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.
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.
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.
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 dependent on antigen dose. For example, at high antigen concentrations, cytokine production but not cell proliferation is diminished (104). Furthermore, PD-1 deficiency (similar to CTLA4) results in autoimmune phenomena, including splenomegaly, B cell expansion with increased serum immunoglobulins, lupus-like glomerulonephritis, arthritis, and autoimmune cardiomyopathy (105). The exact phenotype varies dependent on the background strain in which the knockouts are generated. PD-1 ligation is sufficient to downregulate suboptimal CD28-mediated signaling (103). Thus, after T cell activation both CTLA4 and PD-1 are upregulated and serve to contain the T cell response. Both molecules therefore appear to play important roles in the maintenance of tolerance. Although PD-1 deficiency leads to some modulation of thymic selection (106), it appears to play a more prominent role in peripheral rather than central tolerance. The possible expression of PD-L on parenchymal cells (which can upregulate class II MHC and present antigens to T cells, but do not express B7) suggests that PD-1–PD-L signaling may to some extent underlie the tolerogenic capacity of these nonprofessional APC. By way of contrast, early reports suggest that B7RP-1 (the ICOS ligand) is upregulated on parenchymal cells, such as renal tubular epithelial cells, after activation (for example, by IFN-γ in vitro) (98). The net effect of signaling through these different pathways on T cells present in inflamed tissues will therefore be complex, and the balance may dictate the final outcome of the immune response.
The TNF–TNF-R Pathways (Figure 3)
The CD154-CD40 interaction is the prototypic pathway of the TNF–TNF-R superfamily of molecules, representing one of a series of receptor-ligand interactions that are important in T cell activation and T cell–B cell interactions. These pathways have the capacity to both provide direct T cell costimulation and interact with other costimulatory pathways such as CD28-B7 and ICOS–B7RP-1. The TNF–TNF-R superfamily contains a number of member pairs, including 4–1BB-4–1BBL, CD30-CD30L, CD134-CD134L, and CD70-CD27. Each of these molecular receptor-ligand interactions has been reported to have unique costimulatory functions. These will be discussed individually. Although other newly described members of the family, such as LIGHT-HVEM (107), also exist, there are as yet limited data on their role in autoimmunity and transplantation and they will not be considered further in this review.
The 4-1BB–4-1BBL Pathway.
4-1BB (CD137, ILA), a member of the TNF-R family, exists as both a 30-kD monomer and a 55-kD homodimer (108). 4–1BBL is a member of the TNF family and exists as a disulfide-linked homodimer (109). 4–1BB is primarily expressed on activated CD4+ and CD8+ T cells (108) as well as on activated NK cells (110). 4–1BB expression peaks 2 to 3 d after cell activation (108,111). 4–1BBL is expressed on mature DC (112) and on activated B cells and macrophages (113). However, due to the initial low levels of expression of 4–1BBL after activation (85) it has been suggested that this pathway would not play a major role in the initiation of the immune response. Furthermore, anti–4-1BB monoclonal antibody has a greater effect on previously activated T cells than on resting T cells, preventing the cells from undergoing AICD (111). Like CD95 (Fas), another TNF-R member, 4–1BB is involved in induction of lymphocyte apoptosis. Although 4–1BB induces expression of CD95 on resting primary T and B cells, induction of apoptosis by 4–1BB is independent of CD95 because anti-CD95 antibody fragments do not block 4–1BB-induced apoptosis (114).
Stimulation of 4–1BB induces higher levels of CD8+ than CD4+ T cell proliferation (115) and appears to be critical for CD8+ T cell survival (116). Furthermore, 4-1BBL–deficient mice have an impaired ability to generate CTL responses to influenza virus (117). However, 4–1BB participates in promoting IL-2 production by resting CD4+ T cells, confirming that 4–1BBL can also play a role in antigen-specific CD4+ T cell responses (118). Moreover, anti–4-1BB monoclonal antibody can induce helper T cell anergy and effectively block T cell–dependent B cell responses (119).
