (502), which induced the proliferation of normal B cells cultured with dextran sulfate
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or the leukemic B cell line, BCLl. Further experiments demonstrated that this factor was different from IL-4 and that it could not synergize with anti-Ig to induce the proliferation of normal B cells (502). It was later demonstrated that TRF had BCGF I1 activities copurified, suggesting that TRF and BCGF I1 might be the same lymphokine (503-505). It also became evident that TRF/BCGF I1 had even more complex biological activities. Sanderson et al. (506, 507) found that a lymphokine called eosinophil differentiation factor (EDF) and BCGF I1 were identical. More recently, other activities have been ascribed to TRFIBCGF II/EDE These activities include the ability to increase IL-2R expression on antigen-stimulated thymocytes, inducing them to become Tc cells in the presence of IL-2 (508), and to enhance IL-2R expression on activated B cells (509-511). The cloning and expression of cDNA for TRF/BCGF II/EDF (503, 505) confirmed that all three activities were mediated by a single molecule, which has now been designated IL-5. IL-5 is secreted by activated Th2, but not Thl, cells (260), although this point remains controversial (263). a. Effect oflL-5 on B Cells. Although both IL-4 and IL-5 have multiple biological effects, IL-5 in contrast to IL-4, appears to act primarily on activated B cells (512, 513), inducing their proliferation and differentiation into IgM-secreting cells. Thus, in the context of a cognate T cell/B cell interaction, IL-5 should act on B cells already activated by antigen, IL-4, and T h cells and induce these activated B cells to proliferate and secrete IgM (300, 301, 500-502, 512, 514). In this sense, IL-5 might induce both antigen-specific and polyclonal responses (262, 325). IL-5 is a good candidate for the “nonspecific” activity associated with the non-MHC-restricted,nylon-wool-adherentclass of T h cells described in earlier studies (253, 254). In addition to its ability to induce IgM secretion, IL-5 has also been reported to induce differentiation of TNPABCs into IgM- and IgG-secretingcells (500, 515). IL-5 has been reported by Mosmann and Coffman (283) and Murray et al. (324) to have enhancing effects on IgA secretion by LPS-stimulated B cells. It is not yet clear whether IL-4 and IL-5 act sequentially on the same B cells or whether different B cells respond to each lymphokine. Nonetheless, the fact that IL-4 acts primarily on resting B cells, whereas IL-5 acts primarily on activated B cells (512, 513), suggests that the presence of both lymphokines would result in enhanced proliferation and differentiation of B cells activated by T cells and antigen. IL-5 synergizes with IL-4 in a number of experimental models (300, 514, 516, 517). Recently, another interesting biologic activity of IL-5 has been described. Harada et al. (510) have observed that recombinant (r)IL-5 can induce the expression of IL-2 receptors (Rs) and responsiveness to IL-2 in B cells from dinitrophenyl
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(DNP)-carrier-primed mice. It could be postulated that the biological significance of the induction of functional IL-2Rs by IL-5 on B cells is that such cells would then acquire responsiveness to IL-2, a lymphokine secreted by Thl cells. This would provide a means of regulating Ig production by both subsets of T h cells and suggests that the temporal order of lymphokine activities on B cells might be IL-4 IL-5 IL-2. In addition to the B cell stimulatory effects of IL-4 and IL-5, as noted earlier, LeClercq and colleagues (442, 518) have reported that certain clones of T h cells secrete a lymphokine, termed B cell activation factor (BCAF). BCAF can act on small, resting B cells, inducing polyclonal proliferation and differentiation in the absence of T h cells. Whether production of BCAF is restricted to a particular T h subset (Thl versus Th2) and what the relative frequency of this subset is remain subjects of investigation. Nonetheless] this lymphokine is another candidate for the “nonspecific,”non-MHC-restrictedhelper activity described in earlier reports. 6. Effect of IL-5 on T Cells. No effects of IL-5 on the proliferation of resting or activated T h cells have been reported. However, IL-5 can induce the generation of Tc cells from peanut agglutinin-positive (PNA+)thymocytes in the presence of IL-2 (508). This activity is probably mediated by the IL-5-mediated induction of IL-2R expression on thymocytes followed by acquisition of responsiveness to IL-2. c. Effect of IL-5 on Other Cells. As previously mentioned, IL-5 is identical to the EDF described by Sanderson (503, 506, 507). I L 5 induces the formation of colonies of eosinophils from bone marrow cultures and induces their proliferation. Eosinophils express FceRs and are capable of binding the Fc portion of IgE, after which they release a number of mediators of inflammation. In addition, the importance of eosinophils in the antibody-mediated killing of parasitic organisms has been demonstrated (519-521). In conclusion, IL-5 can potentiate B cell proliferation and differentiation and, in conjunction with IL-4, is involved in local immunity (via IgA production). IL-5 might also play a role in the immune responses to certain parasitic organisms.
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C. B CELLACTIVATIONBY Thl CELLS-THEROLES OF IL-2 AND IFN-7 1. IL-2
a. Effects of IL-2 on B Cells. IL-2 (originally T cell growth factor, or TCGF) was described as an activity in the SN of mitogen-activated T cells that induced the proliferation of T cells (289, 522). The role
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of IL-2 in the proliferation of activated T cells or T cell lines and in the development of Tc cells has been extensively studied (289, 522-526). The role played by IL-2 in B cell proliferation and differentiation is less clear. Early studies (527-530) suggested that IL-2, in combination with other factors, acted synergistically to induce a primary anti-SRBC response in vitro. These helper factors were later identified as IFN-y and IL-1 (527). In contrast, other studies suggested that B cells lacked IL-2Rs and could not respond to IL-2 (279, 531, 532). More recently it has become clear that, although resting B cells express very few, if any, I L - ~ R sat , least some activated B cells express functional IL-2Rs (532-537). Zubler et al. (533) reported that B cells stimulated with a mixture of LPS and anti-Ig expressed IL-2Rs and responded to IL-2, while B cells stimulated with LPS alone did not. Activated B cells (537) can respond to low doses (0.1-10 U/ml) of IL-2 and this effect is mediated via high-affinity IL-2Rs (533, 535-538). In other studies using higher doses of IL-2 (100-1000 U/ml), a greater range of responsiveness by B cells has been demonstrated. Nakanishi et al. (516) reported that although IL-2 did not display significant “TRF” activity at doses lower than 50 U/ml, it induced proliferation and IgM secretion in cultures of anti-Ig-stimulated B cells at doses >lo0 U/ml and in combination with IL-4 and a second T cell-derivedfactor. It has been suggested that the effect at high IL-2 concentrations is not mediated via “classical” high-affinity IL-2Rs (538, 539). In this regard, a number of laboratories (540-546) have reported that high-affinity IL-2Rs (& = 1 X lo-” M) are composed of at least two different subunits, both of which can bind IL-2, but with different affinities. One (p55) is the “classical”Tac antigen (human). It binds IL-2 M). The other subunit (p70-75) with low affinity (& = 1 X M). Together, p55 binds IL-2 with intermediate affinity ( K d =1 X and p75 form a high-affinity IL-2R (Kd = M) (547, 548). Some T cell lines express only low-affinity (p55) or intermediate-affinity (p75) IL-2Rs (540-544). Moreover, the presence of intermediate-affinity IL-2Rs (p75) on resting T cells (549-551), lymphokine-activated killer cells (LAK) (550), natural killer (NK) cells (550, 552), activated human B cells (537, 538, 551), and macrophages (551) has been reported. Although most of the biological effects of IL-2 are thought to be induced via receptors of high affinity (p75 + p55) (278, 279, 553), it has been reported that receptors of intermediate affinity (i.e., p75) can transmit signals after binding IL-2 (547-552). The activation of NK and LAK cells is probably mediated via p75 (550, 552). Saiki et al. (538) and Tanaka et al. (537) have reported that large human tonsillar B cells or B cells stimulated with Stufihylococcus aureus (Cowan I) (SAC) express
