Role of the Programmed Death-1 Pathway in Regulation of Alloimmune Responses In Vivo1

Programmed death-1 (PD-1), an inhibitory receptor up-regulated on activated T cells, has been shown to play a critical immunoregulatory role in peripheral tolerance, but its role in alloimmune responses is poorly understood. Using a novel alloreactive TCR-transgenic model system, we examined the functions of this pathway in the regulation of alloreactive CD4+ T cell responses in vivo. PD-L1, but not PD-1 or PD-L2, blockade accelerated MHC class II-mismatched skin graft (bm12 (I-Abm12) into B6 (I-Ab)) rejection in a similar manner to CTLA-4 blockade. In an adoptive transfer model system using the recently described anti-bm12 (ABM) TCR-transgenic mice directly reactive to I-Abm12, PD-1 and PD-L1 blockade enhanced T cell proliferation early in the immune response. In contrast, at a later time point preceding accelerated allograft rejection, only PD-L1 blockade enhanced T cell proliferation. In addition, PD-L1 blockade enhanced alloreactive Th1 cell differentiation. Apoptosis of alloantigen-specific T cells was inhibited significantly by PD-L1 but not PD-1 blockade, indicating that PD-1 may not be the receptor for the apoptotic effect of the PD-L1-signaling pathway. Interestingly, the effect of PD-L1 blockade was dependent on the presence of CD4+CD25+ regulatory T cells in vivo. These data demonstrate a critical role for the PD-1 pathway, particularly PD-1/PD-L1 interactions, in the regulation of alloimmune responses in vivo.

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title: T‐cell costimulatory pathways in allograft rejection and tolerance
abstract: The destiny of activated T cells is critical to the ultimate fate of immune response. After encountering antigen, naïve T cells receive signal 1 through the T‐cell receptor (TCR)‐major histocompatibility complex (MHC) plus antigenic peptide complex and signal 2 through ‘positive’ costimulatory molecules leading to full activation. ‘Negative’ T‐cell costimulatory pathways, on the other hand, function to downregulate immune responses. The purpose of this article is to review the current state of knowledge and recent advances in our understanding of the functions of the positive and negative T‐cell costimulatory pathways in alloimmune responses. Specifically, we discuss the functions of the CD28:B7 and the tumor necrosis factor receptor (TNFR):tumor necrosis factor (TNF) family of molecules in allograft rejection and tolerance. We address the following important questions: are T‐cell costimulatory pathways merely redundant or do they provide distinct and unique functions? What are the important and unique interactions between the various pathways? And, what are the effects and mechanisms of targeting of these pathways in different types and patterns of allograft rejection and tolerance models?

title: Alternative T‐Cell Costimulatory Pathways in Transplant Rejection and Tolerance Induction: Hierarchy or Redundancy?
abstract: Since the original proposal of a two-signal model for lymphocyte activation (1), it has become firmly established that costimulatory signals mediated primarily via the conventional CD28-B7 and CD154-CD40 pathways, are necessary for full T-cell activation and play a crucial role in mediating allograft rejection (2). In several transplantation models, CD28-B7 or CD154-CD40 blockade has been shown to prevent acute rejection, induce long-term allograft survival and prevent the development and progression of chronic rejection. However, these findings are not reproducible in all strain combinations and models, such as those involving islet, small bowel and skin allografts (3,4), or in primate transplantation (5,6). Moreover, CD28or CD154-deficient animals are still capable of rejecting allografts acutely, albeit at a slower tempo, as well as developing chronic rejection (7,8). Therefore, there are CD28and CD154-independent mechanisms that can initiate alloimmune responses, such as those mediated by CD4 and CD8 effector T cells, NK cells and memory cells that are less reliant on CD28 and CD154 signaling for activation, or effectors utilizing ‘alternative’ costimulatory ligands. These mechanisms may, of course, not be mutually exclusive. It has recently been appreciated that various newly discovered members of the CD28 and TNF-TNF-Receptor superfamilies are capable of acting as ‘alternative’ costimulatory pathways, and thus regulating T-cell immune responses (9) . The roles of these individual pathways in alloimmune responses are only now being unraveled (10–13). One such receptor-ligand pair is the molecule 4–1BB (or CD137) which binds its ligand 4–1BBL. Unlike CD28, but resembling Inducible Costimulator (ICOS) (a T-cell receptor and CD28 superfamily member) and OX40 (or CD134, a TNF-R superfamily member), 4–1BB is not constitutively expressed on T cells, but is up-regulated following initial T-cell activation, suggesting that its main action is not on the initiation of an immune response, but rather on its persistence. It may therefore play an important role in conditions of chronic immune activation. The main cellular targets for 4–1BB are previously activated cells, both CD4 and CD8 T cells, as well as NK cells, in which it promotes proliferation, cellular activation, IL-2 production and cell survival (14,15). In allotransplantation, 4–1BB and 4–1BBL transcripts are found in rejecting murine cardiac grafts (16), and 4–1BB4–1BBL interactions have been shown to regulate alloimmune responses in bone-marrow transplantation models (17,18). Interestingly, 4–1BB-deficient animals have an unaltered time course of skin transplant rejection (19). The precise effect of 4–1BB ligation appears in part to be dependent on the status of the CD28-B7 pathway, since CD28-deficient T cells appear to be more sensitive to 4–1BB blockade than CD28-expressing cells. However, even CD28/4–1BB-double-deficient mice ultimately reject their grafts, demonstrating yet again that there is some redundancy in pathways promoting T-cell activation (19).

title: New B7 Family Members with Positive and Negative Costimulatory Function
abstract: The B7 family of T‐cell costimulatory molecules has expanded considerably in recent years. Among the new costimulatory molecules discovered are inhibitory and activating pathways. Both ligands and receptors often have multiple binding partners, adding to the complexity of T‐cell regulation. Some B7 molecules also exhibit reverse signaling, affecting activation of both antigen‐presenting cells and T cells. An increased understanding of these pathways of T‐cell regulation results in promising new therapeutics because T‐cell interference can be better targeted to specific states of activation or location. This will decrease side‐effects such as systemic immunosuppression and increase efficiency. Targeting B7 molecular pathways for either inhibiting or increasing cell‐mediated immunity has so far shown promising results in models of autoimmunity, transplant rejection and tumor immunotherapy.

title: The role of T-cell costimulatory activation pathways in transplant rejection.
abstract: Organ transplantation is now common. For patients with end-stage cardiac, hepatic, or pulmonary failure, it can be  For patients with diabetes mellitus, pancreatic transplantation offers the chance of a cure and the arrest or reversal of diabetic complications such as neuropathy and retinopathy. In 1996 more than 19,410 organ and tissue transplantations were performed in the United States, and 53,755 patients were on waiting lists for transplantation. Over the past two decades, the development of new immunosuppressive drugs with improved efficacy and decreased toxicity has led to substantial improvement in the survival of patients and in short-term graft survival . . .

title: The Role of CTLA‐4 in the Regulation and Initiation of T‐Cell Responses
abstract: The generation of an effective immune response involves antigen-specific T-cell expansion and difTerentiation of effector function. This process involves at least two signals: one mediated by interaction of the T-cell antigen receptor (TCR) with specific antigen in association with major histocompatibility complex (MHC) molecules, and the second an antigen-independent, costimulatory signal. Over the past few years it has become established that the primary source of costimulatory signals is the interaction between CD28 on the T cell with members of the B7 family (CD80, CD86) on the antigen-presenting cell (APC). For several years the functional role of the enigmatic CD28 homologue CTLA-4 in T-cell activation has been both obscure and controversial. In this article we review recent results from our laboratory and others that provide compelling support for an inhibitory role for CTLA-4 in the regulation of T-cell responses. We discuss these results in the context of the two-signal model of T-cell activation, and propose that T-cell responses are regulated by a complex and dynamic interplay of TCR, CD28 and CTLA-4 signals at both early and late stages of T-cell activation. We also discuss the potential offered by emerging information concerning CTLA-4 for the development of new strategies for the manipulation of immune responses in tumour immunolherapy and other clinical situations.