After repeated stimulation, human CD4+ T cells proliferate negligibly in response to anti-CD3 and anti-CD28 monoclonal antibodies (mAb) but show enhanced responses to combined anti-CD3, anti-CD28, and anti–4-1BB mAb (120). These data suggests that 4–1BB plays a later role in the immune response than CD28 and that 4–1BB functions to perpetuate the immune response after CD28 downmodulation. Whereas anti–4-1BB mAb preferentially stimulate CD8+ T cell proliferation, CD28 ligation exerts a more significant proliferative effect on CD4+ cells (115,121). The 4–1BB may, however, be subdominant, because response of nai[Combining Diaeresis]ve CD8+ T cells is dependent on 4–1BB only when CD28 molecules are absent (85). Furthermore, whereas CD28 plays a role in initial T cell expansion, 4-1BB–4-1BBL exerts its effects by sustaining established CD4+ and CD8+ T cell responses and enhancing cell division and T cell effector function (122). 4-1BB–4-1BBL interaction contributes to the development of an allogeneic Th2 response by CD4+CD28− T cells (123). However, treatment of activated human T cells with an intact CD28 pathway using anti–4-1BB promotes a Th1 response (120). Therefore, the T cell response and phenotype that results is dependent on not only the ligation of 4–1BB but on the status of the CD28-B7 pathway. Because 4–1BB may function during the later stages of an immune response, possibly to sustain T cell activation after CD28 downregulation, it may be of importance in conditions of chronic immune stimulation.
Treatment with a stimulating anti–4-1BB monoclonal antibody leads to accelerated allograft rejection in both murine heart and skin transplantation models (115). CD28 or 4-1BBL–deficient mice reject both MHC and minor antigen-incompatible skin grafts without delay, and CD28/4–1BB double-deficient mice experience prolonged graft survival for both mismatches (although all of the grafts were eventually rejected within 30 d) (117). Tan et al. (124) reported that both 4–1BB and 4–1BBL transcripts were expressed in rejecting grafts using a murine cardiac transplant model. They also demonstrated that 4–1BB promotes CD8+ T cell proliferation by both enhancing signals through the IL-2 receptor and by other IL-2–independent mechanisms.
Interestingly, transfection of either B7–1 or B7–2 into certain lymphoma cell lines does not render the lines immunogenic, but the additional transfection of 4–1BBL results in a tumor that is highly immunogenic and can confer long-lasting protection against subsequent challenge with parental tumor in vivo (125). Furthermore, the 4-1BBL–expressing tumors were capable of priming CTL responses against 4-1BBL–transfected as well as parental tumors in the absence of CD28, although cytokine production was lower, resulting in a weaker CTL recall response and reduced ability to survive challenge with parental tumor (126).
In primary mixed lymphocyte reactions, a significant reduction in the response was observed when either 4–1BBFc or CTLA4Ig was added to the cultures, although CTLA4Ig had the greater effect (127). In other strain combinations, 4-1BB–alkaline phosphatase conjugate (4-1BB–AP) and CTLA4Ig added individually profoundly blocked proliferation of alloreactive T cells, and the combination of the two completely abrogated the response (128). Furthermore, measurement of CTL responses demonstrated that CD28+ T cells killing allogeneic target cells were only moderately inhibited by blocking of 4-1BB–4-1BBL interaction, whereas CD28-deficient T cell killing of the allogeneic target was completely blocked by inclusion of 4-1BB–AP in the cultures (117). Thus, 4–1BBL and CD28 may play redundant roles in allogeneic CTL responses. The functions of the 41BB-4–1BBL in costimulation of CD8+ T cells makes it an attractive target for investigation in models where conventional T cell costimulatory blockade of B7 and CD154 are not optimally effective (37).
The CD30-CD30L Pathway.
