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Role of the PD-1 Pathway in the Immune Response

2012; Elsevier BV; Volume: 12; Issue: 10 Linguagem: Inglês

10.1111/j.1600-6143.2012.04224.x

1600-6143

Leonardo V. Riella, Alison M. Paterson, Arlene H. Sharpe, Anil Chandraker,

Cancer Immunotherapy and Biomarkers

American Journal of TransplantationVolume 12, Issue 10 p. 2575-2587 Comprehensive ReviewFree Access Role of the PD-1 Pathway in the Immune Response L. V. Riella, Corresponding Author L. V. Riella Schuster Family Transplantation Research Center, Renal Division, Brigham and Women's Hospital & Children's Hospital Boston, Harvard Medical School, Boston, MALeonardo V. Riella, lriella@rics.bwh.harvard.eduSearch for more papers by this authorA. M. Paterson, A. M. Paterson Department of Microbiology and Immunobiology, Harvard Medical School and Department of Pathology, Brigham & Women's Hospital, Boston, MASearch for more papers by this authorA. H. Sharpe, A. H. Sharpe Department of Microbiology and Immunobiology, Harvard Medical School and Department of Pathology, Brigham & Women's Hospital, Boston, MASearch for more papers by this authorA. Chandraker, A. Chandraker Schuster Family Transplantation Research Center, Renal Division, Brigham and Women's Hospital & Children's Hospital Boston, Harvard Medical School, Boston, MASearch for more papers by this author L. V. Riella, Corresponding Author L. V. Riella Schuster Family Transplantation Research Center, Renal Division, Brigham and Women's Hospital & Children's Hospital Boston, Harvard Medical School, Boston, MALeonardo V. Riella, lriella@rics.bwh.harvard.eduSearch for more papers by this authorA. M. Paterson, A. M. Paterson Department of Microbiology and Immunobiology, Harvard Medical School and Department of Pathology, Brigham & Women's Hospital, Boston, MASearch for more papers by this authorA. H. Sharpe, A. H. Sharpe Department of Microbiology and Immunobiology, Harvard Medical School and Department of Pathology, Brigham & Women's Hospital, Boston, MASearch for more papers by this authorA. Chandraker, A. Chandraker Schuster Family Transplantation Research Center, Renal Division, Brigham and Women's Hospital & Children's Hospital Boston, Harvard Medical School, Boston, MASearch for more papers by this author First published: 17 August 2012 https://doi.org/10.1111/j.1600-6143.2012.04224.xCitations: 266AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Understanding immunoregulatory mechanisms is essential for the development of novel interventions to improve long-term allograft survival. Programmed death 1 (PD-1) and its ligands, PD-L1 and PD-L2, have emerged as critical inhibitory signaling pathways that regulate T cell response and maintain peripheral tolerance. PD-1 signaling inhibits alloreactive T cell activation, and can promote induced regulatory T cell development. Furthermore, the upregulation of PD-L1 on nonhematopoietic cells of the allograft may actively participate in the inhibition of immune responses and provide tissue-specific protection. In murine transplant models, this pathway has been shown to be critical for the induction and maintenance of graft tolerance. In this review, we discuss the current knowledge of the immunoregulatory functions of PD-1 and its ligands and their therapeutic potential in transplantation. Abbreviations: BCR B cell receptor CTLA-4 cytotoxic T lymphocyte-associated antigen 4 DC dendritic cell EAE experimental autoimmune encephalomyelitis MHC major histocompatibility complex mOVA membrane-bound chicken OVA NOD nonobese diabetic PD-1 Programmed cell death-1 PD-L1 Programmed cell death-1 ligand TCR T cell receptor TLR Toll-like receptor Introduction T cells play a major role in coordinating the immune response against alloantigens during organ transplantation. While T cell activation depends on the initial antigen-specific signal provided to T cell receptors via the antigen-loaded MHC complex, additional signals provided by costimulatory molecules fine-tune this response, determining its strength, nature and duration. Some costimulatory interactions potentiate the activation and proliferation of naïve T cells, while others inhibit T cell