The inability to reproducibly induce robust and durable transplant tolerance using CD28-B7 pathway blockade is in part related to the persistence of alloreactive effector/memory CD8(+) T cells that are less dependent on this pathway for their cellular activation. We studied the role of the novel T cell costimulatory pathway, CD27-CD70, in alloimmunity in the presence and absence of CD28-B7 signaling. CD70 blockade prolonged survival of fully mismatched vascularized cardiac allografts in wild-type murine recipients, and in CD28-deficient mice induced long-term survival while significantly preventing the development of chronic allograft vasculopathy. CD70 blockade had little effect on CD4(+) T cell function but prevented CD8(+) T cell-mediated rejection, inhibited the proliferation and activation of effector CD8(+) T cells, and diminished the expansion of effector and memory CD8(+) T cells in vivo. Thus, the CD27-CD70 pathway is critical for CD28-independent effector/memory CD8(+) alloreactive T cell activation in vivo. These novel findings have important implications for the development of transplantation tolerance-inducing strategies in primates and humans, in which CD8(+) T cell depletion is currently mandatory.
Inducible costimulator (ICOS), a CD28/cytotoxic T lymphocyte antigen 4 (CTLA-4) family member, is expressed on activated T cells. ICOS ligand, a B7 family member, is constitutively expressed on B cells, macrophages, and dendritic cells and is up-regulated on antigen-presenting cells (APCs) and some nonlymphoid tissues by tumor necrosis factor alpha (TNFalpha) or lipopolysaccharide (LPS). Thus, ICOS: ICOS ligand (ICOSL) blockade could reduce alloreactive T cell-APC interactions responsible for graft-versus-host disease (GVHD) and bone marrow (BM) graft rejection. ICOS blockade, achieved with ICOS-/- mice or anti-ICOS monoclonal antibody (mAb) administration, resulted in significant inhibition of GVHD in multiple strain combinations whether mediated by CD4+ and/or CD8+ T cells, alloantigen-specific T-cell receptor (TCR) transgenic (Tg) T cells, or CD28-, T helper 1 (Th1)-, or Th2-deficient T cells. Anti-ICOS significantly delayed GVHD mortality even when mAb infusions were delayed until day 5 after transplantation. ICOS blockade reduced the number of alloantigen-specific effector cells but did not prevent their activation. Imaging of green fluorescent protein-positive (GFP+) effectors indicated that ICOS blockade inhibited expansion of GVHD-causing effector T cells in secondary lymphoid and GVHD target organs. Engraftment rates were significantly higher in ICOS-/- versus wild-type (WT) mice receiving allogeneic BM, and ICOS blockade significantly inhibited expansion of host antidonor alloantigen-specific BM graft-rejecting T cells. These data suggest that the ICOS pathway may be a beneficial therapeutic target for GVHD inhibition, GVHD therapy, and BM graft promotion.
Negative costimulatory signals mediated via cell surface molecules such as CTLA-4 and programmed death 1 (PD-1) play a critical role in down-modulating immune responses and maintaining peripheral tolerance. However, their role in alloimmune responses remains unclear. This study examined the role of these inhibitory pathways in regulating CD28-dependent and CD28-independent CD4 and CD8 alloreactive T cells in vivo. CTLA-4 blockade accelerated graft rejection in C57BL/6 wild-type recipients and in a proportion of CD4(-/-) but not CD8(-/-) recipients of BALB/c hearts. The same treatment led to prompt rejection in CD28(-/-) and a smaller proportion of CD4(-/-)CD28(-/-) mice with no effect in CD8(-/-)CD28(-/-) recipients. These results indicate that the CTLA-4:B7 pathway provides a negative signal to alloreactive CD8(+) T cells, particularly in the presence of CD28 costimulation. In contrast, PD-1 blockade led to accelerated rejection of heart allografts only in CD28(-/-) and CD8(-/-)CD28(-/-) recipients. Interestingly, PD-1 ligand (PD-L1) blockade led to accelerated rejection in wild-type mice and in all recipients lacking CD28 costimulation. This effect was accompanied by expansion of IFN-gamma-producing alloreactive T cells and enhanced generation of effector T cells in rejecting allograft recipients. Thus, the PD-1:PD-L1 pathway down-regulates alloreactive CD4 T cells, particularly in the absence of CD28 costimulation. The differential effects of PD-1 vs PD-L1 blockade support the possible existence of a new receptor other than PD-1 for negative signaling through PD-L1. Furthermore, PD-1:PD-L1 pathway can regulate alloimmune responses independent of an intact CD28/CTLA-4:B7 pathway. Harnessing physiological mechanisms that regulate alloimmunity should lead to development of novel strategies to induce durable and reproducible transplantation tolerance.