CD30 was originally described as a marker of Reed-Sternberg cells in Hodgkin lymphoma (129). CD30 is expressed by activated but not by resting B or T cells (129–133). It has been proposed that CD30 is preferentially expressed on Th2 cells (130), although this is not universally accepted (134). T cell expression of CD30 is dependent on the presence of CD28 costimulatory signals or exogenous IL-4 during primary T cell activation (131). CD30L is a transmembrane protein of the TNF family that is expressed by T and B lymphocytes, macrophages, and a variety of hematopoietic cells and tumors (135,136). Using activated splenocytes, it was demonstrated that CD30L is expressed primarily on CD4+ T cells, with peak expression at days 1 and 2, whereas CD30 is expressed primarily on CD8+ T cells, with peak expression on days 4 and 5 (130). The CD30L has been reported to act both as a costimulator for the proliferation of T cells and as a mediator of cytotoxicity through induction of apoptosis (135,136). Mice deficient in CD30 showed a mild impairment in thymic negative selection, and activation-induced death of thymocytes after CD3 crosslinking is impaired both in vivo and in vitro (137).
Although function of CD30-CD30L interaction is largely unknown, in vitro studies have shown that it has effects on both cell activation and cell death (130,131,133,135,138,139). Gruss et al. (135) demonstrated that CD30-CD30L interaction enhance or reduce proliferation of many different CD30+ human lymphoma cell lines. Using lymph node cells, Gilfillan et al. (131) showed that CD30 signaling has a costimulatory effect during a secondary stimulation with anti-CD3. In addition to this role in cell proliferation, CD30 signaling also regulates T cells by inducing apoptosis. Lee et al. (139) reported that in combination with signals transduced by the TCR, CD30 signaling induces Fas-independent cell death in T cell hybridomas. Moreover, Telford et al. (133) showed that CD30-regulated, Fas-independent apoptosis occurred in murine CD8+ T cells after cessation of TCR signals. Several reports have shown that CD30-CD30L interaction induces immune deviation to Th2. Stimulation of CD30 by plate-bound agonistic anti-CD30 directly signaled for IL-5 but not IFN-γ production by CD30+ CTL lines (130). In addition, costimulation of peripheral blood mononuclear cells with agonistic anti-CD30 antibody resulted in preferential development of antigen-specific T cell lines and clones showing a Th2-like profile of cytokine secretion. Furthermore, blockade in bulk culture of CD30-CD30L interaction shifted the development of antigen-specific T cells toward Th1-like phenotype (140). These observations suggest that CD30 triggering of activated Th cells by CD30L-expressing APC may represent an important costimulatory signaling for the development of Th2-type responses. However, contrary to the in vitro observation, in vivo blockade of CD30L could not abrogate murine experimental leishmaniasis, a Th2-mediated disease (141).
CD30 signaling limits the proliferative potential of autoreactive CD8+ effector T cells and protects the body against autoimmune diabetes mediated by CD8+ T cells in mice (142). Furthermore, transplantation of CD30-deficient mice, both MHC class I and class II disparate skin or heart grafts, were rejected faster than control animals (143). This could be due to impaired apoptosis of alloreactive T cells or due to an imbalance of the alloimmune response toward a Th1 phenotype. Further studies are required to explore the effects and mechanisms of CD30-CD30L blockade in experimental models of autoimmunity and transplantation.
The CD134-CD134 Pathway.
CD134 (OX40) was originally identified as a cell surface antigen on activated rat CD4+ T cells. The human, rat, and mouse CD134 genes were subsequently cloned and shown to belong to the TNF-R superfamily (144–146). CD134 ligand (OX40L) is a type II membrane protein with limited homology to TNF and has been shown to bind to and costimulate CD134+ T cells in vitro (147,148). When CD134 is engaged by anti-CD134 monoclonal antibody or CD134L it generates a costimulatory signal that can be as potent as CD28 (149). Engagement promotes effector and memory-effector T cell functions by upregulating IL-2 production and increasing the life span of effector T cells.
Expression of CD134 is restricted to activated T cells in humans and rodents (145,150). Expression of CD134L has been documented on activated murine B cells (145,151), human dendritic cells (152), human vascular endothelial cells (153), and HTLV-1-transformed T cells (154). Both in vitro and in vivo activation of nai[Combining Diaeresis]ve T cells results in transient expression of CD134 with a peak at 24 to 48 h and down regulation by 96 to 120 h (155). Although CD134 expression is augmented by CD28, it can occur independently (156).
