Immunotherapeutic Strategies Directed at the Trimolecular Complex

Immunotherapeutic Strategies Directed at the Trimolecular Complex

ADVANCES IN IMMUNOLOCY, VOL. 56 lmmunotherapeutic Strategies Directed at the Trimolecular Complex AMITABH GAUR A N D C. GARRISON FATHMAN Deportment o...

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ADVANCES IN IMMUNOLOCY, VOL. 56

lmmunotherapeutic Strategies Directed at the Trimolecular Complex AMITABH GAUR A N D C. GARRISON FATHMAN Deportment of Medicine, Division of Immunology ond Rheumatology, Stanford University Medical Center, Stanford Colifornio 94305

1. Introduction

T cell activation requires interaction among the components ofthe ternary complex: the T cell receptor, MHC gene products, and the nominal peptide antigen. Strategies aimed at inducing unresponsiveness in T cells have targeted each of the components of the activating complex. Prevention and treatment of autoimmune disorders as well as induction of transplantation tolerance are incentives to continue the effort in evolving strategies for establishing specific immune unresponsiveness. This review recapitulates earlier experience in preventing the formation of the ternary complex and discusses some newer attempts to induce unresponsiveness in experimental animals. The three components of the complex serve as independent targets for development of strategies aimed at disrupting the trimolecular complex (Fig 1). II. Target 1 : The T Cell

The earliest attempts at immunotherapy targeting the T cell used anti-lymphocyte serum and monoclonal antibodies directed at the Thy1 antigen, a marker for all T cells, to eliminate or downregulate T cell activation (Like et al., 1979; Maki et al., 1981, 1992; Ledbetter and Seaman, 1982; Cobbold et ul., 1983). The development of hybridoma technology (Kohler and Milstein, 1975; Galfre et al., 1979) made available reagents which could target a specific cell population. Among the target molecules on T cells are the CD3 complex and the CD8 and CD4 molecules. Antibodies to the nonpolymorphic regions of the T cell receptor and to the variable regions of the @-chainof the T cell receptor (TCR)have been used for immunotherapy. T cell vaccination and TCR peptide are the most recent entries into this field of T celldirected imniunotherapy. 219 Copyright 0 1994 b y Academic Press, Inc All rights of reproduction in any Corm reserved.

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Antibodies to *TCR a / p -TCR Vp

*MHC blocking peptides/analogues

f

Antigen Presenting Cell *Antibodies to MHC (la)

FIG.1. Schematic diagram showing the components of the ternary complex. Boxed legends show ways to disrupt the interactions between the components.

A. ANTI-CD3 ANTIBODY The T cell receptor consists of seven polypeptide chains which form the T cell receptor complex. The cup-chains are involved in direct interaction with the antigen MHC complex. The other chains ( y 8 & t 2 ) ofthe complex have important functions in signal transduction. Engagement of these polypeptide chains on the surface b y monoclonal antibodies can result in antigen-independent activation of the T cell. Antibody to the C D 3 complex, OKT3, was among the first monoclonal antibody to be developed for human T cell-surface antigens (Kung et al., 1979). It was quickly employed therapeutically for reversal of acute renal allograft rejection (Cosimi et al., 1981) and then evaluated in multicenter studies for its use in renal allograft rejection (Thistlethwaite et d.,1984; Ortho Study, 1985). Soon it was used as the only immunosuppressive therapy in patients receiving cadaver kidney transplants (Vigeral et d.,1986). The mode of action of antiC D 3 antibody (OKT3) in vivo was initially thought to be the removal

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of T cells from circulation evidenced by a sharp decline in T cell numbers following anti-CD3 administration. However, the elimination of T cells did not seem to be the prime factor in the success of OKT3 therapy. The antibody does not eliminate T cells for long and CD3-negative T cells reappear. Antibody binding to the T cell surface causes capping, internalization, and shedding of the CD3 complex resulting in a modulated expression of the T cell receptor complex and diminshed antigen recognition and response functions (Chatenoud et al., 1982). The use of OKT3 antibody in reversing transplantation rejection and its attendant side effects and problems has been discussed in detail elsewhere (Transplantation Proceedings, 1987). Antibody to the mouse CD3 was developed and like OKT3 found to inhibit antigen-specific cytolysis by T cells (Leo et al., 1987; Havran et al., 1987). The ability of the mouse CD3 antibody (145.2~11)to induce unresponsiveness was studied in uiuo. For up to 5 weeks after antibody administration cells from the recipient adult mice were completely unresponsive in CTL assays. The mechanisms of unresponsiveness were investigated (Hirsch et al., 1988).Though there was a substantial depletion of T cells from the periphery, spleen and lymph nodes still contained T cells. Anti-CD3 antibody seemed to be acting through mechanisms other than depletion of T cells possible surface modulation of the CD3 complex or delivery o f a “suppressive” or negative signal to T cells (Hirsch et ul., 1988). In a different study, injections of anti-CD3 antibody were given neonatally and the effects were studied in adult mice (Rueff-Juy et at., 1989). Although there was almost complete depletion of T cells in the peripheral organs, there was no significant decrease in thymocytes. However, there was complete suppression of T cell functions. A decrease in “bright” CD3+ cells was seen which was correlated with loss of function. The reappearance of function was correlated with a critical threshold of bright CD3’ cells. No difference in levels of mRNA transcripts for alp TCR and C D ~ was E observed between control and treated mice suggesting no apparent feedback mechanism acting on surface modulation of CD3.9 (Rueff-Juy et al., 1989). Studies mentioned thus far have demonstrated depletion of peripheral T cells following administration of the antibody without affecting the number of thymocytes. However, other studies have shown antiCD3-induced apoptosis in developing thymocytes 40 hr after the antiCD3 injection. Almost complete depletion of double-positive thymocytes was seen and the single-positive CD4+ subset was affected more than the CD8’ cells (Shi et al., 1991). Apoptosis of immature T cells seen in uiuo was confirmation of similar observations in vitro with

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thymic organ cultures (Smith et al., 1989; McConkey et al., 1989; Tadakuma et al., 1990) or with a leukemic T cell line (Takahashi et al., 1989). Though anti-CD3 antibody has been utilized to induce immunosuppression in uiuo, it is interesting to note that the same antibody can induce activation of T cells in uiuo. Anti-CD3 is a potent stimulator of T cells when added to cultures in uitro of either human (Van Wauwe et al., 1980) or murine T cells (Leo et al., 1987; MarusicGalesic et al., 1988). The antibody caused activation of T cells in uiwo was evidenced by induction of interleukin-2 (IL2)receptor and production of colony stimulating factor (CSF). At the doses studied the net result was immunosuppression; however, lower doses might be useful in inducing activation in immunocompromised hosts (Hirsch et al., 1989). The ability of anti-CD3 to induce activation in uiuo is an important caveat, especially when the antibody for immunosuppressive therapeutic effects is administered, and may account for some of the “side affects” observed at least in the early phase of the treatment regimen. Anti-CD3 induces activation of T cells when given intravenously. Release of various cytokines including tumor necrosis factor (TNF), interferon-y (IFNy), IL2, and IL3 in the circulation has been observed. Side effects caused by its activating potential have been discussed (Alegre et al., 1990; Chatenoud and Bach, 1992). Interestingly, the activation potential of anti-CD3 was associated with the intact antibody. F(ab’)2 fragments of the antibody may be more useful as immunosuppressive agents since they lack mitogenic properties (Hirsch et al., 1991). F(ab’)2fragments ofthe antibody given to thymectomized mice resulted in prolonged (up to 3 months) and marked impairment of CD4+ T cell functions including reduced proliferation and IL2 secretion to mitogenic stimulus. IL2 supplementation in uiuo restored T helper functions as evidenced by rejection of skin allografts. CD8+ T cells were not affected by the F(ab’)2 treatment (Hirsch et ul., 1991).Nonmitogenic F(ab’)2portions were shown to prevent lethal murine graft versus host disease (GVHD) in fully allogeneic bone marrow transplant recipients. Both depletion of cells and modulation of CD3/TCR complex were observed in the CD4+ subset. CD8’ T cells were again affected only to a limited degree (Blazar et al., 1993). These results demonstrated the ability of the F(ab’)2portion to induce specific unresponsiveness in the CD4+ subset of T cells without evoking activation-linked side effects associated with the intact antibody treatment. Since CD4+ helper T cells have been implicated in the initiation of different autoimmune diseases, nonmitogenic F(ab’)2 portions of anti-CD3 antibody have been tried in a few experimental models of