title: Complexities of CD28/B7: CTLA-4 costimulatory pathways in autoimmunity and transplantation.
abstract: Recent advances in the understanding of T cell activation have led to new therapeutic approaches in the treatment of immunological disorders. One attractive target of intervention has been the blockade of T cell costimulatory pathways, which result in more selective effects on only those T cells that have encountered specific antigen. In fact, in some instances, costimulatory pathway antagonists can induce antigen-specific tolerance that prevents the progression of autoimmune diseases and organ graft rejection. In this review, we summarize the current understanding of these complex costimulatory pathways including the individual roles of the CD28, CTLA-4, B7-1 (CD80), and B7-2 (CD86) molecules. We present evidence that suggests that multiple mechanisms contribute to CD28/B7-mediated T cell costimulation in disease settings that include expansion of activated pathogenic T cells, differentiation of Th1/Th2 cells, and the migration of T cells into target tissues. Additionally, the negative regulatory role of CTLA-4 in autoimmune diseases and graft rejection supports a dynamic but complex process of immune regulation that is prominent in the control of self-reactivity. This is most apparent in regulation of the CD4(+)CD25(+)CTLA-4(+) immunoregulatory T cells that control multiple autoimmune diseases. The implications of these complexities and the potential for use of these therapies in clinical immune intervention are discussed.

title: B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion
abstract: The B7 family members B7-1 and B7-2 interact with CD28 and constitute an essential T-cell co-stimulatory pathway in the initiation of antigen-specific humoral and cell-mediated immune response. Here, we describe a third member of the B7 family, called B7-H1 that does not bind CD28, cytotoxic T-lymphocyte A4 or ICOS (inducible co-stimulator). Ligation of B7-H1 co-stimulated T-cell responses to polyclonal stimuli and allogeneic antigens, and preferentially stimulated the production of interleukin-10. Interleukin-2, although produced in small amounts, was required for the effect of B7-H1 co-stimulation. Our studies thus define a previously unknown co-stimulatory molecule that may be involved in the negative regulation of cell-mediated immune responses.

title: Engagement of the Pd-1 Immunoinhibitory Receptor by a Novel B7 Family Member Leads to Negative Regulation of Lymphocyte Activation
abstract: PD-1 is an immunoinhibitory receptor expressed by activated T cells, B cells, and myeloid cells. Mice deficient in PD-1 exhibit a breakdown of peripheral tolerance and demonstrate multiple autoimmune features. We report here that the ligand of PD-1 (PD-L1) is a member of the B7 gene family. Engagement of PD-1 by PD-L1 leads to the inhibition of T cell receptor–mediated lymphocyte proliferation and cytokine secretion. In addition, PD-1 signaling can inhibit at least suboptimal levels of CD28-mediated costimulation. PD-L1 is expressed by antigen-presenting cells, including human peripheral blood monocytes stimulated with interferon γ, and activated human and murine dendritic cells. In addition, PD-L1 is expressed in nonlymphoid tissues such as heart and lung. The relative levels of inhibitory PD-L1 and costimulatory B7-1/B7-2 signals on antigen-presenting cells may determine the extent of T cell activation and consequently the threshold between tolerance and autoimmunity. PD-L1 expression on nonlymphoid tissues and its potential interaction with PD-1 may subsequently determine the extent of immune responses at sites of inflammation.

title: PD-L2 is a second ligand for PD-1 and inhibits T cell activation
abstract: Programmed death 1 (PD-1)–deficient mice develop a variety of autoimmune-like diseases, which suggests that this immunoinhibitory receptor plays an important role in tolerance. We identify here PD-1 ligand 2 (PD-L2) as a second ligand for PD-1 and compare the function and expression of PD-L1 and PD-L2. Engagement of PD-1 by PD-L2 dramatically inhibits T cell receptor (TCR)-mediated proliferation and cytokine production by CD4+ T cells. At low antigen concentrations, PD-L2–PD-1 interactions inhibit strong B7-CD28 signals. In contrast, at high antigen concentrations, PD-L2–PD-1 interactions reduce cytokine production but do not inhibit T cell proliferation. PD-L–PD-1 interactions lead to cell cycle arrest in G0/G1 but do not increase cell death. In addition, ligation of PD-1 + TCR leads to rapid phosphorylation of SHP-2, as compared to TCR ligation alone. PD-L expression was up-regulated on antigen-presenting cells by interferon γ treatment and was also present on some normal tissues and tumor cell lines. Taken together, these studies show overlapping functions of PD-L1 and PD-L2 and indicate a key role for the PD-L–PD-1 pathway in regulating T cell responses.

title: B7-Dc, a New Dendritic Cell Molecule with Potent Costimulatory Properties for T Cells
abstract: Dendritic cells (DCs), unique antigen-presenting cells (APCs) with potent T cell stimulatory capacity, direct the activation and differentiation of T cells by providing costimulatory signals. As such, they are critical regulators of both natural and vaccine-induced immune responses. A new B7 family member, B7-DC, whose expression is highly restricted to DCs, was identified among a library of genes differentially expressed between DCs and activated macrophages. B7-DC fails to bind the B7.1/2 receptors CD28 and cytotoxic T lymphocyte–associated antigen (CTLA)-4, but does bind PD-1, a receptor for B7-H1/PD-L1. B7-DC costimulates T cell proliferation more efficiently than B7.1 and induces a distinct pattern of lymphokine secretion. In particular, B7-DC strongly costimulates interferon γ but not interleukin (IL)-4 or IL-10 production from isolated naive T cells. These properties of B7-DC may account for some of the unique activity of DCs, such as their ability to initiate potent T helper cell type 1 responses.

title: Development of lupus-like autoimmune diseases by disruption of the PD-1 gene encoding an ITIM motif-carrying immunoreceptor.
abstract: PD-1, a 55 kDa transmembrane protein containing an immunoreceptor tyrosine-based inhibitory motif, is induced in lymphocytes and monocytic cells following activation. Aged C57BL/6(B6)-PD-1(-/-) congenic mice spontaneously developed characteristic lupus-like proliferative arthritis and glomerulonephritis with predominant IgG3 deposition, which were markedly accelerated by introduction of a Fas mutation (lpr). Introduction of a PD-1 null mutation into the 2C-TCR (anti-H-2Ld) transgenic mice of the H-2(b/d) background resulted in the chronic and systemic graft-versus-host-like disease. Furthermore, CD8+ 2C-TCR+ PD-1(-/-) T cells exhibited markedly augmented proliferation in vitro in response to H-2d allogenic cells. Collectively, it is suggested that PD-1 is involved in the maintenance of peripheral self-tolerance by serving as a negative regulator of immune responses.

title: Autoimmune dilated cardiomyopathy in PD-1 receptor-deficient mice.
abstract: Dilated cardiomyopathy is a severe pathology of the heart with poorly understood etiology. Disruption of the gene encoding the negative immunoregulatory receptor PD-1 in BALB/c mice, but not in BALB/c RAG-2-/- mice, caused dilated cardiomyopathy with severely impaired contraction and sudden death by congestive heart failure. Affected hearts showed diffuse deposition of immunoglobulin G (IgG) on the surface of cardiomyocytes. All of the affected PD-1-/- mice exhibited high-titer circulating IgG autoantibodies reactive to a 33-kilodalton protein expressed specifically on the surface of cardiomyocytes. These results indicate that PD-1 may be an important factor contributing to the prevention of autoimmune diseases.