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p75 and can proliferate and differentiate into Ig-secreting cells in the presence of high concentrations ( >lo0 U/ml) of IL-2. Although it is not clear whether munne B cells resemble human B cells in this regard, these results suggest that resting B cells might express intermediate affinity IL2Rs under certain conditions and that they might be able to proliferate and/or differentiate in response to high concentrations of IL-2. When B cells are activated by certain stimuli, the expression of p55 might be induced on cells already expressing p75, resulting in the formation of high-affinity IL-2Rs (547, 548). Such cells would be expected to respond to IL-2 at much lower concentrations when compared to cells expressing only p75. The previously described IL-2R-inducing activity of IL-5 thus becomes very important. It could be hypothesized that IL-5 would induce the expression of p55 on B cells that already express p75, leading to the expression of high-affinity IL-2Rs. According to this hypothesis, B cells would have to first encounter IL-5 (secreted by a Th2 cell) before they could respond to IL-2 (secreted by a Thl cell). Thl cells would therefore amplify responses to IL-5. On the other hand, at least some Thl clones can activate B cells via cognate interaction and in the apparent absence of Th2 cells or their SNs. Whether T cell/B cell interaction can substitute for IL-5 in rendering B cells responsive to IL-2 or whether some Thl clones do secrete IL-5 and IL-2 (262, 263) remains to be determined. In conclusion, IL-2 appears to have both proliferative and differentiative effects on B cells, although additional lymphokines might be required for the optimal expression of such activities. In addition, the relative concentration of I L 2 is probably important in determining which B cells will be most responsive to 1L-2, although large (or activated) B cells are the most likely candidates. b. Effect of IL-2 on T Cells. The proliferative activity of IL-2 on T cells has been well documented (289, 522, 523). In the case of Thl and Th2 cells, clones of both types respond poorly to IL-2 when they are in a "resting" state (277, 280). However, after antigenic or mitogenic stimulation, responsiveness to IL-2 is greatly enhanced (277, 280). This is paralleled by an increase in the expression of IL-2Rs as determined by immunofluorescence or by the binding of radiolabeled IL-2 (278-280, 553). IL-2 is an autocrine growth factor for Thl cells in vitro (285). Interestingly, although Th2 cells utilize IL-4 as their autocrine growth factor, following stimulation they upregulate their levels of IL-2% in much the same manner as Thl cells (277, 280). In fact, IL-2 is better than IL-4 as a growth factor for Th2 cells (280, 285, 286). Such observations suggest that for an optimal Th2 response to occur, IL-2 would have to be present and that, in vivo, IL-2 would play an important role in the proliferation of activated Th2 cells. As noted above, responsiveness
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to IL-2 and IL-4 decreases with time after antigenic or mitogenic stimulation, rendering the T h cells unresponsive in the absence of further stimulation (280). c. Effect of ZL-2 on Other Cells. In addition to its effects on T and B cells when used at high concentrations, IL-2 augments the cytotoxicity of human monocytes (554) and the activity of NK and LAK cells (550, 552). As mentioned earlier, these results suggest that IL-2 might be acting on such cells via p75 (550, 552). They also show that the effects of IL-2 are not confined exclusively to lymphocytes. 2 . ZFN-y
a . Effect of IFN-y on B Cells. IFN-y, a lymphokine secreted by activated Thl (75, 260), Tc (555), and NK cells (556), has a variety of roles in the immune response. IFN-y has both antiviral and antiproliferative activities (557). It also plays a major role in macrophage activation (558-564) and it regulates the activities of cells involved in both humoral and cellular responses (557). IFN-y synergizes with IL-2 and IL-1 in the generation of anti-SRBC antibody responses (527) and promotes Ig secretion by resting or activated murine and human B cells (527, 565, 566). These observations suggest that IFN-y has a differentiation function (565, 566). Paradoxically, recent studies have suggested that IFN-y has potent inhibitory effects on B cell activation, proliferation, and differentiation (262, 266-270, 321). In this regard, IFN-y is inhibitory for IL-4. For example, IFN-y inhibits the IL-4-mediated expression of class I1 antigens (267, 269, 270) and CD23 molecules (FceR) (476) on resting B cells. IFN-y also has inhibitory effects on the proliferation of B cells stimulated by anti-Ig and IL-4 (266) and affects B cell differentiation by inhibiting the IL-4-mediated secretion of IgGl and IgE (268, 271, 300, 321). At the same time, it promotes the secretion of IgG2, (265, 567-569) in LPS-stimulated or antigen plus Thl-stimulated B cells. IFN-y-containing SNs from T h l cells (or inflammatory T cell clones) appear to have a suppressive effect on the Ig production of Th2-stimulated B cells (263, 300). Stevens et al. (265) recently reported that Thl, but not Th2, cells induce lgGza secretion by antigen-specific B cells after antigen stimulation and that this activity can be partially inhibited by antibodies against IFN-y. Interestingly, IgM secretion was not affected by IFN-y. Zn uiw, IFN-y inhibits the secretion of IgGl and IgE in mice treated with anti-IgD antibodies (569). IFN-y also enhances IgGza secretion in mice immunized with Brucella abortus (569). These results suggest that, in addition to its pleiotropic effects on other cells, IFN-y can regulate B cell function by acting both as a B cell differen-
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tiation factor, especially for the synthesis of IgGza and as an antagonist of the early IL-4-mediated effects on B cells. It could be hypothesized that, in vivo, the isotype(s) of Ig ultimately secreted after stimulation by a particular antigen will be determined by the relative numbers of activated T h l versus Th2 cells and the secretion of IL-4 versus IFN-y. Antigens that induce predominantly IgGza responses might preferentially activate Thl cells and antigens that induce I&, and/or IgE might activate Th2 cells. The molecular mechanisms underlying the antagonistic effects of IFN-y on the IL-4-induced activities on B cells are not clear. However, binding studies have demonstrated that IFN-y does not compete for IL-4 in binding to IL-4Rs (489-492), indicating that these two lymphokines do not compete for the same surface receptor. 6. Effect of IFN-y on T Cells. In addition to its antagonistic effects on the IL-4-induced responses of B cells, IFN-y has now been reported to be inhibitory to the IL-4- and IL-2-mediated proliferation of Th2, but not T h l , cells (280, 299). Fernandez-Botran et al. (280) reported that the IFN-y-mediated inhibition of Th2 proliferation was only partial (at doses below 100 Wml) and was mediated by an antiproliferativeeffect rather than a decrease in cell viability. These results suggest that IFN-y can regulate the action of Th2 cells not only by selectively decreasing their proliferation, but by antagonizing the effects of IL-4. c. Effect of IFN-y on Other Cells. As already mentioned, IFN-y plays a major role in the activation of macrophages for cell-mediated cytotoxicity and antimicrobial activity (559-564). In addition, IFN-y induces the increased expression of class I1 molecules and FcyRs on macrophages (559-562). Moreover, IFN-y-treated macrophages secrete increased levels of IL-1 (570), suggesting that IFN-7 probably enhances the APC and accessory cell function of macrophages. Finally, IFN-y might also be involved in direct lymphokine-mediated cytotoxicity of certain types of cells, since it has been shown to synergize with tumor necrosis factor (TNF) and lymphotoxin (LT), other lymphokines secreted by Thl cells (557, 564). In conclusion, Th2 cells support humoral responses, as well as both the humoral and cellular components of immediate hypersensitivity reactions and immune responses against certain parasites. The activities of Thl cells (IL-2, IFN-7) are related more closely to the development of immune responses against viruses, other intracellular organisms, and cell surface antigens to which cell-mediated cytotoxic responses are elicited.
