The Role of Novel T Cell Costimulatory Pathways in Autoimmunity and Transplantation
Authors: Akira Yamada, Alan D. Salama, Mohamed H. Sayegh
Journal: Journal of the American Society of Nephrology (2002)

The CD28-B7 and CD154-CD40 pathways have been described as the critical costimulatory pathways for T cell activation. Blockade of these pathways has been reported to regulate both autoimmune and alloimmune responses in experimental models and in human disease. However, studies have indicated that inhibition of these pathways is insufficient to reproducibly induce long-lasting immunologic tolerance in experimental autoimmunity and transplantation models. This suggests that host immune reactivity toward the autoantigens or graft may persist despite optimal blockade of these pathways. These findings may be explained by the presence of immune mechanisms that are known to be relatively resistant to CD28-B7 and/or CD154-CD40 blockade, such as those involving CD8+ T cells (in some transplant models), primed or memory T cells, and natural killer (NK) cells (in autoimmunity and transplantation). Alternatively, other costimulatory pathways may provide the necessary second signals for complete T cell activation. These two possibilities are of course not mutually exclusive. The recent discovery of new members of the CD28-B7 family, inducible costimulator (ICOS), its ligand, B7RP-1, as well as programmed death–1 (PD-1) and its ligands, PD-L1 and PD-L2, have therefore been of major interest. Furthermore, recent data have demonstrated that other molecules belonging to the tumor necrosis factor (TNF) superfamily and their receptors (TNF-R), including 4–1BB, CD30, CD134 (OX40), and CD27, and their respective ligands, 4–1BBL, CD30L, CD134L, and CD70, also act as efficient costimulatory molecules for T cells. The important role that these newly discovered pathways play in regulation of T cell responses in both autoimmunity and transplantation is only now becoming apparent. In some cases, these pathways may be subdominant (or redundant) and exert potent effects on T cell reactivity only in the absence of or after suboptimal costimulation through CD28-B7 and CD154-CD40. However, in other cases these pathways can play a pivotal role in T cell activation or differentiation that may be dependent on the particular stage of the ongoing immune response. Finally, there are important yet complex interactions between these novel T cell costimulatory pathways and both the CD28-B7 and CD154-CD40 pathways, which determine the outcome of a particular immune response in vivo. In this review, we summarize the biology of these pathways, highlight their roles, their hierarchy of dominance and interactions, and finally promote ideas regarding their therapeutic manipulation for the treatment of autoimmune diseases and as immunotherapy in transplantation. T Cell Activation T cells require two collaborative but distinct signals for full activation (1,2) (Figure 1). The first signal (signal one) is provided by the engagement of the T cell receptor (TCR) with its specific peptide antigen, bound to the MHC molecules on the surface of antigen-presenting cells (APC). The second costimulatory signal (signal two) is provided by engagement of T cell surface receptors with their specific ligands on APC (Figure 1). Signaling through the TCR alone without signal two can lead to a state of T cell unresponsiveness that is termed anergy or to apoptosis. Importantly, not all costimulatory molecules provide a "positive" signal; some provide "negative" signals that result in physiologic termination of immune responses (3) (Figure 1). The balance between positive and negative T cell costimulatory signals plays a critical role in protecting the organism against invading foreign antigens and preventing the development of autoimmunity. Figure 1. : (A) Postive signaling pathways. T cell activation requires two signals. Signal one, the ligation of the T cell receptor with its antigen, which is presented on the surface of MHC molecules on antigen-presenting cells (APC), and signal two, the ligation of costimulatory molecules on T cells with their respective ligands on APC. Through a series of secondary signals, the T cell subsequently undergoes proliferation, cytokine production, and further differentiation into its effector state. (B) Negative signaling pathways. Some costimulatory signals can also lead to negative T cell signaling, resulting in cellular anergy, loss of proliferative capacity, and reduction of cytokine production. These pathways may also be involved in the generation of regulatory cells.The Conventional T Cell Costimulatory Pathways The CD28/CTLA4-B7 Pathway The CD28-B7 T cell costimulatory pathway is one of the best characterized and is critical for T cell activation (4–7) (Figure 2). CD28, present on T cells, has two known ligands, B7–1 (CD80) and B7–2 (CD86), both of which are expressed primarily on activated APC, such as dendritic cells, macrophages, and B cells. When