activation and promote regulation (1, 2). The founding members of the B7:CD28 costimulatory family are the CD28 and CTLA-4 coreceptors that both bind to the B7–1 (CD80) and B7–2 (CD86) molecules. CD28 acts as a strong positive costimulatory receptor and CTLA-4 as a potent coinhibitory receptor. The programmed death 1 (PD-1) receptor: PD-Ligand (PD-L) pathway is another major receptor–ligand network that functions primarily to provide a coinhibitory signal. PD1:PD-L interactions maintain peripheral tolerance and are exploited by tumors and viruses that cause chronic infection to evade immune eradication. As such, this pathway has emerged as a potential therapeutic target for either enhancing or dampening the immune response. This review will summarize our current understanding of the immunoregulatory functions of the PD-1:PD-L pathway and its therapeutic potential, focusing on its relevance to the field of transplantation. Structure and Expression of PD-1 and its Ligands The inhibitory receptor PD-1 (CD279) is a cell surface molecule with a single immunoglobulin (Ig) superfamily domain and a cytoplasmic domain containing two tyrosine-based signaling motifs: a tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM) (Figure 1) (3). PD-1 has two ligands, PD-L1 (B7-H1; CD274) (4) and PD-L2 (B7-DC; CD273) (5, 6), both of which have Ig-V-like and Ig-C-like extracellular domains and a short intracellular domain. PD-1 is inducibly expressed by T cells and B cells after activation (7) as well as natural killer T (NKT) cells, NK cells, activated monocytes and some subsets of dendritic cells (DCs) [reviewed in (8)]. PD-1 is upregulated after TCR or BCR engagement on naïve lymphocytes and persistent antigen stimulation maintains high PD-1 expression (9). The common γ-chain cytokines (IL-2, IL-7, IL-15 and IL-21), TLRs and interferons also can potentiate PD-1 expression on T cells (10). Expression of PD-1 is in part mediated by the recruitment of the nuclear factor of activated T cells c1 (NFATc1) to the nucleus (11). NFATc1 together with AP-1 and NF-κB constitute the most critical transcription factors activated upon antigen recognition by T cells. Interestingly, the calcineurin inhibitor Cyclosporine A markedly reduces PD-1 expression through its effect on NFATc1 (11). While PD-1 upregulation on naïve T cells peaks at 48 h after anti-CD3 or anti-CD3/anti-CD28 stimulation in vitro (12), PD-1 on allogeneic CD4+ T cells progressively increases over time following skin transplantation in vivo, reaching the highest levels on day 10 posttransplant (13). Determination of the level of PD-1 expression in different immune cell subsets at various time points in transplantation remains to be explored. Last, certain subsets of T cells express high levels of PD-1, including CD4+Foxp3+ regulatory T cells (Tregs) (14), T follicular helper cells (TFH) (15), memory T cells (16) and “exhausted” CD8 cells (17). Figure 1Open in figure viewerPowerPoint Effect of PD-1 signaling in T cells. PD-1 signaling dephosphorylates proximal signaling molecules and augments PTEN expression, inhibiting PI3K and AKT activation. The consequences include decreased T-cell proliferation, cytokine production and cell survival. PD-1 inhibition can be overcome by strong TCR signaling, CD28 signaling or IL-2 signaling (not shown). SHP2, protein tyrosine phosphatase; PI3K, phosphatidylinositol 3-kinase; PTEN, phosphatase and tensin homolog; AKT, serine/threonine protein kinase; PIP3, phosphatidylinositol (3,4,5)-triphosphate. Of the PD-1 ligands, PD-L1 has very broad expression, whereas PD-L2 is inducibly expressed in a more restricted fashion. PD-L1 is expressed constitutively on murine T and B cells, DCs, macrophages, mesenchymal stem cells and bone marrow-derived mast cells (12, 18), and induced to higher levels by inflammation. In addition, it can be upregulated on nonhematopoietic cells, including vascular endothelial cells, epithelial cells, muscle cells, hepatocytes, placental cells and pancreatic islet cells (19, 20). In humans, PD-L1 is mainly an inducible molecule. PD-L2 is upregulated on DCs, macrophages, bone marrow-derived mast cells and on a subset of peritoneal B1 B cells as well as on