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D. OTHERINTERLEUKINS 1. IL-1
IL-1 is a polypeptide secreted by a variety of cells, including monocytes, endothelial cells, and epithelial cells (570-572). Monocytes are still considered to be one of the major sources of this lymphokine (571, 572). A number of agents can induce macrophages to secrete IL-1, e.g., immunological stimuli (activated T cells or their products, immune complexes, and complement components) and microbial stimuli (endotoxin, exotoxins, yeasts, viruses, etc.) (572). IL-1 is probably the most pleiotropic lymphokine, having one of the widest range of activities and cellular targets. One of its activities, pyrogenicity (573), was described as early as 1948; IL-1 was originally called “endogenous pyrogen.” A number of other activities, independently described, copurified with leukocyte endogenous pyrogen. Among those activities was a stimulant of hepatic acute-phase protein synthesis, called leukocytic endogenous mediator (574), a substance from activated monocytes that, together with mitogens, induced thymocyte proliferation [lymphocyte activating factor (575)], and several other substances stimulating cartilage resorption, muscle wasting, collagen and fibronectin synthesis (570-572), etc. After the cloning and expression of cDNA for IL-1 (576, 577), it was established that these and other activities were mediated by the same molecule. IL-1 exists in two biochemically distinct but related forms known as IL-la and IL-lP [(576, 577); reviewed in Ref. 5711. Although amino acid homology between these two forms is limited, both bind to the same cellular receptor and are functionally equivalent. In this section, we shall limit our discussion to the activities of IL-1 on cells of the immune system, especially as they relate to the activation of T and B cells. a. Effect of IL-1 on B Cells. IL-1 has the ability to promote the maturation of B cell precursors (578) and pre-B cells (579). Thus, IL-1 induces the expression of x light chain and membrane Ig in a pre-B cell line that produces only cytoplasmic p chains (579). IL-1 synergizes with other lymphokines or antigens to induce mature B cells to proliferate and/or differentiate (reviewed in Refs. 484, 527, 570, 580-582). For example, Liebson et al. (527) showed that IL-1, in combination with IFN-y, synergized with IL-2 in the induction of an anti-SRBC antibody response. Howard et al. (580) reported that IL-1 potentiated the proliferative response of B cells cultured with IL-4 and anti-Ig, particularly at low cell densities. Stein et al. (484) demonstrated that IL-1 synergized with IL-4 in combination with type I1 TI antigens, in the same manner. Thus, IL-1 can act as a cofactor to potentiate the proliferative responses
i
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of B cells to a variety of stimuli. Although it is still not clear whether IL-1 is a critical factor in B cell responses, it can act in concert with other lymphokines to potentiate their proliferation and/or differentiation. b. Effect of IL-1 on T Cells. IL-1 also acts as a cofactor, along with other stimuli such as antigen, mitogen, and other lymphokines, to stimulate T cell proliferation (287, 288, 575, 583-585). IL-1 has been reported to induce IL-2 secretion and IL-2R expression by activated T cells (570, 572, 583-585). More recently, some Th2 cell lines have been reported to require IL-1 in order to proliferate in response to IL-4 (287, 288, 585). On other Th2 cell lines, IL-1 is not absolutely required, but can synergize with other lymphokines such as IL-2 and IL-4 (280). Such results confirm the role of IL-1 as a cofactor in T cell proliferation and also suggest that there may be heterogeneity among Th2 cells with regard to their absolute requirement for IL-1. Alternatively, the requirement for IL-1 might be related to the state of activation of the T h cell. For example, the need for IL-1 might increase as the state of activation of the T h cell decreases with time after antigenic or mitogenic stimulation. Thus, Kupper et al. (286) reported that antigen-stimulated D.10G4 cells proliferate in response to IL-4 in the absence of IL-1 shortly after stimulation, whereas IL-1 was required to support IL-4-mediated proliferation later on. In contrast, IL-1 does not act as a cofactor for IL-2/IL-4-mediated proliferation of T h l cells (280, 287, 288). Kurt-Jones et al. (288) have shown that Th2 cells express more high-affinity IL-1R than do Thl cells. These results seem paradoxical in light of earlier reports that IL-1 acted by inducing IL-2 release from T h l cells, since Th2 cells, which secrete IL-4 and not IL-2, are more responsive to IL-1. c. Effectof I L l on APCs. Macrophage function is profoundly altered by IL-1. The multiple effects of IL-1 on macrophages include the induction of the synthesis of prostaglandin & (PG&), IL-1, CSFs, other cytokines, and collagenase and the induction of increased cytotoxicity (571). In terms of antigen presentation, it is not clear whether IL-1 is required for T cell activation, although under certain conditions the addition of IL-1 seems to restore the ability of macrophages to induce T cell activation after the depletion or inactivation of APCs (570). IL-1 secretion is only one aspect of accessory cell function; it is possible that different T cells or different activation signals may vary in their dependency on IL-1 stimulation. 2 . IL-3
IL-3 is a lymphokine secreted by activated T cells (75, 260, 586) and some myelomonocytic cell lines such as WEHI-3B (586, 587). Both Thl and Th2 cells secrete IL-3 (75, 260).
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a. Effect of IL-3 on Nonlymphoid Cells. In contrast to the other lymphokines discussed in this section, the major targets for IL-3 are immature myeloid progenitor cells, rather than mature lymphocytes. IL-3 induces the proliferation and differentiation of granulocytes, macrophages, and erythroid, megakaryocytic, and mast cell precursors (586, 588-592). Several mast cell lines or other hematopoietic cells are dependent upon IL-3 for their growth (586, 591, 592). The isolation and expression of IL-3 cDNA has confirmed that the multiple activities on a variety of hematopoietic cell lineages are mediated by IL-3 (591, 592). b. Effect of IL-3 on T and B Cells. The effects of IL-3 on cells of the lymphocytic lineage are controversial. It has been suggested that IL-3 is able to support the in m2ro growth of cells from bone marrow that will generate T and B cells when injected into irradiated recipients (593). It is not clear from these results whether IL-3 acts on multipotential stem cells committed to the lymphoid lineage or whether it programs the cells to enter such a lineage (593). It has been reported by Palacios et al. (594), however, that IL-3 can support the growth of a population of B cell precursors, but not mature B lymphocytes. In addition, a number of clones with characteristics of pro-B and pre-B cells that are dependent on IL-3 for continuous proliferation have been established (306, 595). Similarly, cultures of clones expressing low levels of surface Thy-1 and Ly-1 antigens are able to give rise to mature T lymphocytes after in mvo transfer (prosy cell clones) and to proliferate in response to IL-3 (595). In summary, in addition to both its activities on myeloid precursors and mature mast cells, IL-3 could be involved in the early stages of the maturation of T and B cells. These properties implicate IL-3 (in combination with other factors) as a potential link between the immune response, hematopoiesis, and the recruitment of effector cells such as monocytes, granulocytes, and mast cells.