activated, T cells upregulate CTLA-4, a molecule that is structurally similar to CD28 that also binds both B7–1 and B7–2. Interaction of CD28 with B7–1 and B7–2 provides a positive signal, which results in full T cell activation, including cytokine production, clonal expansion, enhanced T cell survival, and provision of B cell help (8). CTLA-4 has a higher affinity for B7–1 than B7–2, and functions to provide a "negative" signal resulting in physiologic termination of T cell responses (9–11) (Figure 1). The importance of CTLA-4 as a negative regulatory costimulatory molecule for T cells is highlighted by the observation that CTLA-4–deficient mice develop a fatal lymphoproliferative disorder with multiorgan autoimmune disease (12,13). Furthermore, recent evidence suggests that the CTLA-4 negative signaling pathway may be required for induction of acquired tolerance in vivo (14–16). Indeed, it has been hypothesized that CTLA-4 may function as a master switch for peripheral T cell tolerance (17). Therefore, strategies that promote CTLA4-mediated negative signaling could be very useful therapeutically in T cell–mediated diseases. Ligation of CD28 by B7–1 or B7–2 can be blocked by anti-B7–1 or anti-B7–2 monoclonal antibodies, respectively, or by CTLA4Ig, a recombinant fusion protein containing the extracellular domain of CTLA-4 fused to an Ig heavy chain tail. CTLA4Ig binds to both B7–1 and B7–2 with higher affinity than does CD28, and thus acts as a competitive inhibitor of CD28 binding to B7–1/B7–2, resulting in blockade of CD28-B7 costimulation. Figure 2. : The CD28-B7 family of costimulatory molecules. Both CD28 and CTLA4 contain a motif (MYPPPY) that is necessary for binding to B7–1 and B7–2. Other members of the family lack this motif and are therefore prevented from binding these ligands. The net effect on cellular function after stimulation through these pathways is dependent on the temporal expression patterns of these molecules during T cell activation and the combination of positive and negative signals delivered.Blockade of the B7 Pathway in Transplantation. We and others (18–22) have shown that CD28-B7 T cell costimulatory blockade prevents acute allograft rejection and induces donor-specific tolerance in several animal models, although this is not a universal finding in all models or strain combinations. In addition, CD28-B7 blockade prevents development (23–25) and interrupts progression (26,27) of chronic allograft rejection in minor antigen-mismatched transplant models. However, B7 blockade is less effective in preventing chronic vasculopathy in fully allogeneic transplant models, in which chronic administration of CTLA4Ig or co-administration of donor antigen with CTLA4Ig is required to attenuate development of chronic rejection (28,29). Interestingly, while blockade of both B7–1 and B7–2 are necessary to prevent allograft rejection and promote long-term engraftment in acute rejection models (30), selective inhibition of signaling through B7–1 is sufficient for prevention of chronic rejection (27). We have recently reported this in a rat cardiac transplant model, where selective B7–1 blockade was ineffective in preventing acute graft loss but prevented progression of chronic allograft vasculopathy (27). These data are consistent with those of Furukawa et al. (31), which demonstrate that allograft vasculopathy is significantly attenuated in B7–1 knockout and B7–1/B7–2 double knockout but not B7–2 knockout mice compared with wild type littermates. Creation of CD28 and B7–1/B7–2 deficient animals has helped shed light into the functions of the CD28-B7 T cell costimulatory pathways in allograft rejection. It is interesting that although B7–1/B7–2–double deficient recipients fail to reject vascularized allografts (32,33), CD28-deficient animals have been reported to reject allografts with some delay (34,35). It appears that both CD8+ T cells (35) and NK cells (36) play important roles in CD28-independent allograft rejection. This is a clinically relevant observation, because it may explain the mechanisms of resistance to CD28-B7 blockade in some allograft models (37). Whether these CD8+ T cells are dependent on one or more of the new T cell costimulatory pathways for activation remains to be determined (see below). Blockade of B7 Pathway in Autoimmunity. Inhibition of the CD28-B7 pathway has also been shown to be effective in the prevention and treatment of established diverse autoimmune diseases in both experimental animal models and patients. In experimental autoimmune glomerulonephritis (EAG), an animal model of human anti-glomerular basement membrane (GBM) disease, there was significant attenuation of clinical disease, anti-GBM autoantibody production, and renal mononuclear cell infiltration in animals treated with CTLA4Ig (38). Furthermore, selective blockade of B7–1 by a mutant form of CTLA4Ig produced similar disease regulation, demonstrating that B7–1–mediated signaling is central to autoreactive T cell activation