The programmed cell death 1 (PD-1) surface receptor binds to two ligands, PD-L1 and PD-L2. Studies have shown that PD-1-PD-L interactions control the induction and maintenance of peripheral T cell tolerance and indicate a previously unknown function for PD-L1 on nonhematopoietic cells in protecting tissues from autoimmune attack. PD-1 and its ligands have also been exploited by a variety of microorganisms to attenuate antimicrobial immunity and facilitate chronic infection. Here we examine the functions of PD-1 and its ligands in regulating antimicrobial and self-reactive T cell responses and discuss the therapeutic potential of manipulating this pathway.
The past year has seen significant advances in our understanding of the critical roles of negative immunoregulatory signals delivered by the programmed death 1 (PD-1)-PD-1 ligand (PD-L) pathway in regulating T-cell activation and tolerance. Emerging evidence indicates that PD-Ls play an essential role on dendritic cells (DCs), both directly during DC-T cell interactions and indirectly through signaling into the DC. Recent studies point to a novel role for PD-L1 in maintaining tissue tolerance. Finally, PD-1 has recently been shown to be highly expressed on exhausted T cells during chronic viral infection, and blockade of PD-1 or PD-L1 can revive exhausted T cells, enabling them to proliferate and produce effector cytokines.
Mice lacking DNA topoisomerase 3beta are predisposed to a shortened lifespan, infertility, and lesions in multiple organs resulting from inflammatory responses. Examination of the immune system of 6- and 52-week-old top3beta(-/-) mice revealed no significant aberrations in their central and peripheral tolerance or in T lymphocyte activation. However, the older but not the younger cohort shows a high incidence of serum autoantibodies relative to their TOP3beta(+/+) age-mates. The mutant mice also show an increase in numerical aberrations of chromosomes in splenocytes and bone marrow cells, as well as an increase in apoptotic cells in the thymus. Thus, it appears plausible that the inflammatory lesions in top3beta(-/-) mice are caused by the development of autoimmunity as they age: Chromosomal abnormalities in top3beta(-/-) mice might lead to a persistent increase in apoptotic cells, which might in turn lead to the progression of autoimmunity.
BACKGROUND: PD-L1 and PD-L2 are ligands for the inhibitory receptor programmed death-1 (PD-1), which is an important regulator of immune responses. PD-L1 is induced on cardiac endothelial cells under inflammatory conditions, but little is known about its role in regulating immune injury in the heart.
METHODS AND RESULTS: Cytotoxic T-lymphocyte-mediated myocarditis was induced in mice, and the influence of PD-L1 signaling was studied with PD-L1/L2-deficient mice and blocking antibodies. During cytotoxic T-lymphocyte-induced myocarditis, the upregulation of PD-L1 on cardiac endothelia was dependent on T-cell-derived interferon-gamma, and blocking of interferon-gamma signaling worsened disease. Genetic deletion of both PD-1 ligands [PD-L1/2(-/-)], as well as treatment with PD-L1 blocking antibody, transformed transient myocarditis to lethal disease, in association with widespread polymorphonuclear leukocyte-rich microabscesses but without change in cytotoxic T-lymphocyte recruitment. PD-L1/2(-/-) mice reconstituted with bone marrow from wild-type mice remained susceptible to severe disease, which demonstrates that PD-L1 on non-bone marrow-derived cells confers the protective effect. Finally, depletion of polymorphonuclear leukocytes reversed the enhanced susceptibility to lethal myocarditis attributable to PD-L1 deficiency.
CONCLUSIONS: Myocardial PD-L1, mainly localized on endothelium, is critical for control of immune-mediated cardiac injury and polymorphonuclear leukocyte inflammation.