The discovery of new functions for the original B7 family members, together with the identification of additional B7 and CD28 family members, have revealed new ways in which the B7:CD28 family regulates T cell activation and tolerance. B7-1/B7-2:CD28 interactions not only promote initial T cell activation but also regulate self-tolerance by supporting CD4+CD25+ T regulatory cell homeostasis. CTLA-4 can exert its inhibitory effects in both B7-1/B7-2 dependent and independent fashions. B7-1 and B7-2 can signal bidirectionally by engaging CD28 and CTLA-4 on T cells and by delivering signals into B7-expressing cells. The five new B7 family members, ICOS ligand, PD-L1 (B7-H1), PD-L2 (B7-DC), B7-H3, and B7-H4 (B7x/B7-S1) are expressed on professional antigen-presenting cells as well as on cells within nonlymphoid organs, providing new means for regulating T cell activation and tolerance in peripheral tissues. The new CD28 families members, ICOS, PD-1, and BTLA, are inducibly expressed on T cells, and they have important roles in regulating previously activated T cells. PD-1 and BTLA also are expressed on B cells and may have broader immunoregulatory functions. The ICOS:ICOSL pathway appears to be particularly important for stimulating effector T cell responses and T cell-dependent B cell responses, but it also has an important role in regulating T cell tolerance. In addition, the PD-1:PD-L1/PD-L2 pathway plays a critical role in regulating T cell activation and tolerance. In this review, we revisit the roles of the B7:CD28 family members in regulating immune responses, and we discuss their therapeutic potential.
Host defense is dependent on the appropriate induction of immune responses. A central concept in immunology is the ability of the immune system to differentiate foreign from self-antigens. The failure of the immune response to recognize foreign pathogens can result in infection and disease in the host. The inappropriate response of the immune system to self-antigens is equally problematic, leading to autoimmune disease. Central and peripheral tolerance mechanisms control self-reactive T-cell responses and protect peripheral tissues from autoimmune attack. This review examines the roles of B7/CD28 family members, which can augment or antagonize T-cell receptor signaling, in the regulation of central and peripheral T-cell tolerance. We also discuss how B7/CD28 pathways influence both T-cell-intrinsic and -extrinsic mechanisms of regulation.
Both CD28 and ICOS are important costimulatory molecules that promote Ag-specific cellular and humoral immune reactions. Whereas CD28 is generally thought to be the most important molecule in the initiation of a T cell response, ICOS is considered to act during the effector phase. We have investigated the contribution of ICOS to T cell responses in the absence of CTLA-4-mediated inhibition. Mice lacking CTLA-4, which show spontaneous CD28-mediated CD4(+) T cell activation, expansion and differentiation, were treated with antagonistic alphaICOS antibodies. Blocking the interaction between ICOS and its ligand B7RP-1 significantly reduced this aberrant T cell activation and caused a reduction in T cell numbers. In vitro analysis of CD4(+) T cells from treated mice revealed that ICOS blockade significantly reduced Th1 differentiation, while Th2 differentiation was only moderately inhibited. Further in vitro stimulation experiments demonstrated that ICOS is able to induce proliferation of murine CD4(+) and CD8(+) T cells but only in the presence of IL-2. These results indicate that ICOS is not only important for T cell effector function but also contributes to the expansion phase of a T cell response in the presence of CD28 signaling.
The ICOS molecule stimulates production of the immunoregulatory cytokine IL-10, suggesting an important role for ICOS in controlling IL-10-producing regulatory T cells and peripheral T cell tolerance. In this study we investigate whether ICOS is required for development of oral, nasal, and high dose i.v. tolerance. Oral administration of encephalitogenic myelin oligodendrocyte glycoprotein (MOG) 35-55 peptide to ICOS-deficient (ICOS-/-) mice did not inhibit experimental autoimmune encephalomyelitis (EAE), T cell proliferation, or IFN-gamma production, in striking contrast to wild-type mice. Similarly, intranasal administration of MOG(35-55) before EAE induction suppressed EAE and T cell responses in wild-type, but not in ICOS-/-, mice. In contrast, ICOS-/- mice were as susceptible as wild-type mice to high dose tolerance. These results indicate that ICOS plays an essential and specific role in mucosal tolerance and that distinct costimulatory pathways differentially regulate different forms of peripheral tolerance. Surprisingly, CD4+ cells from MOG-fed wild-type and ICOS-/- mice could transfer suppression to wild-type recipients, indicating that functional regulatory CD4+ cells can develop in the absence of ICOS. However, CD4+ T cells from MOG-fed wild-type mice could not transfer suppression to ICOS-/- recipients, suggesting that ICOS may have a key role in controlling the effector functions of regulatory T cells. These results suggest that stimulating ICOS may provide an effective therapeutic approach for promoting mucosal tolerance.