3. IL-6 The multiple activities of IL-6 were independently described before it became evident that they were mediated by the same lymphokine. In the mouse, these activities included a hybridoma growth factor (HGF, or interluekin HP-1); a T cell-derived factor that stimulated the proliferation of B cell hybridomas and plasmacytomas (596, 597); a factor derived from the murine macrophage cell line, P388D1, which also supported the growth of hybridomas and plasmacytomas (598); and a growth factor for hybridomas produced by a variety of other cells, e.g., endothelial cells, peripheral blood cells, and fibroblasts (599). In the human, similar activities have also been described and related to a protein designated as a 26-kDa protein (600, 601), IFN-02 (600, 602), and B cell
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stimulatory factor (BSF)-2 (603). BSF-2 induced Ig secretion in B cells and lymphoblasts and had potent growth-promoting activity for hybridomas and plasmacytomas. Cloning and expression of the cDNA for IL-6 (602, 603) has confirmed that these multiple activities are mediated by the same molecule. IL-6 is secreted by a large variety of cells and has pleiotropic activities on a number of cellular targets. Moreover, the activities of IL-6 resemble those of IL-l, including pyrogen activity and an activity mediating costimulation of thymocytes (599). a. Effect of IL-6on B Cells. The activity of IL-6 as a growth factor for hybridomas and plasmacytomas has already been mentioned. In the human, IL-6 is a late-acting factor that promotes Ig secretion by activated B lymphocytes and lymphoblastoid cell lines (603, 604). In activated, normal B cells, IL-6 induces Ig secretion without proliferation. In contrast, it is a potent proliferative factor for lymphoblasts (604). Taken together, these results suggest that IL-6 acts on B cells after IL-4, IL-5, and IL-2. b. Effect of IL-6on T Cells. IL-6 acts as a cofactor for the generation of Tc cells from murine thymocytes in the presence of IL-2 and IFN-./ (605) and enhances differentiation of murine Tc precursors in primary allogeneic mixed-lymphocyte cultures (606). IL-6 also acts as a T cellactivating factor, promoting the induction of IL-2 expression by T h cells after mitogen stimulation (599, 607), or as a cofactor, inducing T cell proliferation after stimulation with mitogen or anti-TcR antibody (606). In addition, IL-6 is a cofactor required for the expression of IL-2R and IL-2 responsiveness by anti-CD28-activated human T cells (608). Although activated Th cells secrete IL-6, it is not clear whether its production is restricted to a particular T h cell type (Thl or Th2). c. Effect of IL-6on Other Cells. IL-6 can synergize with the IL-3 to promote the growth of hematopoietic progenitor cells (599, 607). Thus, IL-6, like IL-1, may function as a link between immune responses and inflammatory responses. Thus, its pleiotropic activities range from the enhancement of antibody secretion, T cell activation, and generation of Tc cells to mediating fever, acute-phase protein production, stimulation of hematopoiesis, and recruitment of effector cells. E. CONCLUSIONS In Sections I11 and IV, we discussed the two types of functionally different T h cells and the role of lymphokines in the regulation of T cell/B cell function. Several conclusions can be drawn: (1) The functional differences between the two T h cell subtypes can be accounted for, at least in part, by the lymphokines that each type of T h cell secretes after antigenic stimulation. (2) Lymphokines are pleiotropic, acting on
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different cell types and on cells not directly involved in the immune response. (3) Different lymphokines act at different stages of T cell/B cell development, although overlaps are common. (4) One lymphokine can induce a particular cell type to express receptors for a second lymphokine, thereby making cells responsive to the effects of the second lymphokine. ( 5 ) Alternatively, a particular lymphokine can antagonize the effects of another lymphokine on the same cell. (6)Lymphokines can act on lymphoid and/or myeloid cell precursors, regulating their maturation and recruitment. Although Thl versus Th2 cells have different effects on B cells and other cells in d r o , their role in vivo is less well understood. Nonetheless, studies in vivo have confirmed the requirement for IL-4 and IFN-y in IgE (487) and IgG2, (569) responses, respectively. In light of our current knowledge of T h cells and lymphokines, it is probable that as the immune system has evolved, it has developed a very complex set of interactions among its components that enable it to respond efficiently to a similarly complex variety of antigenic insults (i.e., extra- and intracellular organisms, malignant cells, and soluble toxins). Hence, the nature of the antigenic stimulus probably determines the nature of the immune response against it. Therefore, different types of antigenic challenges might preferentially activate a particular type of T h cell, i.e., Th2 cells seem more efficient at providing help to B cells (humoral immunity) and, because of the roles of IL-4 and IL-5 in IgE and IgA production, they may be important in hypersensitivity, immunity against certain parasites, and local immunity; Thl cells seem to be more efficient at mediating DTH reactions and cytotoxic responses against cellular targets or intracellular organisms. Again, lymphokines such as IFN-y and IL-2 may be involved. In Fig. 3 we have schematically summarized the activities of the lymphokines secreted by Thl and Th2 cells with special reference to their role in B cell activation, proliferation, and differentiation and the functions of effector cells. Additional experimentation is required in order to understand how different T h cells are generated and activated in vivo. It remains to be determined how activated Thl or Th2 cells and/or their lymphokines induce B cell activation and how lymphokines produced by one T h cell subset regulate the activities of the other T h subset or its lymphokines. Such information would not only be invaluable in the understanding of how immune responses are normally regulated, but also on how certain aspects of the immune response (i.e., hypersensitivity, antibody production, and cytotoxicity) could be potentially suppressed or enhanced in mvo for therapeutic purposes.
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FIG.3. Regulation of the immune response by T h cell subsets and their secreted lymphokines. B cells can be specifically activated by physical interactions with Thl or Th2 cells. Alternatively, large B cells can be nonspecifically activated by lymphokines. Of the lymphokines released by ThZ cells, IL-4 can function by inducing B cells to express higher levels of class I1 and CD23 molecules, inducing their entry into cycle (GI), enabling them to proliferate to a variety of stimuli and programming them to secrete IgG, and IgE following stimulation. IFN-7, secreted by Thl cells, can antagonize all the effects of IL-4 and can instead program the B cells to secrete IgG2,. IL-5 acts on cycling B cells to induce their continued proliferation, differentiation, and expression of IL-2Rs. IL-2 (from Thl cells) could also act on such cells via IL-2R to induce their proliferation and differentiation. IL-6 functions as a late-acting factor, stimulating proliferation and/or Ig secretion by activated B cells and plasma cells. IL-3 plays a role in the maturation of bone marrow precursors and immature lymphoid cells, although the latter is controversial. IL-1 and IL-6 can act as cofactors in the presence of other stimuli or lymphokines to enhance maturation, activation, and proliferation of T and B cells. Finally, these lymphokines also have effects on the maturation, proliferation, and activation of nonlymphoid cells such as mast cells, eosinophils, or macrophages, which have important roles in the effector phase of immune responses. The effector cells induced by each T h cell subset and their activities are listed in the far right-hand column.