Programmed death-ligand (PD-L)1 and PD-L2, newer B7 superfamily members, are implicated in the negative regulation of immune responses and peripheral tolerance. To examine their function in alloimmunity, we used the murine model of orthotopic corneal transplantation. We demonstrate that PD-L1, but not PD-L2, is constitutively expressed at high levels by the corneal epithelial cells, and at low levels by corneal CD45+ cells in the stroma, whereas it is undetectable on stromal fibroblasts and corneal endothelial cells. Inflammation induces PD-L1 up-regulation by corneal epithelial cells, and infiltration of significant numbers of PD-L1+CD45+CD11b+ cells. Blockade with anti-PD-L1 mAb dramatically enhances rejection of C57BL/6 corneal allografts by BALB/c recipients. To examine the selective contribution of donor vs host PD-L1 in modulating allorejection, we used PD-L1-/- mice as hosts or donors of combined MHC and minor H-mismatched corneal grafts. BALB/c grafts placed in PD-L1-/- C57BL/6 hosts resulted in pronounced T cell priming in the draining lymph nodes, and universally underwent rapid rejection. Allografts from PD-L1-/- C57BL/6 donors were also significantly more susceptible to rejection than wild-type C57BL/6 grafts placed into BALB/c hosts, primarily as a result of increased T cell infiltration rather than enhanced priming. Taken together, our results identify differential roles for recipient vs donor PD-L1 in regulating induction vs effector of alloimmunity in corneal grafts, the most common form of tissue transplantation, and highlight the importance of peripheral tissue-derived PD-L1 in down-regulating local immune responses.
CTLA-4-deficient mice develop a lethal autoimmune lymphoproliferative disorder that is strictly dependent on in vivo CD28 costimulation. Nevertheless, it is not known whether there is a specific site on the CD28 molecule that is required for induction of autoimmunity. Using CTLA-4-deficient mice expressing CD28 molecules with various point mutations in the CD28 cytosolic tail, the present study documents that in vivo costimulation for induction of autoimmune disease strictly requires an intact C-terminal proline motif that promotes lymphocyte-specific protein tyrosine kinase Lck binding to the CD28 cytosolic tail, because point mutations in C-terminal proline residues (Pro-187 and Pro-190) completely prevented disease induction. In contrast, in vivo costimulation for disease induction did not require either an intact YMNM motif or an intact N-terminal proline motif, which, respectively, promote phosphoinositide 3-kinase and IL2-inducible T cell kinase binding to the CD28 cytosolic tail. Thus, in vivo CD28 costimulation for induction of autoimmune disease is strictly and specifically dependent on an intact C-terminal proline motif that serves as a lymphocyte-specific protein tyrosine Lck kinase binding site in the CD28 cytosolic tail.
PD-1, an inhibitory receptor expressed on activated lymphocytes, regulates tolerance and autoimmunity. We tested the role of PD-1:PD-1 ligand (PD-L) interactions in cross-presentation and the generation and control of CD8(+) responses against self-Ag. Ag-naive PD-1(-/-) OVA-specific OT-I CD8(+) T cells exhibited exacerbated responses to cross-presented Ag in mice expressing soluble OVA under the control of the rat insulin promoter (RIP-ova(high)). Following adoptive transfer into RIP-ova(high) recipients, PD-1(-/-) OT-I T cells expanded in the pancreatic lymph node. In contrast to wild-type OT-I cells, PD-1(-/-) OT-I T cells secreted IFN-gamma and migrated into the pancreas, ultimately causing diabetes. Loss of PD-1 affected CD8(+) cells intrinsically, and did not significantly alter the responses of wild-type OT-I T cells adoptively transferred into the same RIP-ova(high) recipient mouse. PD-1:PD-L interactions also limited CD8(+) effector cells, and PD-L1 expression on parenchymal tissues protected against effector OT-I T cell attack. Finally, we found that the loss of PD-1 on effector OT-I cells lowers the threshold for Ag recognition in peripheral tissues. These findings indicate two checkpoints where PD-1 attenuates self-reactive T cell responses: presentation of self-Ag to naive self-reactive T cells by dendritic cells in the draining lymph node and reactivation of pathogenic self-reactive T cells in the target organ.