in this model. Differential effects of signaling by B7–1 or B7–2 have also been demonstrated in other autoimmune models, including lupus nephritis in the MRL-lpr/lpr mice, experimental autoimmune encephalomyelitis (EAE), and diabetes in susceptible nonobese diabetic (NOD) mice. Combined blockade of B7–1 and B7–2 in MRL mice attenuated lupus-like renal disease and was associated with suppressed autoantibody production. However, deficiency or inhibition of B7–1 or B7–2 alone resulted in similar levels of pathogenic autoantibodies. Only in the animals lacking B7–2 was there diminished renal Ig deposition and attenuated pathology (39). The B7–1–deficient animals developed more severe nephritis despite similar autoantibody levels, further demonstrating the lack of correlation between antibody titer and disease (40). In EAE, treatment of animals with anti-B7–1 antibody prevents the development of disease, whereas anti-B7–2 antibody exacerbates it (41), although this is not a universal observation in all models (42,43). In the NOD mice, anti-B7–2 treatment suppresses diabetes, but anti-B7–1 antibody alone or in combination with anti-B7–2 antibody accelerates disease. Furthermore, only early treatment with anti-B7–2 prevents the development of diabetes, but it interestingly has no effect on the inflammatory insulitis (44). B7 costimulation signaling through CD28 is also implicated in the development of collagen-induced arthritis, autoimmune thyroiditis, autoimmune uveitis, and myasthenia gravis (39,40,45–48). However, CD28-B7 blockade may not completely abrogate disease, but rather diminishes severity and alters T cell and antibody phenotypes. In experimental myasthenia, for example, CD28 deficiency renders animals less susceptible to disease, but only deficiency of CD154 (see below) confers complete disease resistance (45). Furthermore, although CD28 deficiency protects animals from EAE, disease can be induced after second immunization with antigen, suggesting that alternative pathways can be used for full T cell activation (49). B7 blockade by CTLA4Ig has been studied in patients in a phase I trial as treatment for severe psoriasis vulgaris (50,51) and in phase II trials for therapy of rheumatoid arthritis. CTLA4Ig is currently undergoing trials in other patient groups, including those with multiple sclerosis and lupus nephritis and in renal transplant recipients (8,52). There are currently more preparations of CTLA4Ig that are being tested clinically. In addition, there are several preparations of humanized anti-B7–1 and anti-B7–2 monoclonal antibodies. Importantly, the experimental animal data showing distinct functions of B7–1 and B7–2 in regulating the autoimmune response in various disease models underscores the need to design tailor-made therapeutic strategies in humans with various autoimmune diseases. The CD154-CD40 Pathway There has recently been much interest in studying the role of CD154 and its ligand CD40 in the process of allograft rejection and in the regulation of autoimmune disease (8,53). CD154 is expressed on activated T cells, and CD40 is expressed on APC, including B lymphocytes. CD154-CD40 interaction provides a bidirectional signal for T and B cell activation, thus underlying its importance in T cell–B cell collaboration. CD40 signaling of B cells is critical for Ig switching, and the absence of CD154 characterizes the hyper IgM X-lined syndrome (54). It has been questioned, however, whether CD154 acts directly to transduce a costimulatory signal to the T cell, or indirectly, as ligation of CD40 on APC is a strong inducer of B7 expression (55,56). CD154-CD40 Blockade in Transplantation. Larsen et al. (57) have shown that blocking this pathway with an antibody to CD154 is efficient in preventing acute graft rejection in a mouse cardiac allograft model. Our group (58) reported similar results and demonstrated downregulation of B7–1 expression in cardiac allografts of animals treated with anti-CD154. In our study (58) and in a study by Parker et al. (59) using islet transplantation, coadministration of donor cells synergizes with CD154 blockade to prolong graft survival and induce donor-specific tolerance. In addition, this strategy resulted in the prevention of chronic rejection (60), although others have found contradictory data. CD154 blockade alone was found not to prevent the development of chronic rejection (61,62), and Shimizu et al. (63) recently showed that CD154-deficient animals develop chronic allograft vasculopathy despite long-term allograft survival. In these cases, it has been suggested that CD154 blockade–resistant CD8+ T cells (61), perhaps through one or more of the new pathways, may play a role in the pathogenesis of chronic allograft rejection. A number of studies have demonstrated synergy between B7 and CD154 blockade with or without donor antigen. Larsen et al. (62) reported that simultaneous inhibition of these two pathways led to prolongation of murine skin allograft survival and prevented