title: Critical Role of the Programmed Death-1 (PD-1) Pathway in Regulation of Experimental Autoimmune Encephalomyelitis
abstract: Experimental autoimmune encephalomyelitis (EAE) is mediated by autoantigen-specific T cells dependent on critical costimulatory signals for their full activation and regulation. We report that the programmed death-1 (PD-1) costimulatory pathway plays a critical role in regulating peripheral tolerance in murine EAE and appears to be a major contributor to the resistance of disease induction in CD28-deficient mice. After immunization with myelin oligodendrocyte glycoprotein (MOG) there was a progressive increase in expression of PD-1 and its ligand PD-L1 but not PD-L2 within the central nervous system (CNS) of mice with EAE, peaking after 3 wk. In both wild-type (WT) and CD28-deficient mice, PD-1 blockade resulted in accelerated and more severe disease with increased CNS lymphocyte infiltration. Worsening of disease after PD-1 blockade was associated with a heightened autoimmune response to MOG, manifested by increased frequency of interferon γ–producing T cells, increased delayed-type hypersensitivity responses, and higher serum levels of anti-MOG antibody. In vivo blockade of PD-1 resulted in increased antigen-specific T cell expansion, activation, and cytokine production. Interestingly, PD-L2 but not PD-L1 blockade in WT animals also resulted in disease augmentation. Our data are the first demonstration that the PD-1 pathway plays a critical role in regulating EAE.

title: The Programmed Death-1 (PD-1) Pathway Regulates Autoimmune Diabetes in Nonobese Diabetic (NOD) Mice
abstract: Programmed death-1 (PD-1) receptor, an inhibitory costimulatory molecule found on activated T cells, has been demonstrated to play a role in the regulation of immune responses and peripheral tolerance. We investigated the role of this pathway in the development of autoimmune diabetes. PD-1 or PD-L1 but not PD-L2 blockade rapidly precipitated diabetes in prediabetic female nonobese diabetic (NOD) mice regardless of age (from 1 to 10-wk-old), although it was most pronounced in the older mice. By contrast, cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) blockade induced disease only in neonates. Male NOD mice also developed diabetes after PD-1–PD-L1 pathway blockade, but NOR mice, congenic to NOD but resistant to the development of diabetes, did not. Insulitis scores were significantly higher and frequency of interferon γ–producing GAD-reactive splenocytes was increased after PD-1–PD-L1 pathway blockade compared with controls. Interestingly, PD-L1 but not PD-L2 was found to be expressed on inflamed islets of NOD mice. These data demonstrate a central role for PD-1–PD-L1 interaction in the regulation of induction and progression of autoimmune diabetes in the NOD mouse and provide the rationale to develop new therapies to target this costimulatory pathway in this disease.

title: Programmed Death-1 Targeting Can Promote Allograft Survival1
abstract: The recently identified CD28 homolog and costimulatory molecule programmed death-1 (PD-1) and its ligands, PD-L1 and PD-L2, which are homologs of B7, constitute an inhibitory regulatory pathway of potential therapeutic use in immune-mediated diseases. We examined the expression and functions of PD-1 and its ligands in experimental cardiac allograft rejection. In initial studies, we found that most normal tissues and cardiac isografts had minimal expression of PD-1, PD-L1, or PD-L2, but intragraft induction of all three molecules occurred during development of cardiac allograft rejection. Intragraft expression of all three genes was maintained despite therapy with cyclosporin A or rapamycin, but was prevented in the early posttransplant period by costimulation blockade using CD154 or anti-inducible costimulator mAb. We prepared PD-L1.Ig and PD-L2.Ig fusion proteins and showed that each bound to activated PD-1+ T cells and inhibited T cell functions in vitro, thereby allowing us to test the effects of PD-1 targeting on allograft survival in vivo. Neither agent alone modulated allograft rejection in wild-type recipients. However, use of PD-L1.Ig administration in CD28−/− recipients, or in conjunction with immunosuppression in fully MHC-disparate combinations, markedly prolonged cardiac allograft survival, in some cases causing permanent engraftment, and was accompanied by reduced intragraft expression of IFN-γ and IFN-γ-induced chemokines. PD-L1.Ig use also prevented development of transplant arteriosclerosis post-CD154 mAb therapy. These data show that when combined with limited immunosuppression, or in the context of submaximal TCR or costimulatory signals, targeting of PD-1 can block allograft rejection and modulate T and B cell-dependent pathologic immune responses in vivo.

title: Stimulating PD-1–negative signals concurrent with blocking CD154 co-stimulation induces long-term islet allograft survival1
abstract: A balanced network of positive and negative T-cell co-stimulatory signals is important in regulating T-cell activation. Blocking CD28, CD154 (CD40L), or both co-stimulatory molecules has been efficacious in preventing acute allograft rejection in certain but not all transplantation models. In the present study, the authors tested the hypothesis that stimulating programmed death 1 (PD-1)–triggered negative signals concurrent with blocking CD154 co-stimulatory signals would facilitate islet allograft tolerance.  The authors used a dimeric PD-L1 immunoglobulin (Ig) fusion protein to stimulate the inhibitory receptor PD-1, and a monoclonal antibody to block CD154. The effects of PD-1 engagement and CD154 blockade on lymphocyte activation were determined by cell proliferation, flow cytometry, and a model of islet transplantation.  PD-L1Ig inhibited the proliferation of both CD4+ and CD8+ T cells stimulated by anti-CD3. The inhibitory effect of PD-L1Ig was enhanced by concurrent blockade of CD154 co-stimulatory signals, as demonstrated by T-cell proliferation and expression of cell surface activation markers. PD-L1Ig and anti-CD154 also synergistically blocked the activation and maturation of antigen-presenting cells. In an islet transplantation model, treatment of recipient C57BL/6 (H-2b) mice with PD-L1Ig and anti-CD154 induced long-term survival of DBA/2 (H-2d) islet allografts, whereas treatment with each reagent alone failed to prevent islet allograft rejection.  These results suggest that engaging the negative receptor PD-1 exhibits critical immunoregulatory effects in the allograft response, and blocking positive co-stimulatory molecules with active delivery of inhibitory signals may represent a novel therapeutic strategy in transplantation.

title: New TCR Transgenic Model for Tracking Allospecific CD4 T‐Cell Activation and Tolerance in Vivo
abstract: We have developed an adoptive transfer model system to visualize the dynamics of alloantigen‐specific CD4+ T‐cell activation in vivo. Using TCR‐transgenic (tg) mice reactive to I‐Abm12, we studied the clonal expansion and differentiation of alloreactive T cells by tracking the fate of adoptively transferred TCR‐tg CD4+ T cells in syngeneic mice transplanted with skin grafts expressing I‐Abm12. Following transplantation, alloantigen‐specific TCR‐tg CD4+ T‐cell expansion was observed initially in the draining lymph nodes followed by the spleen. TCR‐tg CD4+ T cells up‐regulated CD69 and CD25 expression, developed an effector/memory surface phenotype and produced IFN‐γ in response to alloantigen ex vivo. Furthermore, we validate the model system as a means for studying the effects of tolerogenic regimens on alloreactive CD4+ T cells, demonstrating that CTLA4Ig inhibits alloantigen‐dependent clonal expansion and effector function of TCR‐tg CD4+ T cells in vivo. We describe the first model for tracking alloreactive CD4+ T‐cell activation in vivo. It provides a powerful tool for studying CD4+ T‐cell mediated alloimmune responses and mechanisms of tolerance induction in vivo.

title: Positive Selection Through a Motif in the αβ T Cell Receptor
abstract: The two lineages of T cells, αβ and γδ, differ in their developmental requirements: only αβ T cells require major histocompatibility complex recognition, a process known as positive selection. The αβ T cell receptor (TCR), but not its γδ counterpart, contains a motif within the α-chain connecting peptide domain (α-CPM) that has been conserved over the last 500 million years. In transgenic mice expressing an αβ TCR lacking the α-CPM, thymocytes were blocked in positive selection but could undergo negative selection. Thus, the α-CPM seems to participate in the generation of signals required for positive selection.