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autoimmune diseases. The effectiveness of anti-CD3 and its F(ab')2 fragment, in preventing autoimmune diabetes, was compared in the streptozotocin-induced diabetes model in mice. Both intact anti-CD3 and its F(ab')2 fragments were found to suppress insulitis and significantly reduce the occurrence of hyperglycemia as compared to untreated controls. Mice treated with nonmitogenic F(ab')2 did not show any of the signs of morbidity seen in the anti-CD3-treated mice. The depletion of T cells was less pronounced in the case of F(ab')S-treated mice. The cells from treated mice, however, showed reduced activity on challenge with mitogens in vitro (Herold et al., 1992). Apparently, nonmitogenic antibody treatment functioned by rendering the T cells unresponsive by modulating the surface expression of the CD3/TCR complex. In two other murine models of autoimmune diseases, collagen-induced arthritis and Lactobacillus coronary vasculitis (LCA) disease, induction was prevented when anti-CD3 treatment was given at an early stage (Bluestone et al., 1992). B. ANTI-CD4 ANTIBODY In an effort to reach specific populations of the helper/inducer subset of T cells, a specific marker for those cells was used as the target antigen of immunotherapy. Helper T lymphocytes express CD4, a transmembrane glycoprotein (Parnes, 1989)which acts as both an adhesion molecule and a signal transducer through its cytoplasmic tail (Glaichenhaus et al., 1991; Miceli et al., 1991).The use of monoclonal antibodies targeted to CD4 has resulted in subset depletion or inactivation in mice and rats with a concomitant loss of immune function associated with the CD4' T cells. For example, mice depleted of CD4+ helper T cells were unable to mount B cell-dependent IgG responses to a T cell-dependent antigen sheep red blood cells (SRBC) (Cobbold et al., 1984) or to antigens of the herpes simplex virus (Leist et al., 1987). CD4+-depletcd mice lacked DTH responses (Leist et al., 1987) and also exhibited prolonged time in rejecting skin grafts from mismatched donors (Cobbold and Waldmann, 1986).Studies have been carried out on the effects of anti-CD4 treatment in experimental animals by different groups with results very similar to those described above. Using a rat monoclonal antibody GK1.5, directed against mouse CD4 molecule (Dialynas et al., 1983a,b), to treat mice, we examined the effect of such treatment on immune unresponsiveness. BALB/c mice immunized with sperm whale myoglobin did not produce specific antibodies following CD4' T cell depletion with GK1.5 at the time of immunization. This humoral unresponsiveness was long lasting; mice remained unresponsive for more than 4 months despite a

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secondary challenge with sperm whale myoglobin (Goronzy et al., 1986). Similar results were obtained by others using bovine serum albumin or ovalbumin (Wofsy et al., 1985). Interestingly, anti-CD4 treatment 3 or more days following immunization with antigen failed to diminish the immunoglobulin response (Wofsy et al., 1985) suggesting an early requirement for B cell help from CD4+ cells. Benjamin and Waldmann (1986) have shown that antigen immunization at the time of anti-CD4 antibody YTS191.1 (Cobbold et al., 1984) treatment results in specific tolerance to the antigen as evidenced by lack of antibody response on secondary immunization 42 days after the antibody treatment. Responses to other antigens remained unaffected. These and similar observations (Gutstein et al., 1986) indicated the ability of anti-CD4 to induce antigen-specific unresponsiveness. Humoral unresponsiveness following anti-CD4 treatment was not limited to soluble antigens but was also seen in response to alloantigens (Weyand et al., 1989a). Cytotoxic T cell responses induced by CD4+ cells either to allotype dissimilar cells (Weyand et al., 1989a) or to virus-infected cells were also dramatically reduced ( Weyand et al., 198913). Anti-CD4 antibody therapy has been used in experimental animals in an attempt to induce transplantation tolerance. The rationale for this approach came from studies suggesting that CD4+ T cells played a crucial role in rejection of the tissue transplanted across both the MHC “major” or non-MHC “minor” barrier (Mason and Morris, 1986; Steinmuller, 1985; Rosenberg et al., 1987).Work in our laboratory has demonstrated that treatment with anti-CD4 antibody before transplant allowed survival of islet allografts (Shizuru et al., 1987).In these experiments, mice (B6 H-2b) were given a single regimen of treatment with anti-CD4 antibody GK1.5 at the time of allogeneic islet transplant from A/J (H-2a)mice. The recipient mice had been made diabetic by treatment with streptozotocin. Successful retention of the allograft islet transplant was measured by maintenance of normoglycemia in the recipient mice. Our results showed indefinite survival of the engrafted islets of Langerhans (Shizuru et al., 1987) following treatment with anti-CD4. The treatment apparently caused selective depletion of most CD4+ lymphocytes; however, 5%-10% CD4’ cells remained in the spleen and lymph nodes of the treated mice. Cells in the thymus were not depleted by this treatment (Goronzy et al., 1986). In xenogeneic islet grafts (rat to mouse) CD4 antibody treatment of the recipient along with anti-la immunotoxin treatment of the graft substantially increased survival time of the graft (Kaufman et al., 1988). We have also been able to achieve indefinite survival of islet allografts in a rat

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model using the mouse anti-rat CD4 antibody 0x38. Pretransplantation treatment of diabetic ACI rats with OX38 allowed acceptance of Lewis rat islets. In the same study, anti-CD8 antibody treatment did not allow long-term allograft survival. Anti-CD8 antibodies when coadministered with anti-CD4 abrogated transplant tolerance and allowed rejection of the allograft. This result suggested that CD8+ cells were soniehow involved in maintaining tissue-specific unresponsiveness (Seydel et al., 1991).This observation was in contrast to earlier observations of Waldmann and co-investigators, in studies in mice, who did not find a requirement of CD8+ cells for induction of anti-CD4mediated tolerance (Waldmann, 1989). Anti-CD4 antibody has additionally been shown to fiacilitate graft survival in skin and bone marrow transplants in mice (Cobbold et al., 1986). Survival of cardiac allografts improved substantially in mice following anti-CD4 therapy (Mottram et al., 1987; Madsen et al., 1987). The mechanism of anti-CD4-mediated “tolerance” in the case of either soluble antigens or transplantation models is not clear. However, some insights into the potential n-rechanism(s) are beginning to emerge. In some models depletion of CD4’ cells is required as suggested by the inability of the nondepleting chimeric CD4 (Alters et ul., 1989) antibodies to successfully treat niurine EAE (Alters et al., 1990). Also, as discussed earlier, humoral unresponsiveness to soluble antigens and survival of allografts seemed to correlate with the depletive capabilities of the antibody. However, nondepletive regimens have also been shown to induce tolerance to soluble antigens (Qin et al., 1987). Additionally, F(ab’)2 portions of anti-CD4 antibodies, though not capable of depleting CD4+ cells, were able to induce tolerance and immunosuppression (Gutstein and Wofsy, 1986; Carteron et d.,1988). Also high doses of a poorly depleting isotype rat IgG 2a anti-CD4 monoclonal antibody allowed tolerance induction again suggesting that depletion was not always required for induction of tolerance (Qin et al., 1989). Even in depletive regimens, cessation of antibody therapy allows repopulation of CD4+ cells to normal levels in approximately 90 days (Goronzy et al., 1986). We attempted to analyze unresponsiveness of the repopulated cells in B6 mice which had not rejected A/ J islets following anti-CD4 therapy. We observed that the repopulated cells responded as well to spleen cells of the donor in an MLR as to a third party. These data suggested the induction of tissue or antigen-specific tolerance but not general unresponsiveness to the donor. In other systems tissue-specific unresponsiveness has been suggested (Dalln-ran et nl., 1987; Armstrong et d., 1987). In a rat cardiac allograft model, we were again able to see

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unresponsiveness when anti-CD4 (0x38)antibody was used for allograft survival. ACI rats received Lewis rat hearts in the abdomen. Only rats treated with OX38 demonstrated indefinite survival of the allograft. A second transplant of a Lewis heart in a different site was accepted without additional treatment while a third party, BN rat heart, was rejected. BN rat hearts were accepted by naive ACI rats following anti-CD4 therapy. As in the mouse islet transplant model system, we observed apparent donor tissue-specific unresponsiveness in rats following anti-CD4 therapy (Shizuru et al., 1990).Similar results have been reported from other groups of rat cardiac allograft systems (Herbert and Roser, 1988; Roser, 1989).Anti CD4-induced donor-specific unresponsiveness persisted in the absence of the transplanted tissue for at least 90 days (the duration of the study) following removal of the transplanted allograft as evidenced by the nonrejection of a second donor-matched cardiac allograft placed 90 days after the removal of the first tolerated graft following anti-CD4 antibody therapy (Shizuru et aZ., 1990). This long-term unresponsiveness in the transplantation system is almost identical to that observed against soluble antigens given following anti-CD4 treatment (Benjamin et al., 1988). In an attempt to elucidate the mechanism of transplantation tolerance following anti-CD4 therapy, our group (Alters et aZ., 1991) studied islet allograft between MHC-disparate mice. The donor islets came from I-E+ mice A/J (I-Ek).In I-E+ mice, T cell receptor V p l l ' and VP5' T cells are deleted in the thymus. In our system, the presence of the I-E+ islet allograft in CD4-depleted recipient mice did not cause clonal deletion of Vp5 or V p l l T cells. No changes in the kinetics of repopulation ofVP5 or V p l l CD4+ cells were observed in transplanted or sham-transplanted anti-CD4-treated mice. FACS analysis using anti-Vpll antibodies 2 months after grafting and following repopulation of CD4+ cells revealed no decrease in VPll' cells. The percentage of V p l l ' T cells seen in anti-CD4-treated transplanted mice were comparable with those of anti-CD4-treated untransplanted mice. Since clonal deletion was obviously not responsible for unresponsiveness in the graft recipients, we assayed T cell receptor crosslinking using solid-phase immobilized Vpll-specific antibodies. Receptor crosslinking usually stimulates specific T cells to proliferate, whereas lack of proliferation is usually correlated with anergy. In our crosslinking experiments, cells from the anti-CD4-treated and I-E islet-engrafted mice failed to respond to anti-Voll crosslinking. There was no difference in the level of stimulation achieved by control antibodies, antiVp8, or anti-CD3between grafted and control mice. Sorted populations of T cells from treated and grafted mice showed little or no stimulation +

