Vijay K. Vanguri

PD-L1 regulates the development, maintenance, and function of induced regulatory T cells

Both the programmed death (PD) 1–PD-ligand (PD-L) pathway and regulatory T (T reg) cells are instrumental to the maintenance of peripheral tolerance. We demonstrate that PD-L1 has a pivotal role in regulating induced T reg (iT reg) cell development and sustaining iT reg cell function. PD-L1−/− antigen-presenting cells minimally convert naive CD4 T cells to iT reg cells, showing the essential role of PD-L1 for iT reg cell induction. PD-L1–coated beads induce iT reg cells in vitro, indicating that PD-L1 itself regulates iT reg cell development. Furthermore, PD-L1 enhances and sustains Foxp3 expression and the suppressive function of iT reg cells. The obligatory role for PD-L1 in controlling iT reg cell development and function in vivo is illustrated by a marked reduction in iT reg cell conversion and rapid onset of a fatal inflammatory phenotype in PD-L1−/−PD-L2−/− Rag−/− recipients of naive CD4 T cells. PD-L1 iT reg cell development is mediated through the down-regulation of phospho-Akt, mTOR, S6, and ERK2 and concomitant with the up-regulation of PTEN, all key signaling molecules which are critical for iT reg cell development. Thus, PD-L1 can inhibit T cell responses by promoting both the induction and maintenance of iT reg cells. These studies define a novel mechanism for iT reg cell development and function, as well as a new strategy for controlling T reg cell plasticity.

PD-L1 has distinct functions in hematopoietic and nonhematopoietic cells in regulating T cell responses during chronic infection in mice

The inhibitory receptor programmed death 1 (PD-1) is upregulated on antigen-specific CD8+ T cells during persistent viral infections. Interaction with PD-1 ligand 1 (PD-L1) contributes to functional exhaustion of responding T cells and may limit immunopathology during infection. PD-L1 is expressed on both hematopoietic and nonhematopoietic cells in tissues. However, the exact roles of PD-L1 on hematopoietic versus nonhematopoietic cells in modulating immune responses are unclear. Here we used bone marrow chimeric mice to examine the effects of PD-L1 deficiency in hematopoietic or nonhematopoietic cells during lymphocytic choriomeningitis virus clone 13 (LCMV CL-13) infection. We found that PD-L1 expression on hematopoietic cells inhibited CD8+ T cell numbers and function after LCMV CL-13 infection. In contrast, PD-L1 expression on nonhematopoietic cells limited viral clearance and immunopathology in infected tissues. Together, these data demonstrate that there are distinct roles for PD-L1 on hematopoietic and nonhematopoietic cells in regulating CD8+ T cell responses and viral clearance during chronic viral infection.

The Programmed Death-1 Ligand 1:B7-1 Pathway Restrains Diabetogenic Effector T Cells In Vivo

Programmed death-1 ligand 1 (PD-L1) is a coinhibitory molecule that negatively regulates multiple tolerance checkpoints. In the NOD mouse model, PD-L1 regulates the development of diabetes. PD-L1 has two binding partners, programmed death-1 and B7-1, but the significance of the PD-L1:B7-1 interaction in regulating self-reactive T cell responses is not yet clear. To investigate this issue in NOD mice, we have compared the effects of two anti–PD-L1 Abs that have different blocking activities. Anti–PD-L1 mAb 10F.2H11 sterically and functionally blocks only PD-L1:B7-1 interactions, whereas anti–PD-L1 mAb 10F.9G2 blocks both PD-L1:B7-1 and PD-L1:programmed death-1 interactions. Both Abs had potent, yet distinct effects in accelerating diabetes in NOD mice: the single-blocker 10F.2H11 mAb was more effective at precipitating diabetes in older (13-wk-old) than in younger (6- to 7-wk-old) mice, whereas the dual-blocker 10F.9G2 mAb rapidly induced diabetes in NOD mice of both ages. Similarly, 10F.2H11 accelerated diabetes in recipients of T cells from diabetic, but not prediabetic mice, whereas 10F.9G2 was effective in both settings. Both anti–PD-L1 mAbs precipitated diabetes in adoptive transfer models of CD4+ and CD8+ T cell-driven diabetes. Taken together, these data demonstrate that the PD-L1:B7-1 pathway inhibits potentially pathogenic self-reactive effector CD4+ and CD8+ T cell responses in vivo, and suggest that the immunoinhibitory functions of this pathway may be particularly important during the later phases of diabetogenesis.