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VI. Cellular Interactions in Wlvo-A Synthesis
In this section, we combine well-known facts about the immune system and how it responds to different pathogenic organisms with a contemporary view of the cellular interactions and effector mechanisms involved. Much of this discussion will be highly speculative and certainly controversial. Nevertheless, in our view, it is exactly this type of synthesis that is sorely lacking in most reviews on the cellular basis of immunity. As a result, both immunologists and nonimmunologists often have great difficulty (and we are no exception) in understanding and devising strategies to treat dysregulations of the immune system. In this regard, there is perhaps no better example of our ignorance in relating the etiology of an infectious disease to the immune system than in the case of acquired immune deficiency syndrome (AIDS). AIDS is a generally fatal viral disease which continues to be of great concern and confusion to immunologists. We know that the causative agent of the disease is the human immunodeficiency virus (HIV). There is a growing body of literature on the structure of the virus, its heterogeneity, the organization of its genome, and the mechanism by which it enters cells. There is also an increasing number of papers describing the many cell types that are infected with virus and the final decay of the immune system in overt AIDS. However, there is no clear understanding of how the infection progresses from latency to overt disease and how cellular interactions per se play positive versus negative roles in maintaining the latent state versus causing a collapse of the immune system. Thus, a major goal of cellular immunology is to bridge the gap between understanding the immune response to infectious organisms and its successful application to prevention and treatment of disease. This will become possible only when we understand how the immune system works at its most basic level and how the various components of the system interact and are regulated. For the sake of brevity, we will use only a few specific examples in our discussion and will not attempt to reference the many well-known and important papers that provided their factual basis. For this, we refer the reader to one of the many excellent microbiology/ immunology textbooks. Although this synthesis is incomplete and speculative, it brings to light the issues which we must understand and address over the next decade if we are to successfully wage a battle against infectious disease. Figure 4 depicts a hypothetical model of the major cellular interactions involved in the generation of a humoral antibody response. Although many aspects of the model must be further clarified, the object of this discussion is to place the events depicted in the model in the setting of
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a lymphoid organ where anatomical restraints will determine to what extent and by what mechanisms cellular interactions occur. More importantly, this must be viewed in the context of the whole animal, e.g., the trafficking of lymphocytes, other effector cells, and antibodies in the generation, maintenance, and down regulation of the immune response. For the individual to mount an effective immune response against a particular pathogen, a number of cellular and molecular events must be tightly regulated. These include the isotypes of antibody secreted by the progeny of the activated B cells, the generation of memory cells, and the activation of effector cells. An attractive working hypothesis to explain how cellular interactions are initiated and regulated is that the type of antigen and its route of entry will determine the site and mechanism by which it is metabolized by the host. This, in turn, will influence the types, numbers, and perhaps the sequence in which Thl and Th2 cells are activated. Once activated, these T h cells can then regulate, via cell contact and/or release of lymphokines, the isotype of antibody secreted by specific B cells as well as the additional types of effector mechanisms generated, i.e., mast cell activation, recruitment of eosinophils, macrophage activation, DTH, and the generation of Ts and Tc cells. ORGAN A. THELYMPHOID After local invasion of the body by pathogens or other antigens, cells in the draining lymph nodes interact to initiate an immune response. Within the lymph nodes, the B cells are sequestered in follicles, while the T cells (which enter the node through postcapillary venules) take up residence in paracortical areas. Soluble circulating antigen, antigen from the tissues, and antigen engulfed by macrophages enter the lymph node through the afferent lymphatics or through the capillaries. In the node, antigen is ingested by macrophages at the periphery of the cortex, in the germinal centers of the follicle, and in the afferent sinuses, diffuse cortex, and medulla. In contrast to macrophages, dendritic cells may not process antigen efficiently, but may bind antigen fragments released by macrophages. Antigen not ingested by APCs collects in the primary follicle and is retained on the FDCs in close proximity to the follicular B cells. There are several major mechanisms by which antigen from the blood and lymph can initiate cellular interactions in the lymph node. (1)When antigen concentrations are high, the macrophages within the nodes are very effective at ingesting organisms, degrading them in lysosomes, and either presenting antigenic fragments to T h cells or releasing antigen fragments which then bind to dendritic cells or B cells. Because of their
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efficiency in taking up foreign material, macrophages may play a predominant role both as APCs and as a source of released antigen fragments. (2) The dendritic cells in the follicles very effectively capture immune complexes and retain this material on their plasma membrane for significant periods of time. Small antigen-containing portions of the membrane of the FDCs pinch off and presumably can be taken up by B cells in the follicle. FDCs serve as reservoirs for native antigen and hence may be the major source of antigen for the follicular B cells. It is also possible that FDCs present paucivalent epitopes to B cells in a multivalent form, since high antigen concentrations on the surface of FDCs would be operationally multivalent. This would result in more effective cross-linking of sIg molecules on B cells. (3) In contrast to macrophages, antigen-specific B cells have an advantage in capturing antigen because of their clonally expressed sIg receptors and their proximity to FDCs. Hence, when antigen concentrations are low, the B cells may be the more efficient APCs. Furthermore, the B cells may play a role in clonally expanding T cells activated by dendritic cells. Taken together, mechanisms 1-3 above suggest that, in the simplest model, B cells would bind native antigen and T cells would bind processed antigen on the surface of clustered macrophages and dendritic cells at the periphery of the germinal centers. Here, clonal expansion of both the B and T h cells should occur after antigen-specific, MHC-restricted interactions. The mechanism by which the antigen-specificT cells travel
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across the paracortical areas to the germinal centers is not known, but this could be mediated by chemotactic factors secreted by APCs. After interactions between T h cells, B cells, and APCs, the activated T h cells secrete lymphokines that are responsible for the growth of both T cells and B cells as well as for the differentiation of the B cells into antibody-producing cells. Fully differentiated plasma cells migrate to the medulla of the lymph node and secrete antibody into the efferent lymphatics. Antibody secreted from all the regional lymph nodes is collected in the lymphatic system and enters the blood stream via the thoracic duct. In addition, memory cells in the node are probably FIG. 4. T cell/B cell interactions in the immune response; a synthesis. ( a ) Highaffinity sig molecules on B cells capture specific antigen. B cells process antigen, recycle fragments to their surface, and present fragments in association with class I1 molecules. B cells undergo early activation events (altered phospholipid metabolism, changes in membrane potential, increase in size, and increase in expression of class I1 molecules). (b) At the site of infection, or in the draining lymph nodes, antigen concentrations will be at their highest. Therefore, although antigen-specific B cells may not be available to capture antigen, other APCs (such as dendritic cells, macrophages, and Langerhans cells) will do so. These cells will take up antigen nonspecifically, process it, and present antigen fragments in association with class I1 molecules on their surface. In the case of dendritic cells, they will take up antigen fragments released by macrophages. FDCs will bind immune complexes. Resting T cells will be activated and then bind to specific antigen-presenting B cells. Note: T cells which become activated will either go directly to step c or become memory T cells capable of going on to step c at a later time. (c) B cells expressing antigenic fragments in association with class I1 molecules interact with T cells (from step b) expressing TcRs specific for the complex of antigen fragments and class I1 molecules. “Tight” interactions involve various T and B cell-associated surface molecules in addition to class I1 molecules and TcRs, i.e., LST4, LFA-la, and Thy-1. Specific interaction induces reorientation of intracellular components associated with the release of soluble proteins. ( d ) The nature of the antigen probably determines the type of Th cell which is activated. Interaction induces Th cells to secrete lymphokines and up regulates IL-2Rs and IL-4Rs, and cells then become responsive to their growthpromoting effects. Physical interaction also induces B cells to become responsive to the lymphokines secreted by the Th cells. Although lymphokines are nonspecific, their effects are exerted primarily on those cells involved in the antigen-specific response via close cellular interactions. (e) Signals mediated by physical interactions and secreted lymphokines induce B cell activation, proliferation, and differentiation. The programming of switching may be directed by some of the released lymphokines (i.e., IL-4). (Jand g) Secreted lymphokines can also act on cycling B cells, inducing their proliferation and differentiation, thus resulting in an antigen-nonspecificpolyclonal response. (h) Primed (memory) B cells process antigen and present fragments in association with class I1 molecules to T cells. These B cells can focus antigen which is in soluble form or antigen in the form of immune complexes which are presented to them on the surface of FDCs, icoccomes, or macrophages. Physical interaction with T cells is basically similar to that of unprimed B cells except that conjugation occurs very rapidly and requires lower concentrations of antigen. Resting memory T cells may be able to interact directly with B cells and may not require activation by dendritic cells.