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in response to Vpl l-specific immobilized antibody in CD4 or CD8 T cells arguing for anergy induction in both CD8+ and CD4 + populations (Alters et at., 1991). To examine the possibility that suppressor cells in transplanted mice caused the receptor crosslinking unresponsiveness, CD4- and CD8-sorted populations were mixed with normal B6 cells (negatively sorted for CD4 or CD8 cells) in an anti-Vp11 stimulation assay. Neither CD4 nor CD8 cells from tolerant mice affected the response of the normal B6 cells in TCR crosslinking (Alters et al., 1991) ruling out any apparent suppressor populations generated by anti-CD4 treatment. Qin et al. (1989, 1990) reported results similar to ours but using nondepleting anti-CD4 antibodies in generating tolerance or anergy. As reported earlier for soluble antigens, e. g., human y-globulin, tolerance induction in their studies required immunization with the antigen under the umbrella of nondepleting CD4 antibodies. The resulting antigen-specific tolerance lasted for a finite period unless immunizations were repeated which could extend and strengthen the specific tolerance. It was postulated that the reversion to the responsive state was due to the arrival of new thymic emigrants which were not tolerant. This hypothesis was supported by the continued unresponsiveness of mice thymectomized after being tolerized. The establishment of tolerance to minor mismatched skin grafts required anti-CD8 antibodies, in addition to anti-CD4 antibodies. However, in this instance, the tolerance was long lasting presumably because of continuous exposure of thymic emigrants to the foreign antigen expressed on the grafted tissue. In a finding similar to that observed by Alters et al., but using nondepleting regimens of antiCD4 antibody, it was found that the Mls 1" (present on the graft)reactive subset of T cells, Vp6 +,was not deleted from the periphery, but rendered anergic as assessed by their inability to proliferate in uitro to either VP6-specific antibody or Mls1"-expressing stimulator cells (Cobbold et al., 1990). GK1.5 treatment, in animal models of autoimmune diseases, has been shown to be effective in blocking or halting the progression of disease. Treatment with anti-CD4 antibody around the time of immunization with myelin basic protein (MBP) prevented experimental autoimmune encephalomyelitis (EAE) (Brostoff and Mason, 1984). Ongoing EAE was also reversed with anti-CD4 treatment (Waldor et al., 1985; 1987a). Experimental autoimmune myasthenia gravis (Christadoss and Dauphinee, 1986), systemic lupus erythematosus (Wofsy and Seaman, 1985), and collagen-induced arthritis (Ranges et al., 1985) have all been treated with anti-CD4 treatment. Using depletive anti-CD4 antibody treatment, our laboratory has prevented the +

+

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development of spontaneous diabetes mellitus in NOD mice (Shizuru et al., 1988).A striking feature ofthe therapy was the long-term absence of disease even after cessation of antibody administration. N o prevention of IDDM was observed if NOD mice were treated for a short course despite adequate depletion. Though we used depleting antibodies for therapy, Carteron et al. (1989) used F(ab')2 fragment of the anti-CD4 antibody in treating another autoimmune disease, mouse lupus, with success. This nondepletive CD4 antibody was demonstrated to be effective in treating autoimmune diseases but again required long-term treatment.

C. ANTI-^^ TCR ANTIBODY About 90% of peripheral T cells express the alp heterodimer; 10% express the y / 6 T cell receptor. The development of monoclonal antibodies (R73 and H57-597) to a nonpolymorphic determinant in the constant region of the rat (Hunig et al., 1989) or mouse (Kubo et al., 1989) alp T cell receptor generated the opportunity for using these antibodies as immunotherapeutics in animal models. Attempts have been made to use these anti-alp TCR antibodies for prevention and treatment of type I1 collagen-induced arthritis in rats (Goldschmidt and Holmdahl, 1991;Yoshino et al., 1991a).Collagen-induced arthritis, an organ-specific autoimmune disease, shares similarities with human rheumatoid arthritis. Administration of anti-alp TCR antibody, either at the time of immunization, with type 11collagen just prior to development of arthritis, or after development of frank symptoms, resulted in inhibition of disease (Goldschmidt and Holmdahl, 1991). Antibody injections given at the time of immunization or before onset of arthritis resulted in complete prevention of disease for the duration of the treatment. The reversal of the disease symptoms in arthritic animals was notable following antibody administration. These impressive effects, however, did not last and after cessation of the antibody therapy severe arthritis was seen in treated animals. Though the antibody was able to deplete large numbers of T cells, the authors suggest functional blockade of the T cell receptor as the mechanism for antibody action. This hypothesis was based on the presence of fairly significant numbers of antibody-coated T cells in the circulation. Also, thymectomized rats went on to develop severe arthritis after a period of disease inhibition during antibody treatment suggesting functional inhibition rather than depletion being responsible for disease inhibitory effects of the anti-alp antibody. In a different rat model, streptococcal cell wallinduced arthritis, which also has similarities with human rheumatoid

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arthritis, anti-alp antibodies have been demonstrated to be effective in prevention of chronic disease (Yoshino et al., 1991b).Anti-alp treatment results in minimal destruction of cartilage and very low-level inflamation in the synovium of the tarsal joints. In these studies, rapid but incomplete depletion of the alp TCR' T cells was observed following anti-alp injection. However, by Day 3 85-90% of cells were eliminated and several injections maintained depletion. Following withdrawal of the treatment the alp' T cells returned gradually to about 70% of the normal level. This same group has reported success in using monoclonal anti-alp TCK antibody in treating and preventing adjuvant arthritis (Yoshino et al., 1990).Complete prevention ofspontaneous diabetes in the NOD female mice, a model for the human IDDM, was observed following weekly injections (during 8-24 weeks of age) of monoclonal a l p TCR-specific antibody (Kubo et al., 1989). Twice weekly treatment with F(ab')2 fragments was also shown to be successful (Sempe et al., 1991). Cyclophosphamide-induced acute diabetes in male NOD rnice was also prevented by treatment with a1 p TCR-specific monoclonal antibody. Interestingly, anti-alp treatment was also able to reduce the incidence ofinsulitis in 8-week-old NOD female mice following a single injection of 500 pg. Even overt diabetes could be reversed by anti-alp antibody treatment. After six daily injections of the antibody to six overtly diabetic mice all became normoglycemic, three only for a short period. Treatment of ongoing diabetes by this antibody suggests that cell-mediated effectors are one of its target. When the total IgG anti-alp was used, large-scale depletion of cells from the circulating pool was observed but the splenic population remained unaffected. That depletion was not the major mechanism was also shown by the efficacy of the nondepleting F(ab')2 fragments. N o general immunosuppressive effects were observed following antibody treatment; no significant changes were found in the ability of the NOD mice to maintain allogeneic skin grafts (Sempe et al., 1991).

D. ANTI-TCRVp ANTIBODY The knowledge that there existed a bias for expression of Va- or p-

chains in recognition of specific peptide antigens in the context of MHC molecules suggested yet another potential for immunotherapy. Analysis of antigen-reactive T cell clones revealed a limited heterogeneity in the use of Vp or V a genes in response to well-defined antigens. T cell responses to different model antigens showed limited heterogeneity in T cell receptor usage when analyzed using specific DNA probes or specific antibodies to the T cell receptor chains. Specific examples include pigeon cytochrome C (Fink et al., 1986; Winoto et

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al., 1986; Sorger et al., 1987; Matis et al., 1987; Hedrick et al., 1988),C1 h repressor (Lai et al., 1988,1990),and SWM (sperm whale myoglobin) (Morel et al., 1987; Danska et al., 1990). Immunotherapy based on

this limited TCR expression allowed treatment in both mouse and rat EAE, animal models of multiple sclerosis. Despite the difference in the antigenic epitopes and MHC restrictions essentially a single Vp gene was utilized by almost all the T cell clones analyzed from mice or rats (Happ and Heber-Katz, 1988; Burns et al., 1989; Acha-Orbea et al., 1988; Urban et al., 1988).This led Heber-Katz and Acha-Orbea (1989) to propose the V-region disease hypothesis which implicated the T cell receptor, not only in antigen recognition, but also as an effector in the initiation of the disease process by other unknown mechanisms. Restricted T cell receptor usage has also been reported in experimental autoimmune uveoretinitis where retinal S antigen reactive pathogenic T cell lines use rat homologues of the mouse Va2 and Vp8 (Merryman et al., 1991; Gregerson et al., 1991). All the studies on TCR usage in disease discussed above defined restricted TCR expression in recognition of different antigens on the basis of predominance of a given p- or a-chain in the T cell clones or lines established or maintained in uitro. Restricted TCR usage was also detected in uiuo. More than 90% of the proliferative response to the MBP epitope 1-11 was found in the VpS+ CD4+ T cells when lymph node cells of immunized PL/J (H-2") mice were sorted into Vp8' and Vp8- populations (Acha-Orbea et al., 1988). That limited heterogeneity in TCR usage demonstrated by T cell clones is a true reflection of the immune response in viuo was shown by our work examining the DBAl2 response to SWM. DBAl2 mice immunized with SWM or with an immunodominant determinant (aa110-121)mounted a strong T cell proliferative response which was limited to the Vp8' CD4+ T cell population (Ruberti et al., 1991). These findings showed that the limited heterogeneity in TCR usage demonstrated by the T cell clones was representative of the immune response in uivo. One attractive feature of a limited TCR use in response to defined antigens was the possibility that specific immunointervention could be applied to control a given immune response. Antibodies specific to the variable region of either a- or p-chains of the heterodimeric T cell receptor can be of potential use. Monoclonal antibodies specific for p-chain variable regions have been used for prevention and treatment of MBPinduced EAE in rodents. The (PL/J x SJL)F1 mouse generated predominantly Vp8 encephalitogenic T cells in response to immunization with either MBP or its N terminal epitope Acl-11. H-2" mice, PL/J or (PL/J x SJL)Fl, generate predominantly Vp8.2+, 1-A"+