The programmed death 1/programmed death 1 ligand (PD-L) pathway is instrumental in peripheral tolerance. Blocking this pathway exacerbates experimental autoimmune diseases, but its role in autoimmune kidney disease has not been explored. Therefore, we tested the hypothesis that the programmed death 1 ligands (PD-L1 and PD-L2), provide a protective barrier during T cell- and macrophage (Mphi)-dependent autoimmune kidney disease. For this purpose, we compared nephrotoxic serum nephritis (NSN) in mice lacking PD-L1 (PD-L1(-/-)), PD-L2 (PD-L2(-/-)), or both (PD-L1/L2(-/-)) to wild-type (WT) C57BL/6 mice. Kidney pathology, loss of renal function, and intrarenal leukocyte infiltrates were increased in each PD-L(-/-) strain as compared with WT mice. Although the magnitude of renal pathology was similar in PD-L1(-/-) and PD-L2(-/-) mice, our findings suggest that kidney disease in each strain is regulated by distinct mechanisms. Specifically, we detected increased CD68(+) cells along with elevated circulating IgG and IgG deposits in glomeruli in PD-L2(-/-) mice, but not PD-L1(-/-) mice. In contrast, we detected a rise in activated CD8(+) T cells in PD-L1(-/-) mice, but not PD-L2(-/-) mice. Furthermore, since PD-L1 is expressed by parenchymal and hemopoietic cells in WT kidneys, we explored the differential impact of PD-L1 expression on these cell types by inducing NSN in bone marrow chimeric mice. Our results indicate that PD-L1 expression on hemopoietic cells, and not parenchymal cells, is primarily responsible for limiting leukocyte infiltration during NSN. Taken together, our findings indicate that PD-L1 and PD-L2 provide distinct negative regulatory checkpoints poised to suppress autoimmune renal disease.
Pathways in the B7:CD28 family of costimulatory molecules regulate T cell activation and tolerance. B7-dependent responses in Cd28(-/-)Ctla4(-/-) T cells together with reports of stimulatory and inhibitory functions for Programmed Death-1 Ligand 1 or 2 molecules (PD-L1 or PD-L2) have suggested additional receptors for these B7 family members. We show that B7-1 and PD-L1 interacted with affinity intermediate to that of B7-1:CD28 and B7-1:CTLA-4. The PD-L1:B7-1 interface overlapped with the B7-1:CTLA-4 and PD-L1:PD-1 (Programmed Death-1) interfaces. T cell activation and cytokine production were inhibited by the interaction of B7-1 with PD-L1. The responses of PD-1-deficient versus PD-1,B7-1 double-deficient T cells to PD-L1 and of CD28,CTLA-4 double-deficient versus CD28,CTLA-4,PD-L1 triple-deficient T cells to B7-1 demonstrated that PD-L1 and B7-1 interact specifically to inhibit T cell activation. Our findings point to a substantial bidirectional inhibitory interaction between B7-1 and PD-L1 and add an additional dimension to immunoregulatory functions of the B7:CD28 family.
The T cell immunoglobulin mucin (TIM) proteins regulate T cell activation and tolerance. Here we showed that TIM-4 is expressed on human and mouse macrophages and dendritic cells, and both TIM-4 and TIM-1 specifically bound phosphatidylserine (PS) on the surface of apoptotic cells but not any other phospholipid tested. TIM-4(+) peritoneal macrophages, TIM-1(+) kidney cells, and TIM-4- or TIM-1-transfected cells efficiently phagocytosed apoptotic cells, and phagocytosis could be blocked by TIM-4 or TIM-1 monoclonal antibodies. Mutations in the unique cavity of TIM-4 eliminated PS binding and phagocytosis. TIM-4 mAbs that blocked PS binding and phagocytosis mapped to epitopes in this binding cavity. These results show that TIM-4 and TIM-1 are immunologically restricted members of the group of receptors whose recognition of PS is critical for the efficient clearance of apoptotic cells and prevention of autoimmunity.
Many chronic viral infections are marked by pathogen persistence and a generalized immunosuppression. The exact mechanisms by which this occurs are still unknown. Using a mouse model of persistent lymphocytic choriomeningitis virus (LCMV) infection, we demonstrate viral targeting of fibroblastic reticular cells (FRC) in the lymphoid organs. The FRC stromal networks are critical for proper lymphoid architecture and function. High numbers of FRC were infected by LCMV clone 13, which causes a chronic infection, whereas few were infected by the acute strain, LCMV Armstrong. The function of the FRC conduit network was altered after clone 13 infection by the action of CD8(+) T cells. Importantly, expression of the inhibitory programmed death ligand 1, which was up-regulated on FRC after infection, reduced early CD8(+) T cell-mediated immunopathology and prevented destruction of the FRC architecture in the spleen. Together, this reveals an important tropism during a persistent viral infection. These data also suggest that the inhibitory PD-1 pathway, which likely evolved to prevent excessive immunopathology, may contribute to viral persistence in FRC during chronic infection.