title: Allograft Rejection in a New Allospecific CD4+ TCR Transgenic Mouse
abstract: The application of TCR transgenic mice to transplantation immunology is hampered by the limited lines available. Recently, we reported CD4+ T cell receptor (TCR) transgenic mice specific for I‐Abm12 expressed on B6.C.H‐2bm12 mice. Here, we characterized rejection of skin and vascularized cardiac allografts in these mice, which we term ABM (for anti‐bm12). In vivo proliferative experiments reveal that all CD4 T cells in ABM mice react to bm12 antigens. Surprisingly, while ABM mice have accelerated (compared to B6 recipients) rejection of bm12 skin allografts, they, like B6 recipients, fail to acutely reject bm12 cardiac allografts. This is not due to lack of immunogenicity of bm12 hearts, as these grafts are acutely rejected by primed ABM recipients, although not by primed B6 recipients. Lastly, long‐term surviving bm12 grafts in ABM recipients are relatively free from chronic rejection (compared with B6 recipients), which may be due to skewing of the CD4 repertoire towards direct alloreactivity, and consequent lack of CD4 mediated indirect allorecognition as evidenced by the lack of IgG deposition in those grafts. The results indicate that a complex interplay between responder frequency, priming and repertoire dictates the occurrence, or lack thereof, of acute and chronic rejection.

title: Expression of the PD-1 antigen on the surface of stimulated mouse T and B lymphocytes.
abstract: A mAb J43 has been produced against the product of the mouse PD-1 gene, a member of the Ig gene superfamily, which was previously isolated from an apoptosis-induced T cell hybridoma (2B4.11) by using subtractive hybridization. Analyses by flow cytometry and immunoprecipitation using the J43 mAb revealed that the PD-1 gene product is a 50-55 kDa membrane protein expressed on the cell surface of several PD-1 cDNA transfectants and 2B4.11 cells. Since the molecular weight calculated from the amino acid sequence is 29, 310, the PD-1 protein appears to be heavily glycosylated. Normal murine lymphoid tissues such as thymus, spleen, lymph node and bone marrow contained very small numbers of PD-1(+) cells. However, a significant PD-1(+) population appeared in the thymocytes as well as T cells in spleen and lymph nodes by the in vivo anti-CD3 mAb treatment. Furthermore, the PD-1 antigen expression was strongly induced in distinct subsets of thymocytes and spleen T cells by in vitro stimulation with either anti-CD3 mAb or concanavalin A (Con A) which could lead T cells to both activation and cell death. Similarly, PD-1 expression was induced on spleen B cells by in vitro stimulation with anti-IgM antibody. By contrast, PD-1 was not significantly expressed on lymphocytes by treatment with growth factor deprivation, dexamethasone or lipopolysaccharide. These results suggest that the expression of the PD-1 antigen is tightly regulated and induced by signal transduction through the antigen receptor and do not exclude the possibility that the PD-1 antigen may play a role in clonal selection of lymphocytes although PD-1 expression is not required for the common pathway of apoptosis.

title: Expression of Programmed Death 1 Ligands by Murine T Cells and APC1
abstract: Programmed death 1 (PD-1) is a new member of the CD28/CTLA-4 family, which has been implicated in the maintenance of peripheral tolerance. Two ligands for PD-1, namely, B7-H1 (PD-L1) and B7-DC (PD-L2), have recently been identified as new members of the B7 family but their expression at the protein level remains largely unknown. To characterize the expression of B7-H1 and B7-DC, we newly generated an anti-mouse B7-H1 mAb (MIH6) and an anti-mouse B7-DC mAb (TY25). MIH6 and TY25 immunoprecipitated a single molecule of 43 and 42 kDa from the lysate of B7-H1 and B7-DC transfectants, respectively. Flow cytometric analysis revealed that B7-H1 was broadly expressed on the surface of mouse tumor cell lines while the expression of B7-DC was rather restricted. PD-1 was expressed on anti-CD3-stimulated T cells and anti-IgM plus anti-CD40-stimulated B cells at high levels but was undetectable on activated macrophages or DCs. B7-H1 was constitutively expressed on freshly isolated splenic T cells, B cells, macrophages, and dendritic cells (DCs), and up-regulated on T cells by anti-CD3 stimulation on macrophages by LPS, IFN-γ, GM-CSF, or IL-4, and on DCs by IFN-γ, GM-CSF, or IL-4. In contrast, B7-DC expression was only inducible on macrophages and DCs upon stimulation with IFN-γ, GM-CSF, or IL-4. The inducible expression of PD-1 ligands on both T cells and APCs may suggest new paradigms of PD-1-mediated immune regulation.

title: Preferential contribution of B7‐H1 to programmed death‐1‐mediated regulation of hapten‐specific allergic inflammatory responses
abstract: Programmed death‐1 (PD‐1) and its ligands, B7‐H1/PD‐L1 and B7‐DC/PD‐L2, are new CD28‐B7 family members that may be involved in the regulation of immune responses. We examined the roles of these molecules in mouse hapten‐induced contact hypersensitivity (CH). Administration of anti‐PD‐1 mAb at sensitization significantly enhanced and prolonged ear swelling. Treatment with anti‐B7‐H1 mAb, but not anti‐B7‐DC mAb, also enhanced CH reactions. The anti‐PD‐1 mAb treatment at sensitization significantly increased the T cell number of draining lymph nodes (DLN). B7‐H1 was induced on activated T cells and antigen‐presenting cells (APC) in the skin and the DLN, whereas B7‐DC expression was restricted to dendritic cells (DC) in the dermis and the DLN. A particular subset of DC, B7‐H1+B7‐DC–CD86low, was found in sensitized DLN. The blockade of B7‐H1, but not B7‐DC, dramatically enhanced the initial T cell proliferative responses against hapten‐pulsed DLN APC, suggesting the preferential contribution of B7‐H1 to the T cell‐APC interaction. Our results demonstrate the regulatory role of PD‐1 and the differential roles of B7‐H1 and B7‐DC in hapten‐induced immune responses. The PD‐1‐B7‐H1 pathway may play a unique role in regulating inflammatory responses.

title: Following the fate of individual T cells throughout activation and clonal expansion. Signals from T cell receptor and CD28 differentially regulate the induction and duration of a proliferative response.
abstract: A detailed understanding of the effects of costimulatory signals on primary T cell expansion has been limited by experimental approaches that measure the bulk response of a cell population, without distinguishing responses of individual cells. Here, we have labeled live T cells in vitro with a stable, fluorescent dye that segregates equally between daughter cells upon cell division, allowing the proliferative history of any T cell present or generated during a response to be monitored over time. This system permits simultaneous evaluation of T cell surface markers, allowing concomitant assessment of cellular activation and quantitative determination of T cell receptor (TCR) occupancy on individual cells. Through this approach, we find that TCR engagement primarily regulates the frequency of T cells that enter the proliferative pool, but has relatively little effect on the number of times these cells will ultimately divide. In contrast, CD28-costimulation regulates both the frequency of responding cells (particularly at sub-maximal levels of TCR engagement), and more prominently, the number of mitotic events that responding cells undergo. When CD28-stimulation is blocked, provision of IL-2 restores the frequency of responding cells and the normal pattern of mitotic progression, indicating that the other CD28-induced genes are not required for this effect. An unexpected finding was that even at maximal levels of TCR engagement and CD28-mediated costimulation, only 50-60% of the original T cells in culture can be induced to divide. The nondividing cells are heterogeneous for naive versus memory markers, suggesting a more complex relationship between expression of memory markers and the ability to be recruited into the dividing pool. From these studies, we conclude that a stringent checkpoint regulates the participation of activated T cells in clonal expansion, with TCR and CD28 signals having both overlapping and differential effects on the induction and maintenance of T cell responses.