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restricted encephalitogenic T cell clones in response to the N terminal Acl-11 epitope of MBP (Acha-Orbea e t al., 1988; Zamvil et al., 1988). F23.1, a depleting monoclonal antibody specific for all members of the Vp8 gene family (Staerz et al., 1985), was used to treat or prevent EAE in H-2" mice (reviewed by Acha-Orbea et al., 1989; Steinman, 1991). In a remarkable study, monoclonal F23.1 antibody was able to cause complete reversal of the disease process in 13 of 16 animals in which disease was induced by transfer of VP8.2+ encephalitogenic cloned T cells. F23.1 eliminated 98% of the Vp8' T cells from circulation and prevented the induction of disease in 18 of 19 mice following immunization with the Acl-11 encephalitogenic epitope of MBP. Even when guinea pig MBP, containing multiple pathogenic epitopes, was used to induce EAE, administration of anti-VP8 after the development of symptoms resulted in substantial (12 of 19) reversal of the disease (Acha-Orbea et al., 1988). Urban et al. (1988) have also used KJ16, another monoclonal antibody (specific for Vp8.1 and 8.2) (Haskins et al., 1984) in preventing EAE in B1O.PL mice. In the B1O.PL (H-2") mice although 84% of T cell clones responding to the N terminal determinant of MBP were Vp8.2' there were some clones (16 in number) which expressed Vp13. Although treatment in vivo with a Vp8.2specific monoclonal antibody F23.2 (Staerz and Bevan, 1985)resulted in almost complete depletion of Vp8.2 cells, it did not completely eliminate proliferative T cell response to MBP nor did it completely prevent the occurrence of MBP-induced EAE. Although 75% of animals treated with anti Vp8.2 did not develop symptoms, 5 of 20 developed fulminant disease in the treated group. Since 1/6th of the MBP-reactive clones expressed VP13 and not Vp8.2 a depleting antiVp13 monoclonal antibody was also used in these experiments. AntiVp13 treatment alone failed to have any impact on disease progression; however, when given along with Vp8.2 specific antibody, it resulted in a dramatic drop (only 1 of 20) in disease incidence (Zaller et al., 19'30). Also, lymph node cells from double antibody-treated animals did not respond to MBP in a proliferation assay. The same mixture of antibodies was found effective in reversing MBP-induced paralysis in the B1O.PL mice (Zaller et al., 1990). Sakai et al., (1988) attempted the use of Vpl7a-specific monoclonal antibody in suppressing EAE in SJL/J (H-2') mice. These mice respond to aa 89-101 of the MBP utilizing largely Vp17a+ clones. However, about half of the clones do not use Vpl7a+ T cell receptor. Whereas both Vp17a+ and Vp17a- clones were effective in transferring disease in naive animals. In this study, depletion mediated by antiVp17a antibody, KJ23a (Kappler et al., 1987), was effective in sup-

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pressing EAE when caused by transfer of 89-101-specific Vp17a T cell clones but was ineffective when the disease was induced by 89-101specific but Vpl7a-negative T cell clones or MBP or even 89-101 peptide. The presence of another epitope nested within 89-101 epitope which is recognized by VP17a- T cells could account for these results. Padula et al. (1991) reported the use of VP4+ T cells in recognizing a new epitope 92-103 in the SJL mice. Anti-VP4 (KT4), when given along with the MBP-specific cell line (57% cells expressing Vp4 TCR) in an adoptive transfer system, was very effective in preventing the development of disease. Three of four mice remained disease free and the fourth had a mild disease as opposed to all five animals which developed severe EAE in the control antibody group. In Lewis rats, an anti-TCR monoclonal antibody recognizing an idiotope on an MBP 68-88-specific rat T cell clone was also used to cause reduction in MBP-induced EAE incidence and severity in 67% of treated animals (Owahashi and Heber-Katz, 1988).The experiments discussed above demonstrate the efficacy of anti-TCR VP-specific monoclonal antibodies in downregulation of immune response and, in the case of autoimmunity, amelioration of disease. The efficacy of this approach depends on the oligoclonality of the T cells in response to a given antigen. Response of a different clonal population (possibly of a lower affinity) on elimination of the main responding population could nullify the immunosuppressive effect. We have shown that despite the neutralizing of the majority of Vp8' cells by SEB treatment mice still developed EAE following immunization with encephalitogenic Acl-11 epitope of MBP. These data were suggestive of the emergence of an encephalitogenic non-Vp8 population having lower affinity for Acl-11 epitope but capable of causing disease (Gaur et al., 1993a). Also, nested epitopes inducing different T cell clonal populations could pose a problem. In situations where more than one TCR is utilized a cocktail of anti-Vp antibodies could be used but oligoclonality of the response remains an essential requirement for the effectiveness of the anti-Vp approach. This was shown in the case of collageninduced arthritis where restricted Vp usage was indicated in disease induction (Banerjee et al., 1988). Use of different Vp monoclonals did not have the desired immunosuppressive effect, possibly because of lack of TCR restriction in the response (Goldschmidt et al., 1990). The situation is further complicated in human autoimmune diseases where no clear restriction in receptor usage has been observed. Analyses of TCR usage in T cell clones obtained from the synovial fluid of a rheumatoid arthritis patient have resulted in conflicting reports. One study demonstrated oligoclonality in clones expanded in vitro (Sta-

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menkovic et al., 1988) whereas others failed to find any evidence for restricted TCR usage in such T cell clones (Duby et al., 1989).In MS, T cell clones from the cerebral spinal fluid but not from the peripheral blood show limited receptor usage (Hafler et al., 1988).Oksenberg et al. (1993) reported conservation of the junctional sequences of the Vp5.2 from direct polymerase chain reactions (PCRs) of plaques from MS patients. Five different motifs were seen. Interestingly one motif was identical to the V-D-J sequence of an M B P peptide-specific clone isolated from an MS patient. The importance of this finding was underscored by the fact that encephalitogenic rat T cell clones, recognizing a MBP epitope similar to that of the human clone, had same amino acid sequences in the V-D-J region of the TCR p-chain. In another report, Vp usage in the peripheral blood and synovial fluid of rheumatoid arthritis (RA) patients was compared by PCR amplification employing specific Vp oligomers. In the seven RA patients, the frequency of the Vp14' cells in the peripheral blood was extremely low but was significantly increased in the synovial fluid ofthe affected joints. There was no skewing in Vp14' cells in the peripheral blood and synovial fluid of patients with nonrheumatoid arthritis inflammations. The VD-J sequencing of the Vp14 cells showed that 46 to 72% of the Vp14 population in the synovium of HA patients had a single clonotype. The near complete absence of Vp14' cells in the periphery of these patients led the authors to suggest that a superantigen (sharing reactivity with the putative autoantigen at the site of inflammation) could be involved in both elimination of V/314+ cells from the periphery and their oligoclonal expansion in the synovium (Paliard et ul., 1991).The complex etiopathology of autoimmune diseases may not allow such precise but simplistic immunotherapeutic regimens such as the one discussed in this section (Sinha et al., 1990).

E. T CELLVACCINATION In the preceding sections, we have discussed the use of antibodies directed at various proteins on the T eel1 surface in regulating T cell responses, We now examine T cells as effectors of immune regulation. This approach involves the use ofantigen-specific T cell lines or clones as vaccinating agents to elicit "anti-idiotypic" regulator T cells capable of inhibiting the response of the antigen-specific T cell population. As opposed to passive administration of antibodies, this approach involves active participation of the individual's immune system in inducing a regulatory response to the immunizing cells. Using MBP-specific T cell lines for vaccination, Cohen and co-workers were successful in preventing EAE in experimental animals (Ben-Nun et d.,1981).Since

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then, they have tried various vaccination protocols (reviewed by Cohen, 1986,1989) in controlling experimental autoimmune diseases. After activation by antigen myelin basic protein-specific T cells were irradiated (Ben-Nun et al., 1981) and inoculated into Lewis rats. The irradiated cells were able to prevent MBP-induced disease but unable to protect against disease induced by adoptive transfer of MBP-specific T cell lines. Glutaraldehyde crosslinking or pressure aggregation of cell-surface molecules seemed to overcome this problem (Lider et ul., 1987). However, Offner et al. (1989) reported the requirement of both irradiation and pressure treatment of vaccinating T cells to obtain complete protection against active and passively transferred disease (Offner et al., 1989). Even a heterogeneous T cell population like lymph node cells, with few antigen-specific cells, or a nonattenuated subpathogenic dose of a T cell line, was shown to be effective in protecting from active or passive EAE (Lider et al., 1987; Beraud et al., 1989). A nonencephalitogenic T cell clone specific for epitopes other than 72-89 of guinea pig myelin basic protein isolated during the recovery phase of EAE has been used to prevent and treat both active and passive EAE in rats. This clone [expressing Vp8.6 TCR and specific for amino acid 55-69 of GPBP (guinea pig basic protein)] apparently shares a cross-reactive idiotype with encephalitogenic clones, specific for other epitopes. Thus vaccination with this clone downregulates the response of encephalitogenic T cells (Offner et al., 1991a). Besides EAE, T cell vaccination has been used in the prevention of other induced autoimmune diseases including thyroiditis (Maron et al., 1983)and adjuvant arthritis (Lider et al., 1987). Vaccination with irradiated mouse spleen cells primed and activated to thyroglobulin prevented development of EAT (experimental autoimmune thyroiditis) in mice on challenge with thyroglobulin in adjuvant; again such vaccination did not block passive disease induced by adoptive transfer of antigen-specific T cells. The preventative effect of T cell vaccination was not reduced following depletion of CD8+ cells prior to vaccination. Depletion of either CD4+ or CD8+ cells after vaccination did not affect the protective ability of T cell vaccination but depletion of both subsets at the same time abrogated the protective effect of T cell vaccination. This indicated requirement of both subsets to mediate T cell vaccination-induced downregulation of specific immune response (Flynn and Kong, 1991). Experimental autoimmune thyroiditis induced in mice by immunization with thyroglobulin was shown to be prevented by vaccination with a cytotoxic MHC class Irestricted antigen-specific T cell hybridoma. The vaccination was done 3 weeks before induction of disease with the antigen (Roubaty et al.,