germinal center B cells (21). PD-L2 is also expressed on human, but not mouse, vascular endothelial cells and T cells (6, 22). Interferon-γ (IFN-γ) potently upregulates PD-L1, and to a lesser extent PD-L2. IL-4 and GM CSF are the strongest known stimuli for inducing PD-L2 expression (5, 12, 19, 23, 24). In addition to binding PD-1, PD-L1 can also bind the B7–1 (CD80) molecule, thus connecting the PD-1:PD-L1 pathway with the B7–1:CD28/CTLA-4 pathway (25). PD-L2, however, does not bind to B7–1. There are data to suggest that PD-L2 may have another receptor, the identity of which is still unknown (26). Function of PD-1:PD-L Pathway A major role of the PD-1:PD-L pathway is the inhibition of T cell function by engagement of the PD-1 receptor on T cells by PD-L1 or PD-L2 on antigen presenting cells (APCs). APCs transfected with PD-L1 or PD-L2 inhibit T cell responses, while blockade or genetic ablation of PD-L1 or PD-L2 on DCs or other APCs enhances their capacity to stimulate T cell responses in vitro as compared to wild-type (WT) APCs (27, 28). Conversely, T cells that lack PD-1 are hyperresponsive relative to WT T cells (5, 27, 29-31). These inhibitory interactions not only suppress T cells during the priming phase of an immune response in secondary lymphoid tissues, but also modulate effector T cell responses, either during migration to the site of inflammation or in the target tissue itself (8, 32). PD-1 transduces an inhibitory signal when it is bound by its ligands in the presence of TCR or BCR activation (5, 33, 34). Phosphorylation of a tyrosine residue in the ITSM of PD-1 appears to have a key functional role in mediating PD-1 immunoinhibition. Phosphorylation of the ITSM motif leads to the recruitment of SH2-domain containing tyrosine phosphatase 2 (SHP-2), and possibly SHP-1, to the cytoplasmic domain of PD-1, which then down-regulates CD28-mediated PI3K activity and consequently, leads to less activation of Akt (Figure 1) (35). The exact mechanism of PD-1-mediated antagonism of the PI3K pathway is not yet clear (35). PD-1 ligation also inhibits the phosphorylation of other signaling molecules including CD3, ZAP70 and PCK (35). Thus, a major function of PD-1 signaling is to directly inhibit antigen receptor signaling. Signaling through PD-1 exerts major effects on cytokine production by T cells, inhibiting production of IFN-γ, tumor necrosis factor-α and interleukin-2 (IL-2). PD-1 can also inhibit T cell proliferation (5, 36), and inhibit the upregulation of Bcl-xL, an antiapoptotic protein (33). Last, PD1 signaling decreases the expression of the transcription factors GATA-3, Tbet and Eomes, which are associated with T cell effector function (37). However, a strong positive signaling through CD28 and/or IL-2 receptor can overcome PD-1 inhibitory effects on T cell proliferation, differentiation and survival (5, 18, 37, 38). PD-1 signaling has also been implicated in reversal of the “stop signal” that is mediated by TCR signaling (39). This means that in the presence of PD-1, T cells have a shortened dwell time in their interactions with APCs, which can lead to decreased T cell activation and may also favor the induction of Tregs. PD-1 can also inhibit signaling through B cell receptor. The role of PD-1 in controlling antibody production may be directly related to PD-1 on the B cells or secondary to effects of PD-1 on T cells. T cell interactions with B cells involve recognition of antigen by helper T cells, which then stimulate B cell expansion, isotype switching and affinity maturation. Among T cells, TFH have emerged as key supporters of the B cell response (40). TFH express high levels of PD-1 (15, 41), and PD-L1 and PD-L2 are upregulated on germinal center B cells (42). PD-1 has been shown to be important for the regulation of the germinal center B cell response; PD-1−/− BALB/c mice have a reduced number of long-lived plasma cells after immunization with (4-hydroxy-3-nitrophenyl) acetyl-chicken-γ-globulin (42). In contrast, in two immunization models with either keyhole limpet hemocyanin or extract of Schistosoma mansoni eggs in B6 background mice, PD-L1 deficiency led to a significant expansion of TFH cells and enhanced Ag-specific