Although previous studies have shown that altered B7 costimulation plays a critical role in UV irradiation-induced regulation of immunity, the individual roles of the B7 receptors (CD28 and CTLA-4) or the B7 family members (CD80 and CD86) have not been explored. Thus, we investigated CTLA-4 signaling during photocarcinogenesis of chronically UV-B-exposed mice using an antagonistic anti-CTLA-4 Ab. Anti-CTLA-4-treated mice developed significantly fewer UV-induced tumors. Moreover, anti-CTLA-4 treatment induced long-lasting protective immunity because progressively growing UV tumors inoculated into anti-CTLA-4- and UV-treated mice that had not developed tumors were rejected. Next, we used mice deficient for CD80, CD86, or both in photocarcinogenesis studies to assess the relative contributions of these CTLA-4 ligands. Double-deficient mice showed significantly reduced UV-induced skin tumor development, whereas CD86(-/-) mice produced skin cancer earlier compared with CD80(-/-) and control mice. The growth of UV-induced tumors appears to be controlled by UV-induced suppressor T cells, because CD80(-/-)/CD86(-/-) mice had strongly reduced numbers of UV-induced CD4(+)CD25(+) suppressor T cells. In vitro, CTLA-4 blockade inhibited the suppressor activity of UV-induced CD4(+)CD25(+) T cells, suggesting that reduced photocarcinogenesis might be due to decreased numbers or function of suppressor T cells. Together, these data indicate that blocking CD80/86-CTLA-4 signaling induced immune protection against the development of UV-induced skin tumors. Furthermore, CD86-mediated costimulation appears to play a more critical role in the protection against photocarcinogenesis than CD80.
Bacterial carbohydrates have long been considered T cell-independent antigens that primarily induce humoral immune responses. Recently, it has been demonstrated that bacterial capsules that possess a zwitterionic charge motif can activate CD4(+) T cells after processing and presentation by antigen-presenting cells. Here we show that these zwitterionic polysaccharides can prevent T helper 1-mediated fibrosis by signaling for the release of IL-10 from CD4(+) T cells in vivo. IL-10 production by these T cells and their ability to prevent fibrosis is controlled by the inducible costimulator (ICOS)-ICOS ligand pathway. These data demonstrate that the interaction of the zwitterionic polysaccharides with T cells results in modulation of surgical fibrosis in vivo and suggest a previously undescribed approach to "harnessing" T cell function to prevent inflammatory tissue disorders in humans.
The small GTPase Rap1 is transiently activated during TCR ligation and regulates integrin-mediated adhesion. To understand the in vivo functions of Rap1 in regulating T cell immune responses, we generated transgenic (Tg) mice, which express the active GTP-bound mutant Rap1E63 in their T lymphocytes. Although Rap1E63-Tg T cells exhibited increased LFA-1-mediated adhesion, ERK1/2 activation and proliferation of Rap1E63-Tg CD4+ T cells were defective. Rap1E63-Tg T cells primed in vivo and restimulated with specific Ag in vitro, exhibited reduced proliferation and produced reduced levels of IL-2. Rap1E63-Tg mice had severely deficient T cell-dependent B cell responses, as determined by impaired Ig class switching. Rap1E63-Tg mice had an increased fraction of CD4+CD103+ regulatory T cells (Treg), which exhibited enhanced suppressive efficiency as compared with CD4+CD103+ Treg from normal littermate control mice. Depletion of CD103+ Treg significantly restored the impaired responses of Rap1E63-Tg CD4+ T cells. Thus Rap1-GTP is a negative regulator of Th cell responses and one mechanism responsible for this effect involves the increase of CD103+ Treg cell fraction. Our results show that Rap1-GTP promotes the generation of CD103+ Treg and may have significant implications in the development of strategies for in vitro generation of Treg for the purpose of novel immunotherapeutic approaches geared toward tolerance induction.
Positive selection during thymocyte development is driven by the affinity and avidity of the TCR for MHC-peptide complexes expressed in the thymus. In this study, we show that programmed death-1 (PD-1), a member of the B7/CD28 family of costimulatory receptors, inhibits TCR-mediated positive selection through PD-1 ligand 1 (PD-L1):PD-1 interactions. Transgenic mice that constitutively overexpress PD-1 on CD4+CD8+ thymocytes display defects in positive selection in vivo. Using an in vitro model system, we find that PD-1 is up-regulated following TCR engagement on CD4+CD8+ murine thymocytes. Coligation of TCR and PD-1 on CD4+CD8+ thymocytes with a novel PD-1 agonistic mAb inhibits the activation of ERK and up-regulation of bcl-2, both of which are downstream mediators essential for positive selection. Inhibitory signals through PD-1 can overcome the ability of positive costimulators, such as CD2 and CD28, to facilitate positive selection. Finally, defects in positive selection that result from PD-1 overexpression in thymocytes resolve upon elimination of PD-L1, but not PD-1 ligand 2, expression. PD-L1-deficient mice have increased numbers of CD4+CD8+ and CD4+ thymocytes, indicating that PD-L1 is involved in normal thymic selection. These data demonstrate that PD-1:PD-L1 interactions are critical to positive selection and play a role in shaping the T cell repertoire.