The CD134-CD134L pathway appears to be particularly important for regulating the extent of CD4+ T cell expansion in the primary T cell response and thus the ability of T cells to persist as a population over time (157). CD134-deficient T cells secrete IL-2 and proliferate normally during the initial period of activation, but cannot be sustained during the latter phases of the primary response and exhibit decreased survival over time. Mice lacking CD134 generate lower frequencies of antigen-specific CD4+ T cells late in the primary response and lower frequencies of surviving memory cells as compared to wild type animals. Furthermore, CD134 and CD134L-deficient mice demonstrate not only impaired T cell proliferation but also diminished Th1 and Th2 cytokine production (155,158–161). Therefore CD134-CD134L interactions help regulate primary T cell expansion and T cell memory (157). This function may be particularly relevant for chronic autoimmune diseases and development of chronic rejection where prolonged antigen exposure occurs.
Early studies suggested that CD134-CD134L interactions were also necessary for B cell activation and humoral immunity (151,162). However, studies using CD134- and CD134L-deficient mice have demonstrated that CD134-CD134L interactions are not essential (or are redundant) for germinal center formation and antibody responses to antigens or infectious agents (158–161). However, transgenic expression of CD134 on dendritic cells (163) does lead to increased numbers of CXCR-5 CD4+ T cells in B cell follicles (156), which may provide augmented T cell help for B cell function. Furthermore, CD134-deficient mice have been reported to be severely impaired in their ability to generate a Th2 response in response to allergen-induced airway disease (164). These mice also exhibit diminished lung inflammation and significantly attenuated airway hyperreactivity (164). CD134-CD134L is also important in other Th2 CD4+ T cell responses including infections with leishmaniasis (141,165).
CD134-CD134L interactions are critical in autoimmune responses with evidence that signaling through CD134 can break peripheral T cell tolerance (166). Targeting this pathway diminishes disease in EAE (167,168) and in a model of inflammatory bowel disease (169). CD134-Ig administration to mice with colitis ameliorated disease was associated with reduced tissue T cell infiltrates as well as diminished TNF-α, IL-1, IL-12, and IFN-γ production (169). Numerous groups have investigated the effect of CD134 pathway blockade in murine EAE. Administration of anti-CD134L antibody effectively ameliorated EAE in both actively induced and adoptively transferred models (170). Interestingly, anti-CD134L monoclonal antibody treatment did not inhibit the development of pathogenic T cells, their proliferative responses or IFN-γ production as evidenced by restimulation of draining lymph node cells with antigen, and these cells effectively transferred EAE to nai[Combining Diaeresis]ve mice. However, flow cytometric analysis showed that the anti-CD134L antibody treatment inhibited the accumulation of CD134-expressing CD4+ T cells in the spinal cord and the migration of adoptively transferred CD4+ T cells. Interestingly, immunohistochemical analysis revealed prominent CD134L staining on endothelial cells in the inflamed spinal cord. What role this may play in maintenance of the immune response and cell migration remains to be defined. Recently Chitnis et al. (49) from our group demonstrated that anti-CD134L monoclonal antibody therapy protected animals from EAE in CD28-deficient but not wild type mice. Furthermore, using CD134-deficient mice, Ndhlovu et al. (171) reported that abortive T cell priming greatly reduced the clinical manifestations of actively induced EAE associated with a reduction in IFN-γ, IL-2, and IL-6 production.
Although there is CD28-independent costimulation of T cells by CD134L (172), there appears to be synergy between the CD28-B7 and CD134-CD134L pathways. Studies using fibroblast transfectants expressing B7–1 and/or CD134L demonstrated that together CD134L and B7–1 enhance T cell proliferation and cytokine production, especially IL-2 production (155). It is possible that while CD28-B7 costimulation regulates early events, driving cell cycle progression and initial T cell expansion, the CD134-CD134L interaction promotes a more sustained cytokine and proliferative response. This would lead to less cell death and higher frequencies of antigen-specific T cells. Therefore, blockade of this pathway may enhance the ability of B7 and/or CD154 blockade to promote deletional tolerance and may thus prove to be therapeutic importance, especially in stringent transplant models that are relatively resistant to B7 or CD154 blockade alone. In contrast, CD134-transgenic mice develop more severe EAE after a delayed onset, and both CD134-transgenic/CD28-deficient and CD134-transgenic/CD40-deficient mice fail to develop EAE, demonstrating the necessity of these molecules (171).