the development of chronic cardiac allograft vasculopathy. Wekerle et al. (64,65) reported that combined B7 and CD154 blockade may substitute for T cell depletion and irradiation (when high-dose donor bone marrow was used), in the induction of mixed allogeneic chimerism and deletional tolerance in a mouse skin transplant model. Similar observations were reported by Larsen's group (66), which used CD154 blockade and donor bone marrow. Preclinical studies indicating the efficacy of CTLA4Ig and a humanized anti-CD154 monoclonal antibody in primate renal (67,68) and islet (69–71) transplantation models have also been reported. Both these agents have been shown to prolong graft survival, but there are no data to indicate that by themselves they reproducibly induce donor-specific tolerance in primates (72). However, when anti-CD154 monoclonal antibody was used as part of a strategy to induce mixed allogeneic chimerism in a renal transplant model (73), the primates did develop donor-specific tolerance. However, some recipients developed thromboembolic complications that responded to anticoagulation with heparin. Such a complication was also observed in some humans entered in the phase I-II renal transplant trial with the humanized anti-CD154 (Biogen Inc., Cambridge, MA) monoclonal antibody, resulting in premature termination of the trial. The exact mechanisms underlying these complications and the plans for future development of this agent in transplantation remain unclear. Of interest is the interaction between conventional immunosuppressive drugs and costimulatory pathway blockade. Although some drug regimens (containing calcineurin inhibitors) may be detrimental to the effects of T cell costimulatory blockade (57,68,74), others (such as rapamycin) may be beneficial (75). The working hypothesis is that calcineurin inhibitors may inhibit, while rapamycin promotes, activation-induced T cell death (AICD), a mechanism that is required for induction of tolerance by CD154 and B7 blockade (75,76). Calcineurin inhibitors also inhibit expression of CTLA4 (77), which may be necessary for induction of tolerance by T cell costimulatory blockade (30). However, we have recently shown that while rapamycin is indeed synergistic with CD154 blockade, calcineurin inhibitors do not universally impair long-term graft survival in all models (78,79). In our model, late introduction of calcineurin inhibitors to animals treated with CD154 blockade, led to the development of chronic allograft vasculopathy, indicating that this type of strategy may not be clinically desirable in humans (78). These collective observations demonstrate that the interactions between T cell costimulatory blockade and immunosuppressive drugs are complex but extremely important to understand so as to develop clinically relevant strategies to translate into humans. CD154-CD40 in Autoimmunity. In numerous autoimmune diseases, blockade of the CD154-CD40 pathway has been shown to abrogate or suppress disease. This is especially true of diseases in which B cell activation is of fundamental importance, such as systemic lupus erythematosus (SLE) and myasthenia gravis (MG), because the CD154-CD40 pathway is critical in T cell–B cell interaction and activation. For example, in models of SLE, disease may be retarded by a brief treatment course with anti-CD154 antibody (80). In experimental MG, blockade of the CD40 pathway alone renders the animals completely resistant to disease induction (45). Other autoimmune diseases can also be modulated by blockade of this pathway. Using models of spontaneous diabetes in rodents, recurrence of autoimmunity (in transplanted isografts) was diminished after treatment with anti-CD154 antibodies, although the efficacy was greater in rats than mice (81,82). This is consistent with previous observations indicating that CD154 blockade protected NOD mice from developing diabetes when therapy is initiated early but that therapy was ineffective for established disease (83). In EAE, deficiency of CD40 within the central nervous system is sufficient to diminish the intensity and duration of disease, despite the demonstration of adequate T cell activation within the peripheral immune system (84). Although CD154-CD40 blockade alone is highly efficacious in autoimmune disease, as is found in certain transplantation models, there is synergy with blockade of the B7-CD28 pathway. For example, in a model of SLE, CD154-CD40 blockade alone retards disease, but when combined with CTLA4Ig therapy, renal disease may be completely prevented and survival significantly improved (80). Humanized anti-CD154 antibodies are currently undergoing phase I-II testing in autoimmune diseases, including lupus nephritis, although at least one preparation (Biogen Inc.) has been associated with thromboembolic complications, and those trials have been terminated prematurely (see above). Other preparations (IDEC Pharmaceuticals, San Diego, CA) have not been reported to cause similar complications