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generated in the germinal centers by the dividing B cells. Perhaps when the supply of antigen is exhausted, cycling B cells return to their Go state and either remain in the follicles or leave the nodes in the efferent lymph and travel to other areas of the body (e.g., bone marrow). Factors influencing the migration of memory B cells to other locations may include the induction of homing receptors and/or the numbers of memory cells in the nodes, i.e., for a node to return to its resting state, memory cells would be released into the lymph. The isotype of antibody produced by plasma cells (e.g., IgG versus IgA) is probably controlled by the types of memory cells and Th cells in the different lymphoid tissues which are activated. The T cell-derived lymphokines from Thl or Th2 cells could mediate switching or induce B cells already expressing other isotypes to differentiate into plasma cells. Lymphokines would play a major role in regulating the secretion of different isotypes of antibody. Hence, IL-4 and IL-5 (secreted by Th2 cells) would be involved in IgGl, IgE, and IgA antibody production, while IFN-y (secreted by T h l cells) would be involved in IgGza antibody production. The growth and terminal differentiation of plasma cells may also require IL-6. For the Thl and Th2 cells to interact in regulating the B cells, lymphokines from one type of Th cell could up regulate receptors for lymphokines secreted by the other type of T h cell. Since evidence is mounting that Thl and Th2 cells might represent different stages of the same lineage, it is possible that low levels of antigen (on APCs) activate T h l cells and that Th2 cells require repeated antigenic stimulation to be activated. Alternatively, Th2 cells might reside near the follicles while Thl cells would cluster around macrophages in paracortical areas. Furthermore, antigen could induce Thl cells to become Th2 cells. This would imply that the type of T cell activated would depend on the dose and/or rate of clearance (versus persistence) of antigen as well as the location of the different types of T cells in the different areas of the node. For example, in the case of minor viral infections (where antigen levels in the circulation would initially be low) Thl cells, Ts cells, and Tc cells might be preferentially activated, whereas in the case of parasites (where antigen persists), Th2 cells may be activated (or T h l cells would be induced to differentiate into Th2 cells).
RESPONSES AGAINST BACTERIAL ANTIGENS B. THEIMMUNE Bacteria are highly diverse and can enter the host by many different routes. Hence, the type of immune response to bacteria may be dependent on the portal of entry into the body. For example, in the gut and mouth, bacteria must penetrate mucosal or epithelial surfaces to enter the bloodstream. Any type of immune response which prevents
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attachment of bacteria to mucosa or epithelial cells will effectively prevent systemic infection. On the other hand, it is desirable not to damage the protective lining of the gut, mouth, etc. IgA that does not fix complement can fulfill these criteria by preventing attachment of bacteria in the gut without causing tissue damage. Hence, the generation of IgAsecreting plasma cells would be highly desirable in Peyer’s patches. Bacteria that do gain entry into the bloodstream are cleared by macrophages or Kupffer cells or are lysed by circulating opsonizing antibodies (e.g., IgM, IgG) and complement. As a consequence of ingestion of antigen, macrophages can secrete a wide variety of mediators which influence both immunocytes and other cells. Hence, in the case of bacterial infections, three types of immunity are probably operative: (1) Phagocytosis of bacterial microorganisms by macrophages or neutrophils occurring both prior to the generation of an antibody response or, more efficiently, after the coating of these organisms with antibody and/or complement components. The opsonization and digestion of bacteria by macrophages results in destruction of the organism and also allows the macrophages to present fragments of bacterial antigens to T cells. (2) B cells might also be rapidly activated in a polyclonal manner by endotoxin or polysaccharides from the bacterial cell wall. (3) Th cells would be activated by B cell clones presenting TD epitopes from bacterial fragments or bacterial products, e.g., toxins. In the mouse, because antibody-mediated phagocytosis of bacteria, bacteriolysis, and the prevention of attachment of bacteria to mucosal and epithelial surfaces can be carried out by particular antibody isotypes, the secretion of IgGl and IgA antibodies may be dependent upon IL-4 and IL-5 secreted by Th2 cells. Thus, activation of Th2 cells is probably of major importance in the generation of antibodies which protect the host against systemic bacterial infections. In contrast, IgG3 responses (e.g., to bacterial polysaccharides) may be relatively thymus independent.
C. THEIMMUNE RESPONSE TO VIRALANTIGENS Viruses, unlike most bacteria, are obligate intracellular pathogens; their extracellular concentrations may be low in the early stages of infection. Furthermore, during cellular interactions in general, virus might be transferred from one cell to another without reaching high concentrations in the blood. Viruses can be neutralized or eliminated by a wide variety of immune mechanisms, including antibody-mediated neutralization (IgM, IgG, or IgA), complement-mediated neutralization, opsonization by macrophages, and cell-mediated cytotoxicity by Tc cells or NK cells. However, until viral antigens are synthesized and expressed on infected target cells or until virions are present in sufficient quantity
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in the circulation to be ingested by macrophages, degraded, and presented to T cells, humoral immunity, as well as the generation of Tc cells, would not occur. Assuming that sufficient antigenic exposure did occur, class I-restricted Tc cells or NK cells activated by IL-2-secreting Thl cells may play a major role in killing cells either expressing viral antigens and/or actively producing virions. Since Thl cells also secrete IFN-7, this should facilitate IgGza secretion by B cells and, in the mouse, IgGza appears to be protective in the case of some viral infections. Furthermore, IFN-.)I(also secreted by Thl cells) is an effective antiviral agent and is involved in the activation of macrophages. In contrast, in other viral infections where IgA or IgGl play major roles, Th2 cells would be activated by antigen and APCs.