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1990).Monoclonal anti-clonotypic antibody specific for this hybridoma was found to be effective in completely preventing antigen-induced thyroiditis in mice (no reduction in autoantibodies to thyroglobulin was seen in treated mice), suggesting that T cell vaccination may well induce anti-clonotypic antibodies, in addition to anti-idiotypic T cells, to neutralize the pathogenic T cells (Texier et nl., 1992). A CD4+ CD8- T cell line specific for mouse testicular antigens has been shown to suppress the induction of autoimmune orchitis (EAO) in mice when given prior to challenge with the autoantigen. Both cellular and antibody responses were suppressed in an antigen-specific manner in treated mice (Itoh et ul., 1992). Experimental autoimmune neuritis (Taylor and Hughes, 1988)and collagen II-induced arthritis (Kakimoto et nl., 1988) have also been suppressed by vaccinations with antigenspecific T cells. Prevaccination with a subuveitogenic dose of antigenspecific T cells resulted in a marked reduction in pathology of experimental autoimmune uveoretinitis (EAU), on subsequent transfer of a disease-inducing dose of the same uveitogenic T cell line. Actively induced disease was not diminished by such treatment. However, anti-idiotypic or anti-ergotypic responses after treatment were observed and could be implicated in regulating the response of pathogenic T cells (Beraud et ul., 1992). MRL/lpr mice spontaneously develop systemic lupus erythematosus (SLE).Vaccination of young mice with low numbers (0.25 million) ofirradiated CD3’ CD4- CD8- cells isolated from hyperplastic lymph nodes of 6-month-old mice resulted in marked reduction in splenic hyperplasia and lymphadenopathy. Transfer of lymph node cells from vaccinated mice to 2-month-old recipients showed a significant decrease in autoimmune signs as evidenced by decreased proteinuria and increased life span (De Alboran et al., 1992). T cell vaccination seems to induce an anti-idiotypic response mediated by CD4’ T cells which “regulate” antigen-specific T cells. CD8’ anti-idiotypic populations have also been generated capable of lysing the idiotype-bearing T cell in uitro. This could account, in part, for certain regulatory effects seen in uivo (Sun et al., 1988). However, the presence of MBP-specific but avirulent T cells in T cell-vaccinated rats suggests the existence of more than one mechanism. How the CD4 arm of the anti-idiotypic response controls the idiotype-positive T cell is not known. However, the CD4 cells do have a suppressive effect as was shown when cells from vaccinated mice inhibited the proliferative response of the idiotype-positive cells to the specific antigen (Cohen, 1986). T cells used in vaccination are more efficient in generating anti-idiotypic responses if they are activated. Antigen or

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mitogen-activated cells seem more efficient in vaccination protocols to prevent disease (Cohen, 1986). This was demonstrated when 5 x lo7 nonactivated cells (with the same specificity) were less effective than fewer (
F. TCR PEPTIDEVACCINATION In a further refinement of the T cell vaccination approach, Vanden-

bark et uZ. (1989) used synthetic peptides corresponding to the pro-

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posed coniplementarity determining region (CDR2) of the Vp8.2 encephalitogenic TCR in Lewis rats. They were able to demonstrate protection from MBP-induced EAE by prevaccinating rats with a synthetic TCR peptide. The protection was specific as peptides corresponding to the CDR2 of the Vp14 chain did not have any effect. TCR peptide-specific cell lines derived from vaccinated rats were not cytotoxic for Vp8.2-expressing MBP-specific T cell lines, ruling out elimination of MBP-specific T cells as the cause for TCR peptide vaccination induced protection from EAE. The predominant role of T cells induced by this vaccination was demonstrated by prevention of disease in animals receiving TCR peptide-specific cells prior to MBP immunization. Antibodies to the T cell receptor peptides were also generated and were reported to prevent EAE in rats. Polyclonal rat or rabbit antibodies specific for the TCR peptide were administered in rats and drastically reduced the severity of the MBP-induced EAE (Hashim et al., 1990). The antibodies specifically stained Vp8.2expressing T cell lines. The staining was weak and not consistent with recognition of the intact T cell receptor. It may be proposed that processed fragments of the TCR bound to cell-surface MHC molecules are the targets of antibody recognition. In a different experiment, nonapeptides from the junctional region (CDR3)of the p-chain ofan encephalitogenic T cell receptor were used i n controlling MBP-induced EAE in rats (Howell et al., 1989). An 11 amino acid peptide corresponding to part of the J region of the a-chain was also tried but peptides longer than 9 amino acids spanning the V-D-J region were not as effective in preventing disease (Howell et al., 1989). TCR peptides (CDR2) have also been shown to treat ongoing EAE in rats. Aqueous solutions of TCR peptides, given either intradermally or subcutaneously after the onset of clinical symptoms, were able to reduce the severity and duration of disease in rats (Offner et al., 1991b).TCR peptides (CRD2) from a variable region of other p-chains like Vp6 which is very similar to VP8.2 were also effective in treating EAE induced in Lewis rats with an encephalitogenic epitope (aa 85-99) of MBP (Offner et al., 1992). A combination of CDR2 TCR peptides from Vp8.2 and Vp4 given in saline subcutaneously at 4-day intervals was shown to effectively block the progression of MBP-induced relapsing EAE in mice (Whitham et al., 1993). We have adapted this TCR peptide vaccination strategy to a mouse model and studied the response in DBA/2 mice to a model antigen. DBAI2 ( H-2d)mice respond to the iminunodominant myoglobin determinant (110-121) by utilizing mostly Vp8.2' T cells (Rubeiti et al., 1991). Immunization with a synthetic peptide corresponding to the

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putative CDR2 region of the mouse Vp8.2 TCR resulted in significant reduction in the proliferative response of vaccinated mice to the myoglobin determinant (Gaur et al., 1993b). Peptide vaccination did not affect non-Vp8.2 responses as evidenced by a lack of effect on the response to the A repressor C 1 protein 12-26, a response which does not utilize V/38.2+ T cells (Lai et al., 1988,1990).TCR peptide-specific antibodies failed to show any binding to Vp8.2' T cells and were not effective in blocking antigen-driven Vp8.2 T cell responses. While investigating the mechanisms of unresponsiveness induced by TCR peptide vaccination, we noted a significant reduction in the anti-Vp8.2 antibody-induced proliferation (TCR crosslinking-induced proliferation) of cells from TCR peptide-vaccinated mice. This suggested that TCR peptide vaccination was probably inducing unresponsiveness in the T cell population expressing Vp8.2 T cell receptors. The responses to TCR crosslinking by control antibodies remained unaffected in these vaccinated mice (Gaur et at., 1993b).This unresponsiveness in the Vp8.2 population of T cells was not due to depletion as revealed b y FACS analysis of peripheral blood lymphocytes which showed maintenance of similar numbers of Vp8.2' cells in control and TCR peptide-vaccinated mice. Similar effects of TCR peptide vaccination were seen in H-2" (PL/J X SJL )F, mice, the EAE-susceptible strain. H-2" mice develop a proliferative response specific to the TCR peptide following immunization and also exhibit reduced proliferation 0fVp8.2 T cells in receptor crosslinking assay (Gaur et al., 1993b).Investigating further for the probable mediator of TCR peptide-induced anergy we depleted mice or CD8+cells by antibody treatment prior to vaccination with the TCR peptide. Mice depleted of CD8' cells did not demonstrate Vp8.2-specific unresponsiveness following vacination with the Vp8.Z TCR peptide (Gaur et al., 1993b) These results implicated the CD8 cells in TCR peptide-induced unresponsiveness. One scenario would suggest that the TCR peptides obtained by processingofendogenous TCR chains could associate with MHC class I molecules and be presented on the surface of the T cells. The TCR peptide-MHC I complex serves as target for the TCR peptide-specific CD8+ regulator/ suppressor T cells generated by TCR peptide vaccination. Most of the TCR chains synthesized within the T cell are degraded (Minami et al., 1987; Bonifacino et al., 1989) and, as has been shown by Jiang et al. (1991),these fragments of TCR could be colocalized in the endocytic compartment along with class I MHC complexes. This raises the possibility that such complexes could be present on the surface of the T cell and, even without purposeful immunization, serve as components of a natural regulatory circuit for T cell responses. CD8+ T cells have +

+

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been shown to be involved in normal regulation of EAE in mice. Mice lacking CDS' cells had more relapses and developed chronic EAE (Jiang et al., 1992; Koh et al., 1992). The observation that rats immunized with MBP developed a TCR peptide-specific response (Offner et al., 1991b) argues for an endogenous regulatory system wherein the expansion of specific Vp + T cells following antigenic challenge leads to anti-Vp T cell response. If the TCR peptide is associated with the class I MHC molecule, then it should be within the size constraints of the peptides found associated with class I molecules. In the rat, an 11 amino acid peptide contained within the larger 23 amino acid 3961 TCR peptide sequence was found to be the minimal epitope required to obtain the effects of TCR peptide vaccination. The sequence, 44-54, elicits TCR peptide-specific class I-restricted T cells and prevents and treats active and passive EAE in Lewis rats (Vainiene et al., 1992; Hashim et al., 1992). For TCR peptide vaccination to be successful in controlling autoimmunity, as in the case of anti-TCR Vp monoclonal treatment, it requires a single Vp to be predominant in a pathogenic response. This offers advantages over antibody treatment as it evokes an immunoregulatory response after immunization. However, in a cautionary note the same CDR2 peptide from the VpS.2 was shown by Desquenne-Clark et al. (1991) to actually result in exacerbation of EAE in Lewis rats. Using high-performance liquid chromatography (HPLC)-purified peptides, results ranged from suppression to enhancement of disease. Kawano et al. (1991)also report mixed results using TCR peptides in controlling EAU. Retinal S antigen-induced EAU was inhibited in some experiments whereas TCR peptide vaccination-enhanced disease symptoms in interphotoreceptor retinoid-binding protein (IRBP) induced EAU. 111. Target 2: The Peptide Antigen

The minimal-peptide T cell epitope (determinant)of an autoantigen is the most attractive component of the trimolecular complex as it offers the highest specificity of immunoregulation. Through the use of the peptide epitope, T cell clones specific only for the peptide-MHC complex can be targeted for clonal elimination or induction of unresponsiveness. Obviously, only those disease situations where the autoantigen is known and the minimal T cell epitopes have been mapped would be amenable to this strategy. Efforts to identify the autoantigens in various autoimmune disorders are currently underway. In the following sections different approaches aimed at achieving specific unresponsiveness employing the peptide determinant are discussed.