and are currently under investigation. The Novel Costimulatory Pathways Several novel T cell costimulatory pathways have recently been described (85). The ICOS–B7RP-1 and PD-1–PD-L pathways are related to the CD28-B7 family (Figure 2). Furthermore, several new members of the TNF–TNF-R superfamily, of which CD514-CD40 is the prototype, have also been found to be efficient costimualtory molecules (Figure 3). The expression patterns and functions of these pathways are complex and as yet not clearly defined in experimental systems including autoimmunity and transplantation. Few data are available regarding the in vivo expression of the molecules involved and their roles in human disease. In addition, their potential interactions with the CD28/CTLA4-B7 and CD154-CD40 pathways remain incompletely understood. Their roles are being investigated by using a combination of monoclonal antibodies, fusion proteins, and novel gene knockout animals. We have begun to understand how these molecules are regulated during immune responses and what effects they exert. Finally, data are emerging on the interactions between these novel pathways and conventional immunosuppressive agents, which will be important in the planning of future treatment strategies in both transplantation and autoimmunity. Figure 3. : The TNF–TNF-R superfamily of molecules. A number of ligand receptor pairs from this superfamily can act as efficient costimulatory molecules. Through their interactions, both T and B cell activation may occur and result in a variety of cell effector functions.Novel CD28/CTLA4-B7 Family Pathways (Figure 2) The ICOS–B7RP-1 Pathway. The newly discovered CD28 homologue, ICOS, is a T cell costimulatory molecule first reported on activated human T cells (86,87). Human ICOS shares 24% identity (and 39% similarity) with human CD28 and 17% identity (and 39% similarity) with human CTLA-4 (88). The MYPPPY motif, which is required for the binding of CD28 and CTLA-4 to B7 ligands (89), is not conserved in ICOS; instead, it is replaced by a FDPPPF motif. Thus, ICOS does not bind B7–1 or B7–2. Similarly, the L-ICOS ligand, B7RP-1 (which has also been named L-COS, B7h, B7H-2, GL-50) (87,90–93) binds ICOS but not CD28 or CTLA-4. In a similar manner to CD28, signaling through ICOS can result in enhanced T cell proliferation and cytokine production, induce T cell upregulation of CD154, and stimulate T cells to provide help for Ig production by B cells (86). However, ICOS has several properties that are distinct from CD28 and thus make it particularly intriguing. Whereas CD28 is constitutively expressed on T cells, ICOS is induced after TCR engagement and is thus expressed only on activated T cells and resting memory T cells (87), suggesting an important role in providing costimulatory signals to activated T cells (94). This is of some importance because it is known that unlike antigen-inexperienced (nai[Combining Diaeresis]ve) T cells, which require CD28 signaling for proliferation and cytokine production, optimal activation and differentiation of recently activated T cells or memory cells can occur independently of CD28 costimulation (85,95). Expression of ICOS is enhanced by CD28 costimulation, and ICOS upregulation is markedly reduced in the absence of B7–1 and B7–2, suggesting that some of the functions ascribed to CD28 may be due in part to ICOS signaling (96). B7RP-1 expression is still incompletely understood. Early data suggests that it may be constitutively expressed at low levels on antigen presenting cells and certain parenchymal cells (such as renal tubular epithelial cells, prostate epithelial cells and brain tissue) and appears to be upregulated in inflammatory states (97,98). Whereas interferon-γ (IFN-γ) stimulation upregulates both B7RP-1 and B7–1/B7–2 on dendritic cells (DC), TNF-α and lipopolysaccharide (LPS) have differential effects, downregulating B7RP-1 and upregulating B7–1/B7–2 (95). What role this pattern of parenchymal expression plays in regulation of immune responses in normal and diseased tissue remains to be determined. The functional effect of ICOS ligation was demonstrated by using a signaling anti-ICOS monoclonal antibody, which resulted in enhanced T cell proliferation and production of several cytokines (interleukin-4 [IL-4], IL-5, IL-10, IFN-γ, TNF-α, and GM-CSF) (86). ICOS may have a critical role in regulating Th2 cell differentiation. The inducible expression of ICOS and its preferential induction of IL-4 and IL-10 suggest that ICOS may amplify and regulate T helper cell differentiation. Coyle et al. (94) have reported that ICOS is an important costimulatory receptor for both recently activated T cells and for Th2 but not Th1 effector cells. Inhibition of ICOS may be effective in suppressing the function of recently activated T helper cells, inhibiting the secretion of both IL-4 and IFN-γ. However, under circumstances where strong immune deviation occurs, the contribution of ICOS to T cell activation may be restricted to Th2 