The CD27-CD70 Pathway.
CD27, another TNF-R superfamily member has been implicated in T cell activation, T cell development, and T cell–dependent antibody production by B cells (173,174). Its ligand, CD70, is a type II transmembrane glycoprotein belonging to the TNF family. It is found on medullary thymic epithelium and is rapidly induced on both T and B cells after cellular activation. CD70 expression on B cells is enhanced by CD40 signaling and is downregulated by IL-4 (175).
Murine CD70 transfectants exhibit a potent costimulatory activity for anti-CD3–stimulated T cell proliferation, which is inhibited by anti-CD70 far more efficiently than murine CD27-Ig (176). Using knockout animals Hendriks et al. (177) suggested that CD27 makes essential contributions to mature CD4+ and CD8+ T cell functions: CD27-supported antigen-specific expansion (but not effector cell maturation) of nai[Combining Diaeresis]ve T cells independent of the cell cycle-promoting activities of CD28 and IL-2. Primary CD4+ and CD8+ T cell responses to influenza virus were impaired in CD27-deficient mice. Effects of CD27-deficiency were most profound on T cell memory, reflected by delayed response kinetics and reduction in number of CD8+ virus-specific T cell to levels seen in primary responses. Furthermore, in the mixed lymphocyte culture using wild type mice, CD27-CD70 interaction induced the generation of cytotoxic T cells (178).
Two reports indicate that CD70 and CD134L on activated B cells could provide CD28-independent costimulatory signals to T cells (172,176). Moreover, CD27-CD70 interactions complement CD40 ligation on B cells, playing a key role in T-dependent B cell responses, and being responsible for plasma cell differentiation (179). Furthermore, CD27-mediated activation may be involved in the NK-cell–mediated innate immunity against virus-infected or transformed cells expressing CD70 (174).
Nakajima et al. (180) reported that treatment of SJL mice with the anti-CD70 monoclonal antibody prevented EAE. The therapeutic effect was not due to the inhibition of T cell priming and antibody production by B cells or immune deviation, although TNF-α production was suppressed. Two separate groups (181,182) recently reported that coexpression of CD70 and B7–1 on tumor cells enhances antitumor immune responses, and this observation could be applicable for prevention of graft rejection. The findings that the CD27-CD70 pathway is important for CD8+ T cell and NK cell functions as well as memory T cell generation and its interaction with CD154-CD40 (179) suggest a key therapeutic target for prevention of alloantibody-mediated chronic allograft vasculopathy and perhaps induction of tolerance in stringent transplant models. Preliminary work from our group indicates that CD27-CD70 blockade might be particularly effective in promoting long-term allograft survival in CD28-deficient animals in which both CD8+ T cells and NK cells play a key role (35).
Recent advances in our knowledge of T cell activation have suggested that inhibiting T cell costimulatory pathways may be an effective way to promote antigen-specific tolerance of transplants and to prevent or treat autoimmune diseases. Blockade of the B7 and CD154 pathways has already shown great promise in certain rodent and primate transplant models and to a much more limited extent in certain human diseases. However, blockade of these conventional T cell costimulatory pathways may not be sufficient to induce tolerance in more stringent transplant models or to inhibit the primed or memory T cell response in autoimmune diseases. Therefore, understanding the functions and mechanisms of other T cell costimulatory pathways in various immune responses may allow for a more efficacious blockade of T cell responses and provide hope of achieving reproducible, robust tolerance in humans (183). On the basis of the known biology of the pathways we have highlighted and the effects seen after their inhibition, it seems likely that one or more of them may prove to be promising therapeutically (Table 1), possibly with combined B7 and/or CD154 blockade. Further experimental studies will be needed to understand which pathways are critical for particular disease states (especially in patients where data are lacking), at what time points, and how these pathways interact with conventional immunosuppressants as well as with interruption of other costimulatory pathways. Our challenge now is to further precisely define the functions of these pathways and the way they interact during autoimmune disease and after transplantation so that the full potential for therapeutic manipulation can be realized.
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