D. THEIMMUNE RESPONSE TO PARASITES Parasites differ enormously in their organization, complexity, life cycles, and pathogenic mechanisms. From many studies in experimental animals, it has been established that different effector mechanisms are active against different parasites and that it is often difficult to predict which mechanism is the predominant one in a particular parasitic infection. Studies on immunity against parasites have suggested that in many cases the humoral antibody response plays a protective role. In the mouse, the two Ig isotypes most often involved in mediating antiparasitic responses are IgGl and IgE. IgE also plays a major role in antiparasite immunity in humans. Among the effector cells involved in immunity to parasites, eosinophils play a fundamental role in mediating parasite killing through interaction with parasites coated with specific antibody of the IgE and IgGl isotypes. This interaction results in the release of eosinophil granules, which contain substances toxic to parasites. Activated macrophages and neutrophils are also capable of killing parasites and, in some instances, mast cells are also involved. Based on these effector mechanisms, which involve IgGl and IgE antibodies, one would predict that the Th2 cells (which secrete IL-4 and IL-5) play a central role in the regulation of humoral immunity against many parasites. In particular, the persistence of parasites might be sufficient to induce the secretion of the large amounts of IL-4 necessary for the induction of IgE secretion (IgE secretion, in contrast to IgGl secretion, requires very high levels of IL-4 in mitogen-driven systems). IL-5could act by recruiting eosinophils, which would be further activated by factors derived from IL-4-activated mast cells. Mast cells would mediate parasite killing through their interaction with the IgE- and IgG1-coated organisms. IL-5 might also induce IgA secretion in local environments where parasites reside (e.g., gut).
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In addition to protective antibodies, the host can defend itself against some intracellular parasites by the development of DTH responses and the activation of macrophages, both of which correlate with protective immunity and healing of parasite-induced lesions. Thus, Thl cells may be involved in these responses, since Thl cells mediate DTH and secrete IFN-y, which is a major macrophage-activating factor.
E. CONCLUDING REMARKS Based on the considerations described above, information from three areas of research will be important to increase our understanding of which cellular interactions are involved in the protective immune response. These areas include (1) the effect of dose, route, and type of antigen on the activation and regulation of subsets of Thl and Th2 cells: (2) the mechanism by which anatomical barriers influence cellular interactions in vivo; and (3) the cellular and molecular mechanisms by which B cells interact with and are regulated by Thl and Th2 cells and their lymphokines to generate specific antibodies of the appropriate isotypes. The disciplines of cellular and molecular immunology represent powerful tools for further elucidating mechanisms involved in cellular interactions. However, the major insight necessary to further our understanding of the immune response in both physiological and pathological states in the intact animal will involve more information about the pathogens themselves, the nature of protective immunity to different types of pathogens, and the activation and regulation of the immune network to elicit these protective responses. ACKNOWLEDGMENTS We are indebted to Dr. Jonathan Uhr, Dr. Michael Berton, Dr. Mark Till, Ms. Alexis Bossie, and Ms. Tracy Stevens for helpful comments and insights concerning this manuscript. We thank all the fellows and students, past and present, who have worked with us and influenced our thinking. We thank Ms. G.A. Cheek and Ms. N. Stephens for their patient and heroic secretarial help. We thank the editors of this volume for providing us with the painful and unusual opportunity of updating (and backdating) our data base and then putting our thoughts together. We apologize to any individuals whose work we inadvertently omitted from this review and its list of citations (address all complaints to E. Vitetta). The experiments which went on while we were writing this review are supported by NIH Grants AI-11851, AI-12789, and AI-07824 and were carried out by Ms.D. Bryant, Ms. E. Carlton, Mr. Y. Chinn, Mr. J. Harris, Ms.S. Joyner, Ms. L. Le, Ms. M.-M. Liu, Ms. M. McCarthy, Ms. S. Shanahan, Ms. R. Summers, Ms. L. Trahan, and Ms. N. Yen. Although we have not yet had time to analyze the results of these experiments, we hope that they do not disprove too much of what we have presented herein. Finally, for those of you who have read this entire review, we hope we have stimulated your thinking as much as its writing has stimulated ours.
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ADVANCES IN IMMUNOLOGY, VOL. 45
MHC-Antigen Interaction: What Does the T Cell Receptor See? PHlLlPPE KOURILSKY AND JEAN-MICHEL CLAVERIE Unite do Biologie Moleculaire du Gene, U277 INSERM and UAC 115 CNRS. and Unite d’informailquo Scientifique, instltut pbrteur, 75724 Fbris Cedex 15, France
1. Introduction
In the last few years, considerable progress has been made in the understanding of the mechanisms by which T cells specifically recognize antigens presented by antigen-presenting cells (APCs). In most instances, a specific receptor carried on T cells (TcR) interacts with the antigen “presented” by molecules of the major histocompatibility complex (MHC). Since the seminal observations that T cells recognized both native and denatured antigens (Gell and Benacerraf, 1959; Franzl, 1962; Campbell and Garvey, 1963), and that APCs were necessary for T cells to recognize antigens (Mosier, 1967), the phenomenon of antigen presentation has been gradually elucidated. It was recognized, after the work of Ziegler and Unanue (1981), that in most cases the antigen is processed by the APC, such that fragments of antigen, rather than antigen per se, are presented by MHC molecules and are recognized by TcRs. The early experimental work on this major finding has been reviewed in detail (Unanue and Allen, 1987; Buus et al., 1987a,b; Long, 1988). There are numerous examples of synthetic peptides corresponding to defined portions of protein antigens that actually substitute for antigen in welldefined immunological reactions involving T cells. In several instances, it was shown directly that such peptides bind to MHC molecules. So far, however, such biochemical data are rather scarce, and the binding of the processed antigen to MHC molecules is often inferred from functional assays involving recognition by TcRs. It is well known that most MHC molecules are highly polymorphic (reviewed in Charron et al., 1988; Kappes and Strominger, 1988; Long et al., 1988; Parham et al., 1988a; Klein, 1986; Nathenson et al., 1986; Lew et al., 1986a). This is indeed how they were initially discovered as major transplantation antigens (Gorer, 1937; Snell, 1948; Dausset, 1958). This is also how their involvement in antigen presentation was initially perceived through the phenomenon known as MHC restriction. The 107 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
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polymorphism of MHC molecules may influence both antigen presentation and recognition by TcRs in many ways. Polymorphic residues from the peptide-binding site may influence the selection of a given set of fragments from the native antigen as well as their conformation and orientation. Other polymorphic residues may positively or negatively interfere with the recognition of the bound peptide with a given TcR. In the whole organism, the MHC-dependent tolerance to self-antigens will also influence the expression of the T cell repertoire (repertoire selection). Thus, much of the experimental evidence pertains to reactions involving a multimolecular complex, rather than solely antigen and MHC. Accordingly, it is often difficult to interpret experimental data relevant to MHC- antigen interaction without making assumptions on what the TcR actually sees, and how the T cell repertoire was generated. Starting from a complex situation, initially described in a global and imaged, albeit sometimes amazingly precise, fashion, the field has rapidly moved toward better defined and more molecular views. These advances have induced shifts of concepts which lead to a reappraisal of several questions. It is the purpose of this review to survey recent data and to analyze critically their interpretation. We show that the data can fit within a consistent, though hypothetical, framework of MHC-antigen-TcR interactions. It is our bias that the underlying hypotheses, which involve the presentation of self-peptides and their variants (Kourilsky and Claverie, 1986, 1989), may provide a unifying view where paradoxes that have intrigued immunologists for years may plausibly be resolved. To facilitate the reading, we have included a few schematic drawings of MHC molecules and TcRs. II. The T Cell Receptor and Accessory Molecules