helper cells. Indeed, ICOS-Ig administration suppressed Th2 cell–mediated airway hyperreactivity in the absence of suppressive effects on Th1-mediated alterations in airway functions (94). ICOS costimulation is involved in both alloimmune responses and those to nominal antigens, because ICOS–B7RP-1 blockade with ICOS-Ig fusion protein suppressed proliferation of T cell responding to allogeneic DC as well as to tetanus toxoid in vitro (90). In vivo studies have suggested complex interactions between ICOS and the CD28-B7 and CD154-CD40 pathways. Inhibition of ICOS in CD28-deficient mice further reduced Th1/Th2 polarization in murine viral and parasitic infection models (99). Blocking of ICOS alone had a limited but significant capacity to downregulate T helper cell subset development. In contrast, cytotoxic T lymphocyte (CTL) responses remained unaffected by blocking ICOS. Taken together, these data suggest that ICOS can regulate both CD28-dependent and CD28-independent CD4+ subset responses but not CD8-mediated CTL responses in vivo (99). ICOS-deficient mice exhibit profound deficits in Ig isotype class switching and germinal center formation. Class switching can be restored in ICOS-deficient mice by CD40 stimulation, demonstrating critical interactions between the ICOS–B7RP-1 and the CD154-CD40 pathways (100). Differentiated ICOS-deficient cells are able to produce IFN-γ and IL-10 but fail to express IL-4 upon restimulation. Furthermore, significantly higher numbers of CD4+ ICOS-deficient T cells retain the nai[Combining Diaeresis]ve phenotype (CD62Lhigh) after cellular activation. ICOS-deficient T cells do not proliferate in response to immunogens (such as keyhole-limpet hemocyanin) administered in alum, but they do if the antigen is coadministered with complete Freund's adjuvant (CFA), suggesting that strong inflammatory responses induced by the CFA can bypass the requirement for ICOS. ICOS is not required for Th2 differentiation, but rather regulates IL-4 and IL-13 production by effector cells. In EAE, ICOS-deficient mice developed greatly enhanced disease compared with wild type mice (101). This may reflect impaired production of the regulatory Th2 cytokines IL-4, IL-13, and/or IL-10. Collectively, the above data demonstrate that ICOS stimulation is important in T cell activation and differentiation, and in T cell–B cell interactions. In addition, there are complex, yet important, interactions between the ICOS–B7RP-1 pathway and the CD28-B7 and CD154-CD40 pathways. Indeed, Ozkaynak et al. (97) recently demonstrated that the blockade of ICOS–B7RP-1 pathway effectively inhibited the development of chronic rejection in association with CD154-CD40 pathway blockade, using a murine cardiac transplant model. Furthermore, ICOS blockade prevented acute rejection and, with concurrent donor-specific transfusion or cyclosporine, induced long-term graft survival. The contribution of ICOS to T cell–mediated immune responses and the functional consequences of ICOS inhibition may be critically influenced by both the nature of the immune response and the timing of intervention with ICOS blockade strategies. For example, the effect ICOS blockade had on the development of EAE was in part dependent on the disease stage (induction or effector stage) when it was administered. Treatment with anti-ICOS antibody during antigen priming (days 1 to 10) resulted in worsening of disease, increased IFN-γ production, increased chemokine expression, greater T cell proliferation, and reduced IgG1 antibody levels, all consistent with a greater Th1 response. Delayed treatment (days 9 to 20) produced the opposite effect, with significantly attenuated disease, decreased IFN-γ production, and reduced chemokine expression and cellular infiltration into the target organ (102). Current investigations are actively aimed at exploring the functions and mechanisms of ICOS–B7RP-1 interactions in various transplantation and autoimmune models. PD-1 and its Ligands, PD-L1 and PD-L2. The newest member of the CD28 superfamily to be described is PD-1. Like CD28, ICOS, and CTLA4, it is a transmembrane protein of the Ig superfamily, and like CTLA4 it possesses only a single V-like domain and an immunoreceptor tyrosine–based inhibitory motif (ITIM) within its cytoplasmic tail (Figure 2). It shares 23% homology with CTLA4, but it lacks the MYPPPY motif required for B7–1 and B7–2 binding. PD-1 receptor is found on activated T and B cells as well as myeloid cells such as macrophages. It binds two known ligands, PD-L1 and PD-L2, found on professional APC, such as DC and monocytes, but also found constitutively on certain parenchymal cells (in the heart, lung, and kidney) as well as on a subpopulation of T and B cells (103,104). In an analgous manner to CTLA4, engagement of PD-1 by its ligands results in a negative regulatory effect, with inhibition of downstream cellular signaling events, diminished cellular proliferation, and cytokine production. However, some of these effects are