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A. PEPTIDEDETERMINANT AS A TOLEROGEN Intact proteins or their peptide determinants have been shown to induce specific unresponsiveness if given in a nonimmunogenic fashion. Exposure to peptide antigen at the neonatal stage usually leads to antigen-specific unresponsiveness in the adult animal as shown in case of sperm whale myoglobin (Young and Atassi, 1983), self-MHC peptides (Benichou et al., 1990),and hepatitis B core antigen peptides (Milich et al., 1989). The inability to mount antigen-specific response on subsequent antigenic challenge in adult life could be due to elimination of antigen-specific T cell clones (Nossal, 1983; Gammon et al., 1986a), clonal anergy (Gammon et al., 1986b), or generation of specific suppressor or regulator cells (Oki and Sercarz, 1985). This approach, of inducing antigen-specific neonatal tolerance, has been used in an attempt to block development of disease in experimental models of autoimmunity. Collagen-induced arthritis in rodents serves as a model for human arthritis. Synthetic peptides corresponding to sequences in Type I1 collagen were injected neonatally to tolerize DBAll mice. At 6-8 weeks of age tolerized mice were immunized with Type I1 collagen to induce arthritis. Of the different peptides tested aa 122-147 (now known as 245-270) provided protection from disease symptoms. Mice tolerized with Type I1 collagen were protected completely in these experiments (Myers et al., 198913). Subsequently, residues important for tolerance induction have been identified in this peptide b y assessing the ability of substituted peptides to induce protection from arthritis in mice (Myers et al., 1992). Myasthenia gravis (MG), a T cell-dependent autoantibody-induced disease, is characterized by neuromuscular dysfunction. Immunization of rodents with acetylcholine receptor results in experimental autoimmune myasthenia gravis (EAMG), a disease similar to MG. Neonatal tolerance to a dominant and discriminatory T cell epitope of acetylcholine receptor (AcHR) achain from Torpedo cdifornica has been reported to confer protection from AcHR-induced disease in a murine model of myasthenia gravis. The region encompassed b y 146-162 ofthe a-chain ofAcHR is immunodominant in the C57BL6 (B6)(H-zb)mice but is unable to provoke disease on its own. However, newborn mice given synthetic peptide corresponding to 146-162 region of the AcHR were resistant to disease induction by AcHR. A reduction in anti-mouse AcHR antibodies was also observed in neonatally tolerized mice. Neonatal injection of intact a-chain of the AcHR was also found to b e effective in preventing clinical signs of EAMG in treated animals (Shenoy et al., 1993). Intraperitoneal administration of the dominant pathogenic N terminal peptide 1-9 (l-9NAc) of MBP in newborn B1O.PL (H-2”) mice

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resulted in protection from peptide-induced EAE in adult life. Protection from disease in tolerized mice correlated with reduction in proliferative T cell response to the peptide (Clayton et al., 1989). Though neonatal treatment with the peptide was able to prevent peptide-induced EAE it did not have any protective effect on the disease induced by immunization with complete MBP. This lack of protection from MBP-induced EAE was apparently due to the immunopathogenic activity of subdominant epitopes other than 1-9NAc in MBP capable of causing active disease symptoms (Clayton et al., 1989). Like tolerance in the neonates, tolerance in adult animals can also be induced in a determinant-specific manner, by selecting a nonimmunogenic formulation and route of antigen delivery. We have attempted to induce MBP-specific unresponsiveness in the adult H-2" (PL/J x SJL)F, mice using two encephalitogenic peptide determinants of MBP. The proliferative T cell response to the acetylated N terminal (Acl-11) determinant is dominant over the middle (amino acid 35-47) determinant of MBP in H-2" mice. Interestingly, the same hierarchy was maintained in the tolerance induction ability of the two determinants. Though both Acl-11 and 35-47 were able to induce tolerance to themselves, the latter was less potent in reducing MBP-specific response. A combination of the two peptides, when given prior to immunization with MBP, was able to reduce the MBP-specific proliferative response to the same extent as did tolerance to native MBP. The same hierarchy of tolerance induction was observed in the diseasepreventing ability of the two peptide determinants. While Acl-1 1was more effective in preventing MBP-induced EAE symptoms as compared to 35-47 peptide the combination of the two was most effective in preventing development of clinical signs of EAE (Gaur et al., 1992). Our findings on the hierarchy of dominance of determinants and its direct relationship with the tolerogenic potential were similar to those obtained by Ria et al. (1990) where, using linked synthetic peptide epitopes, they showed the same hierarchy in immunodominance as tolerogenicity. In clinical disease a patient presents with clinical signs of an autoimmune disease and, thus, already has autoreactive T cells. We used the two peptide determinants to treat ongoing EAE. Mice which were immunized with MBP to induce EAE were given the combination of the two peptides on the day of onset of disease symptoms. Of the seven mice in the group treated, six remained free of disease for over 200 days (after which the study was terminated), whereas all mice in the control group developed severe disease symptoms. Lymph node cells from similarly treated mice demonstrated reduced proliferation to MBP. To ascertain whether the un-

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responsiveness was because of clonal elimination or anergy, exogenous interleukin-2 ( I L 2) was added to the antgen-activated lymph node cell cultures from treated mice. Restoration of an MBP proliferative response by added IL2 suggested that an anergic state of the antigen-specific T cells was responsible for the effectiveness of peptide-mediated therapy of EAE (Gaur et al., 1992). Experimental autoimmune uveoretinitis is a model for human uveitis (Forrester et a,?.,1992). Intraveous injection ofuveitogenic peptides from the IRBP prior to immunogenic challenge with either the peptides or the whole IRBP was able to prevent the development of clinical signs of experimental autoimmune uveoretinitis in adult rats. The protection offered by prior treatment with an intravenous aqueous solution of the peptides was dose-dependent with the higher dose being more potent in conferring protection to an immunogenic challenge by the peptide itself or the whole IRBP. Again, the more uveitogenic peptide (aa 1177-1191 of bovine IRBP) was most effective in preventing EAU induced by whole IRBP (Sasamoto et al., 1992). Peptide determinants coupled to splenocytes administered intravenously serve as efficient tolerogens. Guinea pig MBP(GPMBP) peptide 68-86-linked splenocytes given 2 days after adoptive transfer of EAE with GPMBP-primed T cells were able to arrest the development of EAE in male Lewis rats (Pope et al., 1992). Peptide-linked splenocytes were able to induce specific tolerance in S J U J (H-2') mice. Region 84-104 of MBP is the major encephalitogenic epitope in the H-2Smice. Splenocytes coupled to peptide 84-104 reduced the specific T cell proliferative response but were not effective in blocking adoptively transferred EAE (Tan et al., 1992). However, disease transferred by T cells primed to peptide 91-104 was prevented effectively by administering 91-104-linked splenocytes (Su and Sriram, 1991). Besides the use of peptide determinants for inducing specific unresponsiveness, whole protein antigens have been used in various formulations and routes to induce antigen-specific tolerance in newborn or adult animals. Administration of autoantigens orally has been shown to induce antigen-specific tolerance. Feeding Lewis rats with MBP resulted in antigen-specific unresponsiveness and protected rats from induced EAE. This suppression of MBP-specific T cells was mediated by CD8' T cells which were isolated and demonstrated to transfer suppression both in vitro and i n vivo in naive recipients (Lider et al., 1989b).This suppression does not require cell contact and is achieved by soluble mediators passing through membranes separating effector and target cells (Miller et al., 1991).The soluble factor has been identified as TGFP. TGFp-neutralizing antibodies abrogated suppression