The Novel Costimulatory Programmed Death Ligand 1/B7.1 Pathway Is Functional in Inhibiting Alloimmune Responses In Vivo

The programmed death ligand 1 (PDL1)/programmed death 1 (PD1) costimulatory pathway plays an important role in the inhibition of alloimmune responses as well as in the induction and maintenance of peripheral tolerance. It has been demonstrated recently that PDL1 also can bind B7.1 to inhibit T cell responses in vitro. Using the bm12 into B6 heart transplant model, we investigated the functional significance of this interaction in alloimmune responses in vivo. PD1 blockade unlike PDL1 blockade failed to accelerate bm12 allograft rejection, suggesting a role for an additional binding partner for PDL1 other than PD1 in transplant rejection. PDL1 blockade was able to accelerate allograft rejection in B7.2-deficient recipients but not B7.1-deficient recipients, indicating that PDL1 interaction with B7.1 was important in inhibiting rejection. Administration of the novel 2H11 anti-PDL1 mAb, which only blocks the PDL1–B7.1 interaction, aggravated chronic injury of bm12 allografts in B6 recipients. Aggravated chronic injury was associated with an increased frequency of alloreactive IFN-γ–, IL-4–, and IL-6–producing splenocytes and a decreased percentage of regulatory T cells in the recipients. Using an in vitro cell culture assay, blockade of the interaction of PDL1 on dendritic cells with B7.1 on T cells increased IFN-γ production from alloreactive CD4+ T cells, whereas blockade of dendritic cell B7.1 interaction with T cell PDL1 did not. These data indicate that PDL1 interaction with B7.1 plays an important role in the inhibition of alloimmune responses in vivo and suggests a dominant direction for PDL1 and B7.1 interaction.

Critical Role of Donor Tissue Expression of Programmed Death Ligand-1 in Regulating Cardiac Allograft Rejection and Vasculopathy

Background— Allograft vasculopathy is a major limiting factor in the long-term success of cardiac transplantation. T cells play a critical role in initiation of cardiac allograft rejection and allograft vasculopathy. The negative T-cell costimulatory pathway PD-1:PDL1/PDL2 (programmed death-1:programmed death ligand-1/2) plays an important role in regulating alloimmune responses. We investigated the role of recipient versus donor PD-1 ligands in the pathogenesis of allograft rejection with emphasis on the role of tissue expression in regulating this alloimmune response in vivo. Methods and Results— We used established major histocompatibility complex class II– and class I–mismatched models of vascularized cardiac allograft rejection, blocking anti-PDL1 and anti-PDL2 antibodies, and PDL1- and PDL2-deficient mice (as donors or recipients) to study the role of the PD-1:PDL1/PDL2 pathway in chronic rejection. We also used PDL1-deficient and wild-type mice and bone marrow transplantation to generate chimeric animals that express PDL1 exclusively on either hematopoietic or parenchymal cells. PDL1 but not PDL2 blockade significantly accelerated cardiac allograft rejection in the bm12-into-B6 and B6-into-bm12 models. Although wild-type cardiac allografts survived long term, PDL1−/− donor hearts transplanted into wild-type bm12 mice exhibited accelerated rejection and vasculopathy associated with enhanced recipient T-cell alloreactivity. Interestingly, PDL1−/− recipients did not exhibit an accelerated tempo of cardiac allograft rejection. Using chimeric animals as donors, we show that PDL1 expression on cardiac tissue alone significantly prolonged graft survival compared with full PDL1−/− donor grafts in transplanted wild-type recipients. Conclusions— This is the first report to demonstrate that expression of the negative costimulatory molecule PDL1 on donor cardiac tissue regulates recipient alloimmune responses, allograft rejection, and vasculopathy.

Relapsing–Remitting Central Nervous System Autoimmunity Mediated by GFAP-Specific CD8 T Cells

Multiple sclerosis (MS) is an inflammatory disease of the CNS that causes the demyelination of nerve cells and destroys oligodendrocytes, neurons, and axons. Historically, MS has been thought to be a CD4 T cell–mediated autoimmune disease of CNS white matter. However, recent studies identified CD8 T cell infiltrates and gray matter lesions in MS patients. These findings suggest that CD8 T cells and CNS Ags other than myelin proteins may be involved during the MS disease process. In this article, we show that CD8 T cells reactive to glial fibrillary acidic protein (GFAP), a protein expressed in astrocytes, can avoid tolerance mechanisms and, depending upon the T cell–triggering event, drive unique aspects of inflammatory CNS autoimmunity. In GFAP-specific CD8 TCR-transgenic (BG1) mice, tissue resident memory-like CD8 T cells spontaneously infiltrate the gray matter and white matter of the CNS, resulting in a relapsing–remitting CNS autoimmunity. The frequency, severity, and remissions from spontaneous disease are controlled by the presence of polyclonal B cells. In contrast, a viral trigger induces GFAP-specific CD8 T effector cells to exclusively target the meninges and vascular/perivascular space of the gray and white matter of the brain, causing a rapid, acute CNS disease. These findings demonstrate that the type of CD8 T cell–triggering event can determine the presentation of distinct CNS autoimmune disease pathologies.

Essential role of PDL1 expression on nonhematopoietic donor cells in acquired tolerance to vascularized cardiac allografts.