title: Quantifying the Frequency of Alloreactive T Cells In Vivo: New Answers to an Old Question1
abstract: Alloreactive T cell precursor frequency was measured in vivo using fluorescent dye labeling in combination with novel models based on lymphocyte activation and recovery. CFSE-labeled C57BL/6 (H-2b) spleen and lymph node cells were adoptively transferred to C57BL/6×DBA F1 (H-2b/d) recipients, a parent→F1 MHC mismatch in which only donor cells respond. Recipients were sacrificed at serial time points to assess engraftment efficiency, and the extent of donor cell activation and proliferation. These data were used to calculate alloreactive T cell frequencies that varied 30-fold (0.71 ± 0.31% to 21.05 ± 3.62%), depending upon whether it was assumed that all donor cells injected became established and were capable of responding, or that only those present at later time points (24–72 h) were available to respond. By measuring the number of cells established in the recipient 24 h after transfer, before proliferation, we calculated an in vivo alloreactive frequency of ∼7%. Using CD69 expression at 48 h to quantify activation, we found that 40–50% of the alloactivated CD4+ donor T cells do not divide. Studies of cotransferred congenic and allogeneic cells demonstrated that bystander proliferation does not occur. We conclude that accurate calculations of alloreactive precursor frequency must account for both proliferation and cell engraftment. When this is done, a high percentage of alloreactive T cells exists across an MHC mismatch, but not all alloreactive cells proliferate in vivo. Bystander proliferation is negligible, revealing exquisite specificity to the alloresponse. These data provide a novel approach to quantify alloreactive T cell responses during specific immunomodulatory strategies in vivo.

title: Tracking the Immunoregulatory Mechanisms Active During Allograft Tolerance1
abstract: Immunoregulatory mechanisms dependent on regulatory CD4+ T cells are believed to be critical in the maintenance of peripheral tolerance to allografts. However, a detailed characterization of the effects of these regulatory T cells has been hampered by the absence of a simple means to track and study them. In this work we provide evidence that in a murine model of islet transplantation the interactions between alloaggressive and regulatory T cells can be studied in vitro and in vivo at the single-cell level. The observations made in both an in vitro coculture system and an in vivo CFSE-based adoptive transfer model indicate that lymphocytes from tolerant allograft recipients 1) proliferate weakly to donor strain allogeneic cells but vigorously to third-party strain cells; and 2) suppress the proliferation of naive syngeneic CD4+ and CD8+ T cells to donor tissue in a cell dose- and Ag-specific manner. These effects depend on the presence of CD4+CD25+ T cells and are neutralized by anti-CTLA4 mAb or rIL-2. The principal effect of anti-CTLA4 is directed against the naive, not regulatory, T cell population. These results can be replicated in vivo by transferring lymphocyte populations into transplant recipients, proving that the graft-protecting actions of regulatory T cells are blunted by a rise in the number of allodestructive T cells (pool size model) and depend on the presence of CD4+CD25+ T cells and the integrity of the CTLA4/B7 pathway.

title: Inability to Induce Tolerance Through Direct Antigen Presentation
abstract: Both the direct and indirect antigen presentation pathways are important mechanisms for T cell‐mediated allograft rejection. Studies using knockout mice and monoclonal antibodies have demonstrated that CD4+ T cells are both necessary and sufficient for the rejection of allogeneic tissues, including skin, heart, and islet. Furthermore, combined blockade of the CD28/B7 and CD154/CD40 costimulatory pathways induces tolerance in multiple CD4+ T‐cell dependent allograft models. In this study, we addressed the T‐cell requirement for costimulation in direct antigen presentation. We demonstrated that class II‐specific alloreactive T‐cell receptor transgenic T cells were sufficient to mediate allograft rejection independent of costimulatory blockade. Analysis of the costimulatory capacity of different antigen presenting cell (APC) populations demonstrated that APCs resident within the donor skin, Langerhans cells, are potent stimulators not requiring CD28‐ or CD154‐dependent costimulation for direct major histocompatibility complex (MHC) antigen presentation. These results complement previous work examining the role of costimulation on CD8+ T cells, supporting a model in which the effectiveness of costimulatory blockade in the setting of transplantation may be selective for the indirect pathway of MHC alloantigen presentation.

title: Blockade of Programmed Death-1 Ligands on Dendritic Cells Enhances T Cell Activation and Cytokine Production 1
abstract: Programmed death-1 ligand (PD-L)1 and PD-L2 are ligands for programmed death-1 (PD-1), a member of the CD28/CTLA4 family expressed on activated lymphoid cells. PD-1 contains an immunoreceptor tyrosine-based inhibitory motif and mice deficient in PD-1 develop autoimmune disorders suggesting a defect in peripheral tolerance. Human PD-L1 and PD-L2 are expressed on immature dendritic cells (iDC) and mature dendritic cells (mDC), IFN-γ-treated monocytes, and follicular dendritic cells. Using mAbs, we show that blockade of PD-L2 on dendritic cells results in enhanced T cell proliferation and cytokine production, including that of IFN-γ and IL-10, while blockade of PD-L1 results in similar, more modest, effects. Blockade of both PD-L1 and PD-L2 showed an additive effect. Both whole mAb and Fab enhanced T cell activation, showing that PD-L1 and PD-L2 function to inhibit T cell activation. Enhancement of T cell activation was most pronounced with weak APC, such as iDCs and IL-10-pretreated mDCs, and less pronounced with strong APC such as mDCs. These data are consistent with the hypothesis that iDC have a balance of stimulatory vs inhibitory molecules that favors inhibition, and indicate that PD-L1 and PD-L2 contribute to the poor stimulatory capacity of iDC. PD-L1 expression differs from PD-L2 in that PD-L1 is expressed on activated T cells, placental trophoblasts, myocardial endothelium, and cortical thymic epithelial cells. In contrast, PD-L2 is expressed on placental endothelium and medullary thymic epithelial cells. PD-L1 is also highly expressed on most carcinomas but minimally expressed on adjacent normal tissue suggesting a role in attenuating antitumor immune responses.

title: Molecular Modeling and Functional Mapping of B7-H1 and B7-DC Uncouple Costimulatory Function from PD-1 Interaction
abstract: B7-H1 and B7-DC are ligands for PD-1, a receptor implicated in negative regulation of T and B cell functions. These ligands, however, also costimulate T cell responses. It remains elusive whether or not costimulation is mediated through PD-1. By comparative molecular modeling and site-directed mutagenesis, we found that nonconserved residues between these ligands on the A′GFCC′C′′ face mediate interaction with PD-1. This indicates significant structural heterogeneity of the interactions between PD-1 and its ligands. Importantly, ligand mutants with abolished PD-1 binding capacity could still costimulate proliferation and cytokine production of T cells from normal and PD-1–deficient mice. Our results reveal unique binding characteristics of B7-H1 and B7-DC and provide direct evidence for an independent costimulatory receptor other than PD-1.

title: Requirement for T-cell apoptosis in the induction of peripheral transplantation tolerance
abstract: The mechanisms of allograft tolerance have been classified as deletion, anergy, ignorance and suppression/regulation. Deletion has been implicated in central tolerance, whereas peripheral tolerance has generally been ascribed to clonal anergy and/or active immunoregulatory states. Here, we used two distinct systems to assess the requirement for T-cell deletion in peripheral tolerance induction. In mice transgenic for Bcl-xL, T cells were resistant to passive cell death through cytokine withdrawal, whereas T cells from interleukin-2-deficient mice did not undergo activation-induced cell death. Using either agents that block co-stimulatory pathways or the immunosuppressive drug rapamycin, which we have shown here blocks the proliferative component of interleukin-2 signaling but does not inhibit priming for activation-induced cell death, we found that mice with defective passive or active T-cell apoptotic pathways were resistant to induction of transplantation tolerance. Thus, deletion of activated T cells through activation-induced cell death or growth factor withdrawal seems necessary to achieve peripheral tolerance across major histocompatibility complex barriers.