antibody responses (43). PD-1 deficiency can lead to generation of increased numbers of TFH cells with aberrant phenotypes that lead to dysregulated selection of B cells and antibody diversity in germinal centers (44). Further studies are needed to delineate the functions of this pathway in regulating TFH cell function and B cell responses in the germinal center. Recently described roles for PD-1 expression on DCs and monocytes highlight the possibility that PD-1 signaling may also occur independently of T cell or B cell antigen receptor signaling, possibly by impinging on other receptor signaling pathways (45, 46). For example, PD-1 ligation in monocytes has been shown to stimulate the production of IL-10 during HIV infection, which in turn contributes to reducing T cell function (45). These findings demonstrate that PD-1 expression on a nonlymphocyte population also may influence T cell immune function in HIV infection and this finding may extend to other settings. In addition to PD-1-mediated signaling, there are data to suggest that signals may be transduced by PD-1 ligands. However, the cytoplasmic tail of PD-L1 has no known function. The cytoplasmic domains of human and mouse PD-L2 differ, with the mouse version being only 4 amino acids long and the human bearing 30 amino acids. While this longer form has no known signaling motifs, it is conserved in a number of mammals (but not rodents), which suggests functional significance. Data supporting PD-L1 and/or PD-L2 signaling are primarily based on experiments using soluble PD-1 reagents that engage PD-L1/2 and lead to upregulation of IL-10 production and reduction in DC function (4, 47). The physiological roles of signaling through PD-L1 or PD-L2 remain to be explored. PD-1: PD-L Pathway in Peripheral Tolerance The immune system has the daunting task of responding to foreign antigens, while avoiding self-reactivity. Central tolerance mechanisms prevent the emergence of most self-reactive T lymphocytes from the thymus, but some self-antigen-specific T cells escape into the periphery. To prevent the development of autoimmunity, multiple mechanisms of peripheral tolerance have evolved, including T cell deletion, anergy and the suppressive function of Tregs. Failure of any of these mechanisms might result in autoimmunity. The PD-1:PD-L1 pathway plays a critical role in regulating the delicate balance between protective immunity and tolerance. Initial evidence suggesting an important role for PD-1 in tolerance came from studies using blocking antibodies and knockout mice (27, 48, 49). In contrast to the CTLA-4 knockout mouse, which dies of an aggressive autoimmune lymphoproliferative disease within 3 to 4 weeks of birth (50, 51), the PD-1 knockout mouse is viable on a number of different backgrounds. However, susceptibility to autoimmune disease is exacerbated in PD-1−/- mice on an autoimmune-prone background (52, 53). PD-1−/− and PD-L1−/− NOD mice develop accelerated spontaneous diabetes, which manifests in 100% of male and female mice by 10 weeks (36, 54). Similarly, PD-L1 deficiency on the MRL-Faslpr background results in development of myocarditis and pneumonitis (55). These studies indicate that the PD-1 pathway is critical for self-tolerance. PD-1 regulates both thymic selection and peripheral tolerance. PD-1 plays a role in inhibiting positive selection of thymocytes during the transition from the “double negative” to CD4+CD8+“double positive” stage (56, 57). The role of PD-1 in negative selection is less clear. Two studies using the alloreactive 2C TCR transgenic model showed increased negative selection in the absence of PD-1 (53, 58), while analysis of HY-specific CD4 and CD8 TCR transgenic mice revealed no role for PD-1 in negative selection (59). In addition, PD-1 is critical for peripheral tolerance, restraining the development of self-reactive CD4 and CD8 T cells. PD-1 has an important role in controlling the outcome of initial encounters between naive self-reactive T cells and DCs. PD-1:PD-L1 interactions are required for both the induction and maintenance of CD4 T cell tolerance. For example, the PD-1 pathway is a critical mediator of tolerance induced by administration of