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in both in vitro and in v i m experiments (Miller et al., 1992). A pilot trial of oral tolerization was conducted in 30 multiple sclerosis patients. Bovine myelin protein or a placebo was given orally in a double-blind study. Results of the published trial suggest some beneficial effects to patients receiving myelin orally (Weiner et al., 1993). Importantly, no exacerbation of disease was observed in myelin-fed patients. A controlled study with matched patient groups will be needed to draw definitive conclusions (Weiner et al., 1993). Oral tolerance protocols have also been used with success in the EAU model in rats where retinal S antigen administered orally to animals was able to prevent S antigen-induced EAU. This suppression was again mediated by CD8+ T cells (Nussenblatt et al., 1990). Collagen-induced arthritis was prevented by feeding Type I1 collagen (Zhang et nl., 1990) and diabetes in NOD mice blocked by feeding insulin (Zhang et al., 1991). Besides the oral route of tolerization, CD8+ immunoregulatory cells have been reported to be generated when soluble or nonimmunogenic antigens are used for tolerance induction. Antigen-specific CD8+ regulatory cells have been isolated from the thymus and spleens of mice injected with soluble autologous thyroglobulin and prevent development of autoimmune thyroiditis. These cells appeared to have been generated in the thymus and migrate to the spleen and periphery (Rose and Taylor, 1991). In a murine model of autoimmune orchitis, intravenous administration of soluble testicular antigen was shown to prevent the development of orchitis as judged b y histopathological examination. Protection was adoptively transferrable and found to be mediated by CD8' T cells (Mukasa et al., 1992). Although CD8+ T cells have been implicated in antigeninduced tolerance CD8 + cells were not essential for peptide-induced tolerance (Gaur et ul., unpublished results). To examine the potential role of CD8+ cells in peptide tolerance induction, we depleted CD8+ cells in adult mice by antibody treatment. Mice either depleted or not depleted of CD8 + cells were tolerized equally by a synthetic peptide determinant of sperm whale myoglobin given intraperitoneally emulsified with incomplete Freund's adjuvant (Gaur et al., unpublished results). Both in collagen II-induced arthritis and in autoimmune thyroiditis models where soluble antigen administration resulted in protection from disease, CD4+ T cells were the mediators of antigen-induced suppression (Myers et al., 1989a; Kong et al., 1989; Parish et ul., 1988). In a different strain of mice, Balb/cByJ, susceptible to autoimmune orchitis, CD4+ spleen cells from the disease-resistant strain Balb/cJ were able to suppress the development of EAO in the recipients (Teuscher et al., 1990).

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Besides using peptides alone to induce specific unresponsiveness a complex of soluble MHC class I1 molecule with the pathogenic peptide has also been used to induce specific tolerance. Encephalitogenic peptide 91-103 of MBP complexed with a soluble I-ASmolecule was administered in SJL/J mice and prevented adoptively transferred disease. Active EAE, induced by immunization with another proteolipoprotein peptide, 139-151, was also reduced by treatment with the I-AS-peptide complex. The prevention of disease was due to clonal anergy as demonstrated by in vitro study of the effect of the peptide-MHC complex on the response of a T cell clone specific for MBP peptide 91-103 (Sharma et al., 1991).

B. ANALOGSAS MHC BLOCKERSOR T CELLANTAGONISTS Responses to autoantigens are presumed to be similar to normal immune responses. Thus it seemed logical to block recognition of the disease causing peptide:MHC I1 complex to block activation of selfreactive T cells. Such MHC-T cell receptor blockade has been achieved using peptides with good MHC binding which are not structurally related to the autoantigen. Alternative strategies use known autoantigens as peptides with substitute amino acids to increase MHC binding or to interfere with T cell recognition. In these studies hen egg lysozyme (HEL) peptide 46-61 which is immunogenic in I-Akexpressing mice was used with cells expressing I-Ak or planar membranes bearing I-Ak. Inhibition of MHC class I1 binding and reduction in activation of a T cell hybridoma correlated well for the different competing peptides. One peptide with a sequence which was identical to that of mouse lysozyme was also a good inhibitor of H E L recognition suggesting that the same MHC molecule can bind and compete for autologous and foreign peptides (Babbitt et al., 1986). Competition for the MHC molecule in vivo has been demonstrated using synthetic peptides corresponding to mouse lysozyme (ML) and the HEL sequence 46-62. The mouse sequence was not immunogenic but was capable of preventing the T cell response to the HEL peptide when mice were coimmunized with 30-fold excess of the ML peptide (Adorini et al., 1988). This blockade was allele specific; I-Ek restricted responses were not affected (Adorini et al., 1989). Peptide blockers in soluble form, modified to increase their half-life in circulation, have also been demonstrated to be effective in blocking allele-specific immune responses (Muller et al., 1990).Since autoreactive T cells may recognize endogenous or/and exogenous antigens it was important to assess the ability of MHC II-blocking peptides to effectively prevent both kinds of antigen presentation. Antigen-presenting cells (APCs;

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B cell hybridomas) with appropriate MHC expression were transfected with a construct encoding HEL to generate endogenous lysozyme peptides. HEL-transfected APCs were able to activate a class II-restricted lysozyme-specific T cell hybridoma. Presentation to the T cell was blocked by the addition of I-Ek-bindingmouse lysozyme peptide 46-62. This peptide inhibited the activation induced by endogenous or exogenous stimulator peptides (Adorini et al., 1991). This study demonstrated lack of strict compartmentalization between exogenous and endogenous pathways of antigen presentation. MHC-blocking peptides were then tested in experimental models to block autoimmune diseases. An ideal M HC-blocking peptide would have a high affinity of binding to the disease-linked MHC but lack T cell-activating potential. MBP Acl-11, an encephalitogenic peptide, was substituted with alanine at all positions to determine the relative importance of each residue in either binding to I-A" MHC or activating an Acl-ll-specific T cell clone. This analysis revealed that the residue at position 3 was crucial for T cell activation and the residue at position 4 was important for binding to the MHC. A peptide analog with alanine substitutions at positions 3 and 4 was found to bind I-A" better than Acl-11 and also failed to stimulate Acl-ll-specific T cells. However, the Acl-11 (3A,4A) analogue failed to protect H-2"mice from A c l - l l induced EAE when used in 10-fold excess in coimmunization protocols. Interestingly, another analog Acl-1l(4A) which is better than Acl-11 in MHC binding and in stimulating T cells and, thus, would be expected to worsen disease symptoms, was found to be effective in inhibiting Acl-1 l-induced EAE. Also, treatment with IFA-emulsified Acl-ll(4A) at the time of disease onset resulted in marked reduction in disease incidence and severity (Wraith et al., 1989; Smilek et al., 1991). How Acl-1 l(4A) which is a strong activator of encephalitogenic T cell clones (but does not cause disease on its own) inhibits A c l - l l induced EAE is not clear. Perhaps antigen-induced tolerance (anergy) or suppression in addition to MHC blockade might be possible mechanisms. Sakai et al. (1989) used nonencephalitogenic I-A"-binding competitor peptides from the N terminus of MBP to block AC1-ll-induced EAE. The 20 amino acid nonacetylated, nonencephalitogenic N terminus peptide of MBP was effective in inhibiting EAE at only threefold excess to Acl-11 in coimmunization protocols. The acetylated 9-20 peptide was also effective in blocking Acl-1 l-induced disease. Since these peptides were able to compete for I-A" binding in T cell stimulation assays it was assumed that the disease-inhibiting ability was due to MHC blockade. MHC competitor peptides which are structurally unrelated to the disease-causing peptides have also been successfully used to prevent

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EAE. Peptide 323-339 of ovalbumin binds I-A"and competes for MHC binding with other I-A"-binding peptides in T cell hybridoma stimulation but is not immunogenic in H-2" (PL/J x SJL)F1mice. Coimmunization regimens including ovalbumin peptide 322-339 (at 20- to 25-fold molar excess over Acl-11) resulted in an inhibition of EAE in mice (Gautam et al., 1992). A structurally unrelated peptide that bound with high-affinity I-AS was studied. This KM core extension peptide at 10-fold excess was effective in preventing EAE in SJL/J mice induced by an encephalitogenic peptide, 139-151,of mouse proteolipoprotein (PLP).The protection from disease was transient and could be effected by injecting the competitior peptide a day before encephalitogenic challenge at a different site suggesting MHC blockade as the cause of disease inhibition (Lamont et al., 1990). Mouse lysozyme peptide 49-62, which is nonimmunogenic due to self-tolerance, at a 1000-fold excess was able to suppress the myosin-induced myocarditis in mice (Smith and Allen, 1991). A peptide analog, substituted at three positions, of an arthritogenic peptide (CII 245-270) from Type I1 collagen, capable of binding to 1-Aq, was found to be effective in preventing the onset of CII 245-270induced arthritis at a 320-480 molar excess in DBAll mice. Noteworthy was the inability of the peptide analog to induce tolerance in contrast to the tolerogenic capacity of the CII 245-270 peptide, arguing for MHC blockade by the analog as the reason for the ameliorating arthritic symptoms (Myers et al., 1993). Studies mentioned above presume MHC blockade or competition as the mechanism of peptide analog competitors. However, there is no clear evidence as to how these peptide analogs might be acting by competing for MHC sites in vioo and not by other mechanisms including peptide-specific tolerance and suppression. How a competitor is able to block in oivo all the avaliable MHC molecules of a given allele is not clear. Even if this were to happen based on binding to 0.1% of MHC sites, which is required for T cell activation (Demotz et al., 1990; Harding and Unanue, 1990), still this strategy would block all immune responses (against self- or foreign antigens) restricted by the targeted MHC molecule leading to a generalized immune unresponsiveness. Besides, high doses of the competitor peptide with suitable modifications to increase the half-life of the peptides would be required to block all available MHC molecules. Observations where peptide analog of the antigenic peptide were found to block the activation of the T cell at concentrations 1000- to 10,000-fold less than unrelated MHC competitor peptides having similar affinities of binding to

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the MHC molecule suggest a new and effective approach for controlling autoimmunity. Analogs were made with single amino acid substitutions in the peptide determinants of tetanus toxoid (TT 830-843) and hemagglutinin (HA 307-319). Analogs with varying affinities for HLADR1 were used to inhibit antigen presentation to either TT- or HAspecific T cell clones. Interestingly analogs of the same determinant were most effective in blocking T cell activation by their cognate antigens: HA analogs were good inhibitors for the HA clone and TT analogs for TT-specific clone despite having similar affinities for the DR1 molecule. When APCs were prepulsed with the antigen (HA or TT peptides) only antigen-related analogs (HA or TT) and not unrelated MHC blockers were able to inhibit T cell activation. Unrelated MHC-blocking peptides inhibited T cell activation only when added along with the stimulatory peptide. Mechanisms other than peptide competition for MHC were operative. Based on these results, De Magistris et ul., (1992) proposed that the bimolecular complex, peptide analog-MHC, serves as antagonist for the TCR, i.e., it binds the TCR but does not activate the T cell. Such inactivated state of the T cell is maintained only until the TCR is engaged by the antagonist. Apparently, such antagonist-TCR interaction transduces neither activation or inactivation (anergy) signals to the T cell. Alanine-substituted analogs of an encephalitogenic peptide of MBP were compared with analogs of an arthritogenic peptide from heat shock protein 65kDa (HSP-65) from mycobacteria in their ability to block disease in Lewis rats. Although MBP peptide analog had a higher affinity for the MHC than the arthritogenic peptide analog it was not able to prevent arthritis. Only the arthritogenic peptide analog was able to prevent arthritis on coimmunization with the HSP-65 peptide. Also, preimmunization with this analog prevented arthritis induction by the HSP-65 peptide indicating that mechanisms other than or in addition to competition for MHC are involved (Wauben et al., 1992). In a recent report Ostrov et al. (1993) find that fine changes in the TCR junctional regions correlate with the ability of a peptide analog to serve as an antagonist. Accordingly, antagonism is evident when antigen-interactive residue of the TCR is engaged by the altered residue in the analog.