title: Blocking both signal 1 and signal 2 of T-cell activation prevents apoptosis of alloreactive T cells and induction of peripheral allograft tolerance
abstract: The alloimmune response against fully MHC-mismatched allografts, compared with immune responses to nominal antigens, entails an unusually large clonal size of alloreactive T cells. Thus, induction of peripheral allograft tolerance established in the absence of immune system ablation and reconstitution is a challenging task in transplantation. Here, we determined whether a reduction in the mass of alloreactive T cells due to apoptosis is an essential initial step for induction of stable allograft tolerance with non-lymphoablative therapy. Blocking both CD28–B7 and CD40–CD40 ligand interactions (co-stimulation blockade) inhibited proliferation of alloreactive T cells in vivo while allowing cell cycle-dependent T-cell apoptosis of proliferating T cells, with permanent engraftment of cardiac allografts but not skin allografts. Treatment with rapamycin plus co-stimulation blockade resulted in massive apoptosis of alloreactive T cells and produced stable skin allograft tolerance, a very stringent test of allograft tolerance. In contrast, treatment with cyclosporine A and co-stimulation blockade abolished T-cell proliferation and apoptosis, as well as the induction of stable allograft tolerance. Our data indicate that induction of T-cell apoptosis and peripheral allograft tolerance is prevented by blocking both signal 1 and signal 2 of T-cell activation.

title: Tumor-associated B7-H1 promotes T-cell apoptosis: A potential mechanism of immune evasion
abstract: B7-H1, a recently described member of the B7 family of costimulatory molecules, is thought to be involved in the regulation of cellular and humoral immune responses through the PD-1 receptor on activated T and B cells. We report here that, except for cells of the macrophage lineage, normal human tissues do not express B7-H1. In contrast, B7-H1 is abundant in human carcinomas of lung, ovary and colon and in melanomas. The pro-inflammatory cytokine interferon-γ upregulates B7-H1 on the surface of tumor cell lines. Cancer cell–associated B7-H1 increases apoptosis of antigen-specific human T-cell clones in vitro, and the apoptotic effect of B7-H1 is mediated largely by one or more receptors other than PD-1. In addition, expression of B7-H1 on mouse P815 tumor increases apoptosis of activated tumor-reactive T cells and promotes the growth of highly immunogenic B7-1+ tumors in vivo. These findings have implications for the design of T cell–based cancer immunotherapy.

title: Immunologic Self-Tolerance Maintained by Cd25+Cd4+Regulatory T Cells Constitutively Expressing Cytotoxic T Lymphocyte–Associated Antigen 4
abstract: This report shows that cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) plays a key role in T cell–mediated dominant immunologic self-tolerance. In vivo blockade of CTLA-4 for a limited period in normal mice leads to spontaneous development of chronic organ-specific autoimmune diseases, which are immunopathologically similar to human counterparts. In normal naive mice, CTLA-4 is constitutively expressed on CD25+CD4+ T cells, which constitute 5–10% of peripheral CD4+ T cells. When the CD25+CD4+ T cells are stimulated via the T cell receptor in vitro, they potently suppress antigen-specific and polyclonal activation and proliferation of other T cells, including CTLA-4–deficient T cells, and blockade of CTLA-4 abrogates the suppression. CD28-deficient CD25+CD4+ T cells can also suppress normal T cells, indicating that CD28 is dispensable for activation of the regulatory T cells. Thus, the CD25+CD4+ regulatory T cell population engaged in dominant self-tolerance may require CTLA-4 but not CD28 as a costimulatory molecule for its functional activation. Furthermore, interference with this role of CTLA-4 suffices to elicit autoimmune disease in otherwise normal animals, presumably through affecting CD25+CD4+ T cell–mediated control of self-reactive T cells. This unique function of CTLA-4 could be exploited to potentiate T cell–mediated immunoregulation, and thereby to induce immunologic tolerance or to control autoimmunity.

title: Cytotoxic T Lymphocyte–Associated Antigen 4 Plays an Essential Role in the Function of Cd25+Cd4+ Regulatory Cells That Control Intestinal Inflammation
abstract: It is now clear that functionally specialized regulatory T (Treg) cells exist as part of the normal immune repertoire, preventing the development of pathogenic responses to both self- and intestinal antigens. Here, we report that the Treg cells that control intestinal inflammation express the same phenotype (CD25+CD45RBlowCD4+) as those that control autoimmunity. Previous studies have failed to identify how CD25+ Treg cells function in vivo. Our studies reveal that the immune-suppressive function of these cells in vivo is dependent on signaling via the negative regulator of T cell activation cytotoxic T lymphocyte–associated antigen 4 (CTLA-4), as well as secretion of the immune-suppressive cytokine transforming growth factor β. Strikingly, constitutive expression of CTLA-4 among CD4+ cells was restricted primarily to Treg cells, suggesting that CTLA-4 expression by these cells is involved in their immune-suppressive function. These findings raise the possibility that Treg cell function contributes to the immune suppression characteristic of CTLA-4 signaling. Identification of costimulatory molecules involved in the function of Treg cells may facilitate further characterization of these cells and development of new therapeutic strategies for the treatment of inflammatory diseases.

title: B7/CD28 costimulation is essential for the homeostasis of the CD4+CD25+ immunoregulatory T cells that control autoimmune diabetes.
abstract: CD28/B7 costimulation has been implicated in the induction and progression of autoimmune diseases. Experimentally induced models of autoimmunity have been shown to be prevented or reduced in intensity in mice rendered deficient for CD28 costimulation. In sharp contrast, spontaneous diabetes is exacerbated in both B7-1/B7-2-deficient and CD28-deficient NOD mice. These mice present a profound decrease of the immunoregulatory CD4+CD25+ T cells, which control diabetes in prediabetic NOD mice. These cells are absent from both CD28KO and B7-1/B7-2KO mice, and the transfer of this regulatory T cell subset from control NOD animals into CD28-deficient animals can delay/prevent diabetes. The results suggest that the CD28/ B7 costimulatory pathway is essential for the development and homeostasis of regulatory T cells that control spontaneous autoimmune diseases.

title: PD-1: an inhibitory immunoreceptor involved in peripheral tolerance.
abstract: PD-1 deficiency causes a variety of autoimmune diseases. Inhibitory signaling through the PD-1 receptor might therefore be involved in the regulation of peripheral tolerance. The constitutive expression of PD-1 ligands (PD-L1 and PD-L2) on parenchymal cells of heart, lung and kidney suggests that the PD-1-PD-L system could provide unique negative signaling to help prevent autoimmune diseases.

title: PD‐1:PD‐L inhibitory pathway affects both CD4+ and CD8+ T cells and is overcome by IL‐2
abstract: Programmed death‐1 (PD‐1) is an immunoreceptor tyrosine‐based inhibitory motif (ITIM)‐containing receptor expressed upon T cell activation. PD‐1–/– animals develop autoimmune diseases, suggesting an inhibitory role for PD‐1 in immune responses. Members of the B7 family, PD‐L1 and PD‐L2, are ligands for PD‐1. This study examines the functional consequences of PD‐1:PD‐L engagementon murine CD4 and CD8 T cells and shows that these interactions result in inhibition of proliferation and cytokine production. T cells stimulated with anti‐CD3/PD‐L1.Fc‐coated beads display dramatically decreased proliferation and IL‐2 production, while CSFE analysis shows fewer cells cycling and a slower division rate. Costimulation with soluble anti‐CD28 mAb can overcome PD‐1‐mediated inhibition by augmenting IL‐2 production. However, PD‐1:PD‐L interactions inhibit IL‐2 production even in the presence of costimulation and, thus, after prolonged activation, the PD‐1:PD‐L inhibitory pathway dominates. Exogenous IL‐2 is able to overcome PD‐L1‐mediated inhibition at all times, indicating that cells maintain IL‐2 responsiveness. Experiments using TCR transgenic CD4+ or CD8+ T cells stimulated with antigen‐presenting cells expressing PD‐L1 show that both T cell subsets are susceptible to this inhibitory pathway. However, CD8+ T cells may be more sensitive to modulation by the PD‐1:PD‐L pathway because of their intrinsic inability to produce significant levels of IL‐2.