antigen-coupled fixed splenocytes, which can reverse diabetes in NOD mice (60). PD-1 deficiency on self antigen-specific T cells increases CD8 T cell responses to antigen-bearing resting DCs, and abrogates CD8 T cell tolerance to peripheral self-antigen expression in vivo (30, 31, 61). PD-L2 is required for oral tolerance, as shown by failure of oral tolerance in PD-L2−/− mice (62). The use of T cell depletion therapy is expanding in solid organ transplantation. Upon lymphodepletion, regulatory mechanisms, including PD-1 signaling, prevent the emergence of autoreactive T cells during homeostatic proliferation. In a lymphopenic environment, a subpopulation of PD-1high T cells develop that have skewed TCR repertoires and appear to be preapoptotic (63). It has been speculated that PD-1 may inhibit expansion of potentially pathogenic self-reactive CD8 T cells during homeostatic reconstitution of lymphopenic environments (63). Using an adoptive cell transfer model, Thangavelu et al. demonstrated that the reconstitution of Rag−/− recipients with PD1−/− hematopoietic stem cell (HSC) precursors, but not WT HSC, causes severe autoimmunity, which does not develop when mature WT or PD-1−/− T cells are used as donor cells (59). These findings support a critical role for PD-1 in regulating homeostatic proliferation, in particular of recent thymic emigrants. Therefore, preservation of an intact PD-1 pathway might be important for the prevention of autoimmunity in patients undergoing reconstitution of their immune system after lymphoablation in solid organ and hematopoietic stem cell transplantation. Regulatory T cells Regulatory T cell populations are critical for the maintenance of peripheral tolerance, are potent inhibitors of many immune responses and play an important role in the prevention of graft rejection (64, 65). CD4+Foxp3+ Tregs, the most widely studied suppressive T cell population, are critical for peripheral tolerance as illustrated by the fatal autoimmune condition of scurfy mice and the human immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX), both caused by mutations in the foxp3 gene (66, 67). Foxp3+ Tregs can be divided into “natural” Treg (nTreg) that arise as committed regulatory cells from the thymus, and “induced” Treg (iTreg) that are generated in the periphery by polarization of naïve CD4+ T cells in a TGFβ- and IL-2-dependent fashion (68-76). Foxp3+ Tregs highly express PD-1 and PD-L1 (1) and a role for the PD1:PD-L pathway in the generation of Tregs has been supported by a number of studies (77-81). PD-L1−/− DCs were shown to be defective at supporting the TGFβ-induced conversion of naïve T cells to Tregs in vitro, and conversion and maintenance of iTreg function in vivo was also dependent on PD-L1 (79). PD-L1 engagement of its receptors (PD-1, and possibly B7–1) on naïve T cells leads to iTreg development, at least in part, by inhibiting mTOR/Akt signaling, as shown by experiments using microbeads coated with anti-CD3, anti-CD28 and PD-L1 fusion protein as artificial APCs (77). There are multiple studies that have identified AKT as a strong repressor of Tregs (82-84). It is proposed that AKT diminishes TGF-β-induced Foxp3 expression in a kinase-dependent manner and via a rapamycin-sensitive pathway (82). There is also evidence that weak TCR signals during T cell differentiation, and limited costimulation, can favor Foxp3 induction (85-88). Using human Th1-polarized T cells in a human-into-mouse graft-versus-host disease model (xGVHD), Amarnath et al. showed that PD-L1:PD-1 signaling was critical for the conversion of human Th1 cells into Tregs in vivo, preventing the development of xGVHD (81). The role of PD-1 in Treg conversion was partially mediated by the activation of intracellular SHP1/2 and consequent reduction of STAT1 phosphorylation, thereby abrogating IFN-γ-mediated maintenance of T-bet and favoring Foxp3 expression (81). Thus, the PD-1 pathway may promote tolerance induction in vivo by inducing and sustaining iTreg. The role of PD-L1 in natural or existing Tregs is less well-understood. Antibody blockade studies suggest that this pathway inhibits expansion of established Tregs, as well as effector T cells (89, 