The TcR is a major partner involved in the recognition of antigen as it is presented by the MHC molecules on an antigen-presenting cell. In addition, so-called accessory molecules may also strengthen the interaction between APCs and T cells. Well-documented reviews on TcRs (Kronenberg et al., 1986; Wilson et al., 1988; Davis and Bjorkman, 1988) and accessory molecules are available (P.Anderson et al., 1988; Dustin et al., 1988). Here, we give a brief outline of features relevant to the present discussion. TYPES OF TcRs A. THEDIFFERENT The polymorphic receptor carried by T cells that reacts with antigen presented by MHC molecule is a heterodimer made of two chains assembled in an antibody-likefashion. In adults, one finds predominantly
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TcRs made with CY and 0 chains (the a-P TcR). During ontogeny of the immune system, one finds T cells with a y-6 TcR made with y and 6 chains. These T cells seem to be predominantly found in epithelial tissues such as intestine and skin. Still other receptors may exist, as suggested by recent experiments performed in the chicken by Lahti et al. (1988). Matis et al. (1987a) have shown that a mouse 7-6TcR-mediated reaction can be MHC linked, as are ones mediated by the a-/3 TcRs. Diversity of TcRs is generated in much the same way as in immunoglobulins, through the combinatorial joining of DNA segments and the variable addition of nucleotides at junctions (see in Davis and Bjorkman, 1988; Marrack and Kappler, 1988a). No well-documented case of somatic mutation in TcRs has been reported so far. Accordingly, it has been thought that the overall diversity of TcRs might be less than that of antibodies. However, vastly different estimates (from lo9 to 1030) of theoretical repertoire sizes have been given (see in Davis and Bjorkman, 1988; Marrack and Kappler, 1988a). It is striking, however, that a limited number of variable genes have been found to be used in response to several antigens (cf. Section X,A), so that the actual repertoire size in a given individual may not be this high. Alignment of TcR sequences has led to defined hypervariable regions, much in the same way as in antibodies, with one possible major difference: the TcR CY and 0 sequences display only one well-clusteredregion of hypervariable residues (equivalent to CDR3). Outside this region, the variable residues are more evenly distributed than in antibodies and the regions corresponding to CDRl and CDR2 appear less diverse (Bougueleret and Claverie, 1987). In the absence of direct data on the three-dimensional structure of TcRs, models have been built under the assumption that TcRs display an immunoglobulin Fab-like structure (cf. IX,B). B. THECD3 COMPLEX AND ACCESSORY MOLECULES TcR heterodimers (a-p or y-6) are usually found associated with other molecules in the CD3 complex, which includes at least four additional polypeptide chains (reviewed in Clevers et al., 1988). In addition, a number of so-called accessory molecules participate in the interaction between T cells and APCs. Among these are CD4 and CD8, which determine restriction to a given MHC class (CD4, to class 11; CD8, to class I) and, to a certain extent determine helper T cells and CTL functions, respectively. Thus, CD4+ CD8- helper T cells recognize antigens presented by class I1 molecules, and CD8+ CD4CTLs recognize antigens presented by class I molecules. There are also examples of CD4+ CD8- class 11-restricted CTLs (Braakman et al.,
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1987). Other molecules (ICAM-1, LFA-1, etc.) also participate in cellular interactions (reviewed by P. Anderson et al., 1988; Dustin et al., 1988). Transfection experiments have definitively established that MHCrestricted antigen recognition is carried by the a-0 TcR heterodimer (Dembic et al., 1986; Saito et al., 1987; Kuo and Hood, 1987; Malissen et al., 1988). There is evidence that the CD4 and CD8 molecules are essential when the specific recognition of MHC-presented antigens by TcRs is relatively weak (e.g., Gabert et al., 1987). On the contrary, they appear to be dispensable when this interaction is “strong.” For instance, CD8-independent CTLs can be isolated (for a recent example, see Dembic et al., 1986). The physical relationship of CD4 and CD8 to the CDS complex is difficult to assess at this stage. It has been found recently that CD8 can bind to human class I MHC molecules through a disulfide bond (Blue et a l . , 1988; Bushkin et al., 1988). CD8 is disulfide linked to CD1 on immature thymocytes (Snow et al., 1985). CD4 protein binds to class I1 molecules on B cells (Doyle and Strominger, 1987; Gay et al., 1988). Work in progress will rapidly define more precisely which contacts are made by CD4 and CD8. These accessory molecules may bind not only the MHC molecules of the APCs, which present the specific antigen, but may also bind other MHC molecules. Accordingly, one might have to distinguish between MHC-dependent reactivity (not directly dependent upon antigen) and MHC-restricted recognition of antigen. The mode of action of CD4 and CD8 is not yet fully understood. It has been suggested that CD4 might be involved in signal transduction (Rudd et al., 1988), when brought into close physical proximity of the CDS-TcR complex in class II-restricted antigen recognition (Ledbetter et al., 1988). Since CD4, CD8, and other accessory molecules do not contribute to the intrinsic specificity of interactions involving MHC, antigen, and TcR, they will not, in spite of their immunological importance, be discussed further. 111. The MHC Molecules
The major histocompatibility complex is a chromosomal region that has been extensively characterized in several mammals, particularly man and mouse (Flavell et al., 1986) (where it is named the HLA and the H-2 complex, respectively), and a few other organisms, particularly the rat, the rabbit (6.Kulaga et al., 1987), the swine (cf. Ehrlich et al., 1987; Sachs et al., 1988), and the chicken (6.Guillemot et al., 1988; for a general review, see Klein, 1986). Molecules encoded in the MHC (in short, “MHC molecules”) and relevant to antigen presentation so
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far fall into two classes that differ structurally: class I MHC molecules are made of a polymorphic glycosylated “heavy” chain of about 45 kDa, which spans the cell membrane, associated with a poorly polymorphic, nonglycosylated, 12-kDa “light” chain, called &-microglobulin, not encoded in the MHC. Class I1 molecules are made of two polymorphic chains, a and 0, that both span the cell membrane and associate into a heterodimer. Early biochemical studies involving digestion with proteases suggested that class I heavy chains displayed three external structural domains, while a! and 0 chains of class I1 molecules each exhibited two. This belief was later reinforced by the striking correlation which was found between exons of the cloned genes and these putative domains. Two of these extracellular domains in both class I molecules and class I1 heterodimers display much more variability than the others, which more closely resemble immunoglobulin constant domain, and unambiguously include MHC molecules in the immunoglobulin superfamily (Williams and Barclay, 1988). Thus, it appeared possible that the structure of class I and class I1 molecules was, perhaps, not so different: each could be made, on the extracellular side, of two relatively constant and two more variable domains, albeit connected differently (three domains plus &-microglobulin versus “2 + 2”). Hypothetical molecular models displaying these features were built (Novotny and Auffray, 1984). STRUCTURE OF HLA-A2 A. THECRYSTAL Although the above predictions were grossly correct, the determination at the 3.5-Hi resolution of the crystal structure of the human class I antigen HLA-A2 was illuminating and const