title: PD-L1-deficient mice show that PD-L1 on T cells, antigen-presenting cells, and host tissues negatively regulates T cells.
abstract: Both positive and negative regulatory roles have been suggested for the B7 family member PD-L1(B7-H1). PD-L1 is expressed on antigen-presenting cells (APCs), activated T cells, and a variety of tissues, but the functional significance of PD-L1 on each cell type is not yet clear. To dissect the functions of PD-L1 in vivo, we generated PD-L1-deficient (PD-L1(-/-)) mice. CD4(+) and CD8(+) T cell responses were markedly enhanced in PD-L1(-/-) mice compared with wild-type mice in vitro and in vivo. PD-L1(-/-) dendritic cells stimulated greater wild-type CD4(+) T cell responses than wild-type dendritic cells, and PD-L1(-/-) CD4(+) T cells produced more cytokines than wild-type CD4(+) T cells in vitro, demonstrating an inhibitory role for PD-L1 on APCs and T cells. In vivo CD8(+) T cell responses also were significantly enhanced, indicating that PD-L1 has a role in limiting the expansion or survival of CD8(+) T cells. Studies using the myelin oligodendrocyte model of experimental autoimmune encephalomyelitis showed that PD-L1 on T cells and in host tissues limits responses of self-reactive CD4(+) T cells in vivo. PD-L1 deficiency converted the 129S4/SvJae strain from a resistant to experimental autoimmune encephalomyelitis-susceptible strain. Transfer of encephalitogenic T cells from wild-type mice into PD-L1(-/-) recipients led to exacerbated disease. Disease was even more severe in PD-L1(-/-) recipients of PD-L1(-/-) T cells. These results demonstrate that PD-L1 on T cells, APCs, and host tissue inhibits naïve and effector T cell responses and plays a critical role in T cell tolerance.

title: B7-H1 costimulation preferentially enhances CD28-independent T-helper cell function.
abstract: B7-H1 is a recently described B7-like molecule that costimulates T-cell growth and cytokine secretion without binding to CD28, cytotoxic T-lymphocyte antigen-4 (CTLA-4), and inducible costimulator (ICOS). In this report, a mouse homologue of human B7-H1 is identified, and its immunologic functions are studied in vitro and in vivo. Mouse B7-H1 shares 69% amino acid homology to the human counterpart. Similar to human B7-H1, mouse B7-H1 can be induced to express on macrophages, T cells, and B cells and to enhance T-cell proliferation and secretion of interleukin-10 (IL-10), interferon-gamma, and granulocyte-macrophage colony-stimulating factor but not IL-2 and IL-4. Furthermore, B7-H1 preferentially costimulates CD4+ T cells independently of CD28 and enhances mixed lymphocyte responses to allogeneic antigens. In contrast to B7-1, expression of B7-H1 on murine P815 tumor cells by transfection fails to increase allogeneic and syngeneic cytolytic T-cell responses in vitro and in vivo. Administration of B7-H1Ig fusion protein, however, enhances keyhole limpet hemocyanin- specific T-cell proliferation and 2,4,6-trinitrophenyl-specific immunoglobulin G2a antibody production. The study thus identifies a unique costimulatory pathway that preferentially affects T-helper cell functions.

title: Regulation of PD‐1, PD‐L1, and PD‐L2 expression during normal and autoimmune responses
abstract: Newer members of the B7‐CD28 superfamily include the receptor PD‐1 and its two ligands, PD‐L1 and PD‐L2. Here, we characterize the expression of PD‐1, PD‐L1, and PD‐L2 in tissues of naive miceand in target organs from two models of autoimmunity, the pancreas from non‐obese diabetic (NOD) mice and brain from mice with experimental autoimmune encephalomyelitis (EAE). In naive mice, proteiexpression of PD‐1, PD‐L1, and PD‐L2 was detected in the thymus, while PD‐1 and PD‐L1 were detected in the spleen. PD‐L1, but not PD‐L2, was also detected at low levels on cardiac endothelium, pancreatic islets, and syncyciotrophoblasts in the placenta. In pre‐diabetic NOD mice, PD‐1 and PD‐L1 were expressed on infiltrating cells in the pancreatic islets. Furthermore, PD‐L1 was markedly up‐regulated on islet cells. In brains from mice with EAE, PD‐1, PD‐L1, and PD‐L2 were expressed on infiltrating inflammatory cells, and PD‐L1 was up‐regulated on endothelium within EAE brain. The distinct expression patterns of PD‐L1 and PD‐L2 led us to compare their transcriptional regulation in STAT4–/–, STAT6–/–, or NF‐κB p50–/–p65+/– dendritic cells (DC).PD‐L2, but not PD‐L1, expression was dramatically reduced in p50–/–p65+/– DC. Thus, PD‐L1 and PD‐L2 exhibit distinct expression patterns and are differentially regulated on the transcriptional level.

title: Expression and Regulation of the PD‐L1 Immunoinhibitory Molecule on Microvascular Endothelial Cells
abstract: To evaluate the expression and regulation of a novel B7‐like protein, PD‐L1, the ligand for the immunoinhibitory receptor PD‐1 expressed on activated T‐cells, on microvascular endothelial cells (ECs)

title: Induced expression of PD‐1, a novel member of the immunoglobulin gene superfamily, upon programmed cell death.
abstract: The classical type of programmed cell death is characterized by its dependence on de novo RNA and protein synthesis and morphological features of apoptosis. We confirmed that stimulated 2B4.11 (a murine T‐cell hybridoma) and interleukin‐3 (IL‐3)‐deprived LyD9 (a murine haematopoietic progenitor cell line) died by the classical type of programmed cell death. Assuming that common biochemical pathways might be involved in the deaths of 2B4.11 and LyD9, we isolated the PD‐1 gene, a novel member of the immunoglobulin gene superfamily, by using subtractive hybridization technique. The predicted PD‐1 protein has a variant form of the consensus sequence found in cytoplasmic tails of signal transducing polypeptides associated with immune recognition receptors. The PD‐1 gene was activated in both stimulated 2B4.11 and IL‐3‐deprived LyD9 cells, but not in other death‐induced cell lines that did not show the characteristic features of the classical programmed cell death. Expression of the PD‐1 mRNA in mouse was restricted to the thymus and increased when thymocyte death was augmented by in vivo injection of anti‐CD3 antibody. These results suggest that activation of the PD‐1 gene may be involved in the classical type of programmed cell death.

title: Immunologic tolerance maintained by CD25+ CD4+ regulatory T cells: their common role in controlling autoimmunity, tumor immunity, and transplantation tolerance
abstract: There is accumulating evidence that T‐cell‐mediated dominant control of self‐reactive T‐cells contributes to the maintenance of immunologic self‐tolerance and its alteration can cause autoimmune disease. Efforts to delineate such a regulatory T‐cell population have revealed that CD25+ cells in the CD4+ population in normal naive animals bear the ability to prevent autoimmune disease in vivo and, upon antigenic stimulation, suppress the activation/proliferation of other T cells in vitro. The CD25+ CD4+ regulatory T cells, which are naturally anergic and suppressive, appear to be produced by the normal thymus as a functionally distinct subpopulation of T cells. They play critical roles not only in preventing autoimmunity but also in controlling tumor immunity and transplantation tolerance.

title: Is CTLA-4 a master switch for peripheral T cell tolerance?
abstract: The CD28/CTLA-4 and B7 families of cell surface molecules regulate a complex pathway of T cell signals that profoundly affect peripheral T cell responses. On the one hand, CD28/B7-mediated T cell costimulation is essential for the development and maintenance of T cell responses. In contrast, the prevailing data suggest that the engagement of CTLA-4 with the same B7 ligands down-regulates the initiation and progression of immune responses. This commentary examines the role of CTLA-4-mediated signaling in the regulation of the immune response, leading to the hypothesis that the CTLA-4/B7 interaction plays an essential role in the establishment of peripheral self-tolerance as well as the regulation of normal T cell immunity.