IV. Target 3: The MHC (la) Molecule

Susceptibility and resistance to various autoimmune diseases is linked to expression of a given type of MHC molecule. Individuals

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expressing HLA DR4 have a sixfold higher propensity to develop rheumatoid arthritis (Wordsworth and Bell, 1992). Similar HLA disease associations have been found for other autoimmune diseases like myasthenia gravis and insulin-dependent diabetes mellitus (IDDM) (Bell et al., 1986; Todd et al., 1987, 1988).Animal models of autoimmune diseases also demonstrate the direct relationship of disease susceptibility with the expressed MHC gene products (Steinman, 1992; Acha-Orbea and McDevitt, 1987). The MHC exerts its influence on the disease susceptibility by presumably different ways including facilitating the escape from the thymus through positive selection of selfreactive clones or by deleting suppressor/regulator cells which may be controlling autoreactive cells in the periphery. Expression of MHC class I1 molecules on tissues which normally do not express it may become the trigger for activating autoreactive T cells. This induction of expression of MHC molecules can be initiated by cytokines such as IFNy which could be produced due to some local inflammatory reaction to a pathogen. Transgenic mice expressing cytokines under the control of insulin promoter develop insulitis (Sarvetnick et al., 1990).Another mechanism which could be involved is molecular mimicry wherein shared residues between a pathogen and the MHC gene products could render cells expressing MHC susceptible to attack by pathogen-reactive T cells (Oldstone, 1987). Sequence homology between Epstein-Barr virus (EBV), implicated in susceptibility to rheumatoid arthritis, and HLA-DRP w4 is one such example (Roudier et al., 1988).Presentation ofthe antigen by the MHC molecule appears central to its ability to influence development of autoreactive T cells. Antibodies which could target the MHC gene products specifically seemed appropriate agents for controlling autoimmune diseases. In one of the earliest attempts at regulating autoimmunity animals treated with anti-Ia antibodies showed suppression of overt clinical signs but not histopathological signs of EAE development (Steinman et al., 1981). Repeated injections of monoclonal anti-Ia antibodies after the first attack reduced the mortality due to EAE. Severity and the frequency of relapsing attacks in treated mice were also diminished (Sriram and Steinman, 1983). Monkeys injected with rat monoclonal anti-Ia antibodies showed mitigated EAE signs. Treated animals soon developed anti-rat immunoglobulin and an anti-idiotypic response (Jonker et al., 1988).EAT was completely prevented by anti-Ia (antiI-A) antibody injection at the time of primary immunization with the thyroglobulin. Secondary immunizations with thyroglobulin needed to induce EAT were well tolerated and no disease was produced suggesting in this instance the inhibitory effect of anti-Ia antibodies

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on secondary antigenic challenges (Vladutiu and Steinman, 1987). In the same disease model anti-I-E antibodies were also effective in preventing EAT (Stull et al., 1990) whereas treatment with anti-I-E antibodies exacerbated collagen-induced arthritis in mice. Only antiI-A antibodies were found to inhibit disease in this model (Wooley et al., 198s). EAU in rats (Wetzig et al., 1988; Rao et al., 1989)and EAMG were also treated with anti-Ja antibodies. Response to soluble AcHR was greatly inhibited by anti-Ia antibodies. It was interesting to note that anti-Ia treatment could also affect the effector arm of the immune response as levels of anti-AcHR antibodies were also reduced by the treatment. This inhibitory effect on EAMG was shown to be mediated by cells from anti-Ia-treated mice when they inhibited anti-AcHR antibody production of cells from AcHR-primed animals in an in vitro mixing experiment suggesting that anti-Ia treatment induces generation of specific suppressor cells (Waldor et aE., 1983,1987b). In spontaneously occurring diabetes in NOD mice long-term treatment with a monoclonal antibody specific to the unique NOD MHC, I-Annd,was shown to prevent development of spontaneous diabetes but not insulitis as compared to mice receiving control antibodies. Transfer of spleen cells from anti-Ia-treated mice to NOD recipients prevented the development of diabetes in the adoptively transferred disease model. Depletion of CD4' cells from treated donor cells abrogated the disease inhibitory effect and strongly argues for generation of suppressor cells following anti-Ia treatment (Boitard et al., 1988).However, spleen cells from anti-Ia-treated NZB mice, which were protected from disease (spontaneous lupus-like disease) because of the treatment, failed to inhibit antibody production from B cells of untreated NZB mice pointing to the absence of generation of suppressor mechanism following anti-Ia treatment (Klinman et al., 1986). Similarly no suppressor activity was found in mice treated for EAT by antiIa antibodies (Stull et al., 1990). Anti-Ia antibodies as discussed in this section could be used for modulating immune responses. These antibodies presumably work primarily by affecting the antigenpresenting ability of the MHC which theoretically could be achieved by the following mechanisms: (i) inhibiting peptide-MHC interaction by inducing conformational changes disallowing peptide binding. AS most of the anti-Ia antibodies used are directed to the nonpolymorphic region close to the cell surface it seems unlikely that they are directly inhibiting peptide interaction by steric hindrance. (ii) by elimination of antigen-presenting cells or modulation of MHC expression on the surface of APCs after binding. However, as compared to MHCcompeting peptides which are specifically aimed at blocking the pre-

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sentation of the peptide antigen anti-Ia antibodies can also regulate the immune response by generating specific suppressor cell populations. Anti Ia treatment is not as specific as some other strategies detailed above and could in principle lead to downregulation of all responses dependent on the MHC molecule targeted by the anti-Ia treatment. However, in situations where the autoantigen or the TCR usage is not known as in the case of NOD model it may seem to be a viable alternative. To circumvent the problem of nonspecificity in antiIa treatment Aharoni et al. (1991) developed monoclonal antibodies specific for unique sites generated by binding of an MBP antigen fragment and the self-MHC, I-AS.These antibodies inhibited the proliferative response to the encephalitogenic epitope of the rat MBP without affecting PPD-specific responses in uitro. The antibodies were able to block development of clinical EAE induced by spinal cord homogenate when given in multiple injections around the time of disease induction in SJL/J mice.

V. Conclusion

Among the potential immunotherapeutic strategies outlined in this review the most favored would be the therapy which is most selective in turning off autoantigen-specific T cells while sparing the rest of the immune response. Immunotherapy with peptide antigen would be the best choice. Use of a peptide antigen to induce tolerance or to develop antagonists made by selectively changing crucial residues in the autoantigenic epitope would affect only antigen-specific autoreactive T cells. Problems such as dose and half-life of the peptide need to b e addressed. Synthetic organic molecules mimicking the peptide, with extended stability in circulation, modeled on the structure of the peptide could be potential replacements. Formulation and routes of tolerization which are acceptable need to be worked out. A possibility remains that peptides used as tolerogens in treatment regimens may induce activation of the autoreactive clones and thus exacerbate disease. The duration of unresponsiveness following is also important. Will new T cells joining the circulating pool recreate autoimmunity or maintain normal tolerance to self? Peptide antagonists having the potential to bind but not activate T cells have the advantage of working at lower doses and would be preferred over native sequences. However, the most obvious and currently unfulfilled requirement in this approach is knowledge of the autoreactive determinant. Diseases where the autoantigenic epitope(s) is (are) identified would be amenable to such manipulation. Attempts are currently under way to identify

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the autoantigens in several autoimmune diseases. The availability of the crystal structure of the MHC class I and TI molecules makes possible the modeling of hypothetical candidate peptide antigens which will best fit the antigen-binding groove for a given allele. Peptides synthesized based on such predictions could then be used as tolerogens (or antagonists) in situations where the HLA allelic association with an autoimmune disease is known. In situations where the target antigen remains elusive other approaches such as antibodies to the differentiation markers on T cells could be used. If a restriction in the TCR Vp usage is found and correlated with the disease symptoms anti-TCRVp antibodies seem appropriate as therapeutic tools. Antibodies to the CD4 molecule are currently in clinical trials for treating rheumatoid arthritis and multiple sclerosis. Encouraging results have been obtained; however, the depletion of CD4’T cells, which is required for symptom-ameliorating effects, does lead to concerns regarding compromised immune status of the patients.

ACKNOWLEDGMENTS This work was supported by NIH Grants A1 27989 and DK 43711. We thank Brett Charlton and Peter Krause for help with the computer.

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This article was accepted for publicahon on Y December 1993.