The PD1:PDL1 pathway is an essential negative costimulatory pathway that plays a key role in regulating the alloimune response. PDL1 is expressed not only on antigen‐presenting cells (APCs) but also cardiac endothelium. In this study, we investigated the importance of PDL1 expression on donor cardiac allograft in acquired transplantation tolerance in a fully MHC‐mismatched model. We generated PDL1 chimeric mice on B6 background that expressed PDL1 on either hematopoietic cells or nonhematopoietic cells of the heart. Sham animals were used as controls. These hearts were then transplanted into BALB/c recipients and treated with CTLA4‐Ig to induce tolerance. Cardiac endothelium showed significant expression of PDL1, which was upregulated upon transplantation. While the absence of PDL1 on hematopoietic cells of the heart resulted in delayed rejection and prevented long‐term tolerance in most but not all recipients, we observed an accelerated and early graft rejection of all donor allografts that lacked PDL1 on the endothelium. Moreover, PDL1‐deficient endothelium hearts had significant higher frequency of IFN‐γ‐producing alloreactive cells as well as higher frequency of CD8+ effector T cells. These findings demonstrate that PDL1 expression mainly on donor endothelium is functionally important in a fully allogeneic mismatched model for the induction of cardiac allograft tolerance.

Paradoxical functions of B7: CD28 costimulation in a MHC class II-mismatched cardiac transplant model.

Blockade of the B7: CD28 costimulatory pathway has emerged as a promising therapy to prevent allograft rejection. However, this pathway has also been demonstrated to be important for the generation and maintenance of regulatory T cells. In this study, we investigated the role of the B7: CD28 pathway in the ‘bm12 into B6’ MHC class II‐mismatched vascularized cardiac transplant model of chronic rejection. Allograft rejection was remarkably accelerated in B6 background B7DKO and CD28KO recipients compared with B6 wild‐type (WT) recipients. Allograft rejection was associated with a significantly enhanced Th1/Th2 alloreactivity and marked reduction in the ratio of regulatory T cells to CD4+ effector/memory cells. We noted that administration of anti‐B7‐1 and anti‐B7‐2 mAb prior to transplantation also accelerated allograft rejection. Furthermore, depleting CD25+ cells in B6 WT recipients of bm12 hearts prior to transplant also precipitated rejection at a similar rate. Neither B7/CD28 deficiency nor CD25 depletion affected graft survival in single MHC class I‐mismatched (bm1 into B6) recipients. This study highlights the paradoxical functions of B7: CD28 costimulation in a MHC class II‐mismatched model, in which the B7: CD28 pathway is demonstrated to be important in preventing rejection through the generation and maintenance of Tregs.

Auto-antibody production and glomerulonephritis in congenic Slamf1−/− and Slamf2−/− [B6.129] but not in Slamf1−/− and Slamf2−/− [BALB/c.129] mice

Several genes in an interval of human and mouse chromosome 1 are associated with a predisposition for systemic lupus erythematosus. Congenic mouse strains that contain a 129-derived genomic segment, which is embedded in the B6 genome, develop lupus because of epistatic interactions between the 129-derived and B6 genes, e.g. in B6.129chr1b mice. If a gene that is located on chromosome 1 is altered through homologous recombination in 129-derived embryonic stem cells (ES cells) and if the resultant knockout mouse is backcrossed with B6, interpretation of the phenotype of the mutant mouse may be affected by epistatic interactions between the 129 and B6 genomes. Here, we report that knockout mice of two adjacent chromosome 1 genes, Slamf1(-/-) and Slamf2(-/-), which were generated with the same 129-derived ES cell line, develop features of lupus, if backcrossed on to the B6 genetic background. By contrast, Slamf1(-/-) [BALB/c.129] and Slamf2(-/-) [BALB/c.129] do not develop disease. Surprisingly, Slamf1(-/-) [B6.129] mice develop both auto-antibodies and glomerulonephritis between 3 and 6 months of age, while disease fully develops in Slamf1(-/-) [B6.129] mice after 9-14 months. Functional analyses of CD4(+) T cells reveals that Slamf2(-/-) T cells are resistant to tolerance induction in vivo. We conclude that the Slamf2(-/-) mutation may have a unique influence on T-cell tolerance and lupus.

Viral antigen density and confinement time regulate the reactivity pattern of CD4 T-cell responses to vaccinia virus infection

T-cell recognition of ligands is polyspecific. This translates into antiviral T-cell responses having a range of potency and specificity for viral ligands. How these ligand recognition patterns are established is not fully understood. Here, we show that an activation threshold regulates whether robust CD4 T-cell activation occurs following viral infection. The activation threshold was variable because of its dependence on the density of the viral peptide (p)MHC displayed on infected cells. Furthermore, the activation threshold was not observed to be a specific equilibrium affinity (KD) or half-life (t1/2) of the TCR–viral pMHC interaction, rather it correlated with the confinement time of TCR–pMHC interactions, i.e., the half-life (t1/2) of the interaction accounting for the effects of TCR–pMHC rebinding. One effect of a variable activation threshold is to allow high-density viral pMHC ligands to expand CD4 T cells with a variety of potency and peptide cross-reactivity patterns for the viral pMHC ligand, some of which are only poorly activated by infections that produce a lower density of the viral pMHC ligand. These results argue that antigen concentration is a key component in determining the pattern of KD, t1/2 and peptide cross-reactivity of the TCRs expressed on CD4 T cells responding to infection.