title: The emerging role of CTLA-4 as an immune attenuator.
abstract: Based on the striking phenotype of CTLA-4–deficient animals, the role of CTLA-4 in various human autoimmune diseases has been investigated using a candidate gene approach. Initial linkage between CTLA-4 and autoimmune disease was made in a case/control study involving patients with Grave's disease. By analyzing CTLA-4 alleles identified through variations in an AT repeat in the 3′ untranslated region, Yanagawa et al. 1995xCTLA-4 gene polymorphism associated with Graves' disease in a caucasian population. Yanagawa, T, Hidaka, Y, Guimaraes, V, Soliman, M, and DeGroot,  | PubMedSee all ReferencesYanagawa et al. 1995 demonstrated that CTLA-4 or a closely linked gene could be associated with an increased risk of Grave's disease. Subsequent studies have verified and extended these observations. Using an A/G polymorphism within CTLA-4 exon 1, Donner et al. 1997xCTLA-4 alanine-17 confers genetic susceptibility to Graves' disease and to type 1 diabetes mellitus. Donner, H, Rau, H, Walfish, P.G, Braun, J, Siegmund, T, Finke, R, Herwig, J, Usadel, K.H, and Badenhoop, K.  143–146Crossref | PubMed | Scopus (385)See all ReferencesDonner et al. 1997 were able to confirm a statistical association between Grave's disease and the CTLA-4 polymorphism. Furthermore, these studies demonstrated that CTLA-4 contributes to the risk of Grave's disease independently of human leukocyte antigen status or sex.An association between CTLA-4 and autoimmune diabetes mellitus has also been reported (26xThe CTLA-4 gene region of chromosome 2q33 is linked to, and associated with, type 1 diabetes. Nistic, L, Buzzetti, R, Pritchard, L.E, Van der Auwera, B, Giovannini, C, Bosi, E, Larrad, M.T.M, Rios, M.S, Chow, C.C, Cockram, C.S et al.  1075–1080Crossref | PubMed | Scopus (584)See all References, 32xPanning for gold (genome-wide scanning for linkage in type 1 diabetes) . Todd, J.A and Farrall, M. Hum. Mol. Genet. 1996; 5: 1443–1448PubMedSee all References, 7xCTLA-4 alanine-17 confers genetic susceptibility to Graves' disease and to type 1 diabetes mellitus. Donner, H, Rau, H, Walfish, P.G, Braun, J, Siegmund, T, Finke, R, Herwig, J, Usadel, K.H, and Badenhoop, K.  143–146Crossref | PubMed | Scopus (385)See all References). The same polymorphisms found in Grave's disease were found to be associated with diabetes. Although it remains possible that the involved gene may be a CTLA-4–linked gene such as CD28, the number of independent confirmations that the CTLA-4 gene is associated with these two autoimmune diseases makes it likely that certain CTLA-4 alleles may be risk factors for this disease. Consistent with this hypothesis, the linkage of autoimmune diseases to a CTLA-4 allele with a long AT repeat in the 3′ untranslated region is intriguing. Previous studies have demonstrated that such AT-rich tracks contribute to mRNA instability. If the size of the CTLA-4 AT tract limited the accumulation of CTLA-4 mRNA, the resulting decrease in CTLA-4 expression might account for the observed increase in the risk of autoimmune disease.Based on the strong association with Grave's disease and diabetes mellitus, the linkage of CTLA-4 to additional autoimmune diseases should be explored. However, no matter how extensive the involvement of CTLA-4 in the pathogenesis of human autoimmune diseases, it is clear from the present data that CTLA-4 can play a regulatory role in terminating peripheral immune responses. Disruption of the normal physiologic control provided by the CTLA-4 receptor can contribute to the pathogenesis of autoimmune disease, as demonstrated both by gene knockout studies in mice and by genetic linkage analysis in human autoimmune disorders.‡To whom correspondence should be addressed (e-mail: craig@knapp.uchicago.edu).

title: CTLA4 signals are required to optimally induce allograft tolerance with combined donor-specific transfusion and anti-CD154 monoclonal antibody treatment.
abstract: Sensitization to donor Ags is an enormous problem in clinical transplantation. In an islet allograft model, presensitization of recipients through donor-specific transfusion (DST) 4 wk before transplantation results in accelerated rejection. We demonstrate that combined DST with anti-CD154 (CD40L) therapy not only prevents the deleterious presensitization produced by pretransplant DST in the islet allograft model, it also induces broad alloantigen-specific tolerance and permits subsequent engraftment of donor islet or cardiac grafts without further treatment. In addition, our data strongly indicate that CTLA4-negative T cell signals are required to achieve prolonged engraftment of skin allograft or tolerance to islet allograft in recipients treated with a combination of pretransplant DST and anti-CD154 mAb. We provide direct evidence that a CD28-independent CTLA4 signal delivers a strong negative signal to CD4+ T cells that can block alloimmune MLR responses. In this study immune deviation into a Th2 (IL-4) response was associated with, but did not insure, graft tolerance, as the inopportune timing of B7 blockade with CTLA4/Ig therapy prevented uniform tolerance but did not prevent Th2-type immune deviation. While CTLA4-negative signals are necessary for tolerance induction, Th1 to Th2 immune deviation cannot be sufficient for tolerance induction. Combined pretransplant DST with anti-CD154 mAb treatment may be attractive for clinical deployment, and strategies aimed to selectively block CD28 without interrupting CTLA4/B7 interaction might prove highly effective in the induction of tolerance.

title: The role of CD80, CD86, and CTLA4 in alloimmune responses and the induction of long-term allograft survival.
abstract: Blocking the interaction of the CD28 costimulatory receptor with its ligands, CD80 and CD86, inhibits in vivo immune responses, such as allograft rejection, and in some instances induces tolerance. Previously, we found that CTLA4Ig, which blocks the CD28/CTLA-4 (CD152) ligands CD80 and CD86, can be used to induce transplantation tolerance to vascularized allografts. Recent data suggest that an intact CD152-negative signaling pathway is essential for induction of tolerance to nominal Ags. Here, we show that blockade of CD152 using an anti-CD152 mAb at the time of transplantation prevents the induction of long-term allograft survival by agents that target CD80 and CD86. In contrast, CD152 signals are not required for the maintenance of established graft survival. We also report for the first time that blockade of CD86 alone can induce long-term graft survival. This requires that anti-CD86 mAb is given on the day of transplantation and also depends upon an intact CD152 pathway. This result, plus experiments using CD80-deficient mice, suggests a dominant role for CD80 molecules on donor cells as the relevant ligand for CD152. We additionally find that blockade of CD152 at the time of transplantation does not interfere with the effectiveness of anti-CD154 mAbs, suggesting distinct mechanisms for inhibition of graft rejection by blocking the CD28 vs CD154 pathways.

title: Physiological Mechanisms of Regulating Alloimmunity: Cytokines, CTLA-4, CD25+ Cells, and the Alloreactive T Cell Clone Size1
abstract: The mechanisms underlying physiological regulation of alloimmune responses remain poorly defined. We investigated the roles of cytokines, CTLA-4, CD25+ T cells, and apoptosis in regulating alloimmune responses in vivo. Two murine cardiac transplant models were used, B10.D2 (minor mismatch) and C57BL/6 (major mismatch), into BALB/c recipients. Recipients were wild type, STAT4−/− (Th1 deficient), or STAT6−/− (Th2 deficient) mice. Minor mismatched allografts were accepted spontaneously in ∼70% of wild type and STAT4−/− mice. By contrast, there was significantly shorter graft survival in minor mismatched STAT6−/− mice. Either the adoptive transfer of STAT4−/− splenocytes or the administration of IL-4Fc fusion protein into STAT6−/− mice resulted in long term graft survival. Blocking CTLA-4 signaling accelerated the rejection in all recipients, but was more pronounced in the minor combination. This was accompanied by an increased frequency of alloreactive T cells. Furthermore, CTLA-4 blockade regulated CD4+ or CD8+ as well as Th1 or Th2 alloreactive T cells. Finally, while anti-CD25 treatment prolonged graft survival in the major mismatched combination, the same treatment accelerated graft rejection in the minor mismatched group. The latter was associated with an increased frequency of alloreactive T cells and inhibition of T cell apoptosis. These data demonstrate that cytokine regulation, CTLA-4 negative signaling, and T cell apoptosis play critical roles in regulating alloimmunity, especially under conditions where the alloreactive T cell clone size is relatively small.