90). In the setting of hepatitis C virus infection, it has been suggested that PD-L1 negatively regulates Tregs by blocking STAT5 phosphorylation (89). However, PD-L1 maintains Foxp3 expression and enhances the suppressive capacity of induced Tregs in vitro (77). It may be that the PD-1 pathway exerts distinct effects on induced Tregs and natural Tregs. Further work is needed to clarify this issue as well as the question of whether PD-1 plays a role in regulating the dichotomy between iTreg and Th17 differentiation. In addition, a subpopulation of regulatory allo-specific CD8+ T cells expressing PD-1, which is induced by ICOS: B7h blockade, was found to be important for immune regulation (91). Thus, it appears that the PD-1 pathway may regulate the generation and/or functions of multiple types of Tregs. PD-1:PD-L Pathway in Transplantation Understanding immunoregulatory mechanisms is essential for the development of novel interventions that might help improve long-term graft survival. Coinhibitory signals play a key role in the regulation of the alloimmune response against the transplanted organ. In particular, a number of studies have demonstrated that an intact PD-1:PD-L interaction is important for the induction and maintenance of graft tolerance (13, 32, 92-96). PD-1, PD-L1 and PD-L2 mRNA expression are significantly increased in cardiac allografts during rejection, while expression is minimal in syngeneic grafts or naïve hearts, indicating that upregulation is related to the host alloresponse, rather than a consequence of ischemia/reperfusion injury (97). A similar observation was made in mouse liver transplantation, where allogeneic livers had a significantly higher expression of PD-L1, both in the sinusoidal and parenchymal areas (92). One of the unique characteristics of PD-L1, compared to other costimulatory molecules, is its pattern of broad expression, not only on hematopoietic cells, but also on nonhematopoietic cells such as endothelium, placental trophoblasts and islet cells (1). After transplantation, there is significant upregulation of PD-L1 on endothelial cells in heart allografts (32). The potential function of PD-L1 in the endothelium is particularly intriguing, since the vasculature is the first interface between the immune cells and the target graft, residing in an optimal location for controlling the alloimmune response. Manipulation of the PD-1:PD-L1 Pathway in Alloimmunity Animal models of solid organ transplantation Functions of the PD-1:PD-L1 pathway have been investigated in multiple different murine solid organ transplant models (Table 1), as well as in bone marrow transplantation. Blockade of PD-1 and PD-L1, but not PD-L2, using an antibody approach, significantly accelerated cardiac graft rejection in a fully MHC mismatched allogeneic model (BALB/c into B6), particularly in the absence of CD28 costimulation (98). In less allogeneic models, such as the MHC class II mismatched model (bm12 into B6), blockade of PD-L1 also precipitated rejection, whereas PD-1 and PD-L2 blockade had no effect on graft survival (96, 99). The absence of a significant effect of PD-L2 blockade in transplantation suggests that PD-L1 and PD-L2 may have different roles in tolerance induction, possibly related, in part, to diverse expression of these ligands. For example, PD-L1 expression is high in tolerogenic subsets of DCs, Tregs and mesenchymal stem cells (32, 100, 101), whereas the expression of PD-L2 is mostly restricted to professional APCs. Table 1. PD-1: PD-L1 function in various transplant models Transplanted organ Model Intervention Effect Potential mechanisms Ref. Heart BALB/c → B6 PD-L1.Ig or PD-L2.Ig ± cyclosporine or rapamycin PD-L1.Ig prolongs graft survival in combination with limited immunosuppression ↓ IFN-γ↓ RANTES, MIP-1, IL10, CXCR3, CCR5 (97) BALB/c → B6 1) CTLA-4-Ig+ anti-PD-L1 or 2 mAb 2) CTLA-4-Ig in PD-L1KO or PD-L2KO recipients PD-L1 blockade or PD-L1KO recipient prevents tolerance development (early and late) ↑CD8 eff mem ↑ IFN-γ, IL4 ↓ Tregs in grafts (94) BALB/c → B6 1) Anti-PD-1 mAb + CD154mAb/DST 2) PD-1KO recipients + CD154mAb/DST PD-1 blockade or PD-1KO recipient prevents tolerance development ↑IFN-γ, IL2, IP10, RANTES, CXCR3, CCR5 (95) BALB/c → B6 CD28KO or B7DKO Anti-PD-1 or anti-PD-L1 mAb PD-L1 blockade accelerates rejection at a faster tempo than PD-1 blockade ↑CD4 and CD8eff mem ↑IFN-γ (98) BALB/c PD-L1 chimera → B6 PD-L1 chimeric donor Graft PD-L1 expression on both hematopoietic and nonhematopoietic cells are essential for tolerance ↑CD8 eff mem ↑IFN-γ, GrB (32) Bm12→ B6 B6→ bm12 B6 → bm1 1) Anti-PD-L1 or Anti-PD-L2 mAb 2) PD-L1KO or PD-L2KO recipients 3) PD-L1KO or PD-L2KO donor hearts On bm12 model: PD-L1 blockade accelerates rejection; PD-L1KO donor accelerates rejection ↑CD4 eff mem ↑IFN-γ, IL4 (96) Bm12→ B6 Bm12→ B6 B7–1KO or B7–2KO or PD-1KO Selective blockade of PD-L1: B7–1 pathway via antibody approach (2H11) or dual blockade with anti-PD-L1 mAb (MIH-6) PD-L1:B7–1 blockade exacerbates chronic injury; dual blockade of PD-L1 has a greater deleterious effect. ↓Tregs ↑IFN-γ, IL4, IL6 (99) F344→ Lewis (rat model) Overexpression of PD-L1 on donor heart via adenovirus ± cyclosporine Slight delay in rejection in combination with cyclosporine ↓CD4 graft infiltration (119) Skin Bm12→B6 and bm12→ABM transgenic mice 1) Anti-PD-1, anti-PD-L1 or anti-PD-L2 mAb Only PD-L1 blockade accelerated rejection; CD25-dependent effect ↑ allogeneic T cell proliferation ↑IFN-γ↓ apoptosis of alloreactive CD4 cells (13) mOva skin →B6 Anti-PD-1 and anti-PD-L1 mAb ± anti-CD28 and anti-CD154 mAb PD-1 and PD-L1 blockade accelerated skin rejection ↑ allogeneic T cell proliferation ↑IFN-γ (102) Islet cells Syngeneic islets from WT or PD-L1KO → NOD mice PD-L1 and/or PD-L2 deficiency on islets PD-L1 deficiency accelerates diabetes development PD-L1KO islets: ↑ CD4+ T cells infiltrating islets (36) PD-L1 expressed on islets protects against infiltration of autoreactive T cells ↑IFN-γ, TNF-α Allogeneic islets from DBA/2→ B6 mice PD-L1.Ig and anti-CD154mAb PD-L1.Ig prolonged islet allograft survival ↓ T cell activation (118) Liver B6 → C3H (85% tolerance) 1) Anti-PD-L1 mAb 2) Anti-PD-1 mAb3) PD-L1 deficiency in liver PD1 and PDL1 blockade precipitates rejection; PDL1KO liver prevents tolerance development ↑cytotoxic T cell infiltration ↑GrB, FasL and perforin in grafts (92) DST, donor-specific transfusion; ABM, anti-bm12 (ABM) transgenic (tg) model; NOD, nonobese diabetic mice. PD-L1 plays a critical role in the induction and maintenance of peripheral transplant tolerance. In a fully MHC mismatched cardiac transplant model in which tolerance was induced by multiple doses of CTLA-4-Ig, early administration of an anti-PD-L1 mAb prevented tolerance induction, while delayed administration abrogated graft survival (94). Accelerated rejection was associated with a significant increase in the frequency of CD8+ effector memory T cells and IFN-γ-producing alloreactive T cells in the periphery, while Foxp3+-graft infiltrating Tregs were decreased (94). This finding has been confirmed in PD-L1-deficient recipients. Furthermore, blockade or elimination of PD-L1 also abrogates other tolerogenic strategies, such as anti-CD154 mAb combined with donor-specific transfusion (95). In summary, the PD-1:PD-L1 pathway is key for tolerance development, however, strong TCR signaling might overcome PD-1-mediated inhibition, which may occur in nonimmunosuppressed recipients of fully MHC-mismatched transplanted organs. In order to more finely assess the effects of PD-1 and PD-L1 in allo-specific T cells, our group has used the anti-bm12 (ABM) transgenic (tg) model, in which CD4+ TCR tg cells are specifically reactive to I-Abm12 (13). Following bm12 skin transplantation, both PD-1 and PD-L1 blockade enhanced alloreactive T cell proliferation and Th1-cell differentiation, although PD-L1 blockade led to a greater effect compared to PD-1 and was unique in its capacity to inhibit alloantigen-specific T cell apoptosis. The effects of PD-L1 blockade were dependent on the presence of CD4+CD25+ Tregs in vivo (13). Another group used a single-antigen-mismatched transplant model where allo-specific CD8+ T cells can recognize mOVA skin grafts and be tracked over time (102). Similarly, blockade of PD-1 resulted in rapid expansion of donor-specific T cells and accelerated skin graft loss, even in the presence