Yvette Latchman

PD-L2 is a second ligand for PD-1 and inhibits T cell activation.

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.

Tissue expression of PD-L1 mediates peripheral T cell tolerance

Programmed death 1 (PD-1), an inhibitory receptor expressed on activated lymphocytes, regulates tolerance and autoimmunity. PD-1 has two ligands: PD-1 ligand 1 (PD-L1), which is expressed broadly on hematopoietic and parenchymal cells, including pancreatic islet cells; and PD-L2, which is restricted to macrophages and dendritic cells. To investigate whether PD-L1 and PD-L2 have synergistic or unique roles in regulating T cell activation and tolerance, we generated mice lacking PD-L1 and PD-L2 (PD-L1/PD-L2−/− mice) and compared them to mice lacking either PD-L. PD-L1 and PD-L2 have overlapping functions in inhibiting interleukin-2 and interferon-γ production during T cell activation. However, PD-L1 has a unique and critical role in controlling self-reactive T cells in the pancreas. Our studies with bone marrow chimeras demonstrate that PD-L1/PD-L2 expression only on antigen-presenting cells is insufficient to prevent the early onset diabetes that develops in PD-L1/PD-L2−/− non-obese diabetic mice. PD-L1 expression in islets protects against immunopathology after transplantation of syngeneic islets into diabetic recipients. PD-L1 inhibits pathogenic self-reactive CD4+ T cell–mediated tissue destruction and effector cytokine production. These data provide evidence that PD-L1 expression on parenchymal cells rather than hematopoietic cells protects against autoimmune diabetes and point to a novel role for PD-1–PD-L1 interactions in mediating tissue tolerance.

Regulation of PD‐1, PD‐L1, and PD‐L2 expression during normal and autoimmune responses

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.

Negative co-receptors on lymphocytes.

The past year has seen significant advances in our understanding of critical roles of negative immunoregulatory signals delivered through the B7-CD28 superfamily in regulating T cell activation and tolerance. Structural data on CTLA-4 have provided novel insights into the inhibitory functions of CTLA-4. Initial characterization of the PD-1-PD-1-ligand pathway has revealed that this pathway can downregulate TCR- and CD28-mediated signals. Recent studies indicate that ICOS exerts distinct effects at different phases of an immune response: ICOS can inhibit as well as stimulate T cell responses.

Programmed death-1 (PD-1):PD-ligand 1 interactions inhibit TCR-mediated positive selection of thymocytes.

Positive selection during thymocyte development is driven by the affinity and avidity of the TCR for MHC-peptide complexes expressed in the thymus. In this study, we show that programmed death-1 (PD-1), a member of the B7/CD28 family of costimulatory receptors, inhibits TCR-mediated positive selection through PD-1 ligand 1 (PD-L1):PD-1 interactions. Transgenic mice that constitutively overexpress PD-1 on CD4+CD8+ thymocytes display defects in positive selection in vivo. Using an in vitro model system, we find that PD-1 is up-regulated following TCR engagement on CD4+CD8+ murine thymocytes. Coligation of TCR and PD-1 on CD4+CD8+ thymocytes with a novel PD-1 agonistic mAb inhibits the activation of ERK and up-regulation of bcl-2, both of which are downstream mediators essential for positive selection. Inhibitory signals through PD-1 can overcome the ability of positive costimulators, such as CD2 and CD28, to facilitate positive selection. Finally, defects in positive selection that result from PD-1 overexpression in thymocytes resolve upon elimination of PD-L1, but not PD-1 ligand 2, expression. PD-L1-deficient mice have increased numbers of CD4+CD8+ and CD4+ thymocytes, indicating that PD-L1 is involved in normal thymic selection. These data demonstrate that PD-1:PD-L1 interactions are critical to positive selection and play a role in shaping the T cell repertoire.

PD-L1 and PD-L2 have distinct roles in regulating host immunity to cutaneous leishmaniasis

To compare the roles of programmed death 1 ligand 1 (PD‐L1) and PD‐L2 in regulating immunity to infection, we investigated responses of mice lacking PD‐L1 or PD‐L2 to infection with Leishmania mexicana. PD‐L1–/– and PD‐L2–/– mice exhibited distinct disease outcomes following infection with L. mexicana. In comparison to susceptible WT mice, PD‐L1–/– mice showed resistance to L. mexicana, as demonstrated by reduced growth of cutaneous lesions and parasite burden. In contrast, PD‐L2–/– mice developed exacerbated disease with increased parasite burden. Host resistance to L. mexicana is partly associated with the development of a Th1 response and down‐regulation of the Th2 response. Both PD‐L1–/– and PD‐L2–/– mice produced levels of IFN‐γ similar to WT mice. However, the development of IL‐4‐producing cells was reduced in PD‐L1–/– mice, demonstrating a role for PD‐L1 in regulating Th cell differentiation. This inadequate Th2 response may explain the increased resistance of PD‐L1–/– mice. Although no alterations in Th1/Th2 skewing were observed in PD‐L2–/– mice, PD‐L2–/– mice exhibited a marked increase in L. mexicana‐specific antibody production. Increased Leishmania‐specific IgG production may suppress the healing response through FcγR ligation on macrophages. Taken together, our results demonstrate that PD‐L1 and PD‐L2 have distinct roles in regulating the immune response to L. mexicana.

Ly6Clow Monocytes Differentiate into Dendritic Cells and Cross-Tolerize T Cells through PDL-1

Monocyte-derived dendritic cells are active participants during the immune response against infection, but whether they play a role in maintaining self-tolerance under steady-state conditions is not known. Here we investigated the differentiation of monocytes, their ability to ingest apoptotic cells, and their potential functionality in vivo. We observed that Ly6C (Gr-1)low mature monocytes up-regulate their MHC II level in the spleen, express high levels of PDL-1 (programmed death ligand 1), and are more efficient than Ly6Chigh immature monocytes in the ingestion of apoptotic cells in vivo. Sorted circulating Ly6Clow monocytes were able to cross-present both apoptotic cell-associated OVA and soluble OVA protein. Monocytes containing apoptotic cells can further differentiate into CD11c+CD8α−MHC II+ splenic dendritic cells that maintained high expression of PDL-1. Since wild-type but not PDL-1-deficient peripheral blood monocytes containing apoptotic cell-associated OVA suppressed the response to OVA immunization, PDL-1 expression was required for monocyte-mediated T cell tolerance. These observations demonstrate that Ly6Clow mature monocytes can promote tolerance to self Ag contained in apoptotic cells through a PDL-1-dependent mechanism.

Targeting NKT cells and PD-L1 pathway results in augmented anti-tumor responses in a melanoma model

Invariant or Type 1 NKT cells (iNKT cells) are a unique population of lymphocytes that share characteristics of T cells and natural killer (NK) cells. Various studies have shown that positive costimulatory pathways such as the CD28 and CD40 pathways can influence the expansion and cytokine production by iNKT cells. However, little is understood about the regulation of iNKT cells by negative costimulatory pathways. Here, we show that in vivo activation with α-GalCer results in increased cytokine production and expansion of iNKT cells in the absence of programmed cell death ligand-1 (PD-L1, B7-H1, and CD274). To study whether PD-L1 deficiency on NKT cells would enhance antigen-specific T-cell responses, we utilized CD8+ OT-1 OVA transgenic T cells. α-GalCer enhanced the expansion and cytokine production of OT-1 CD8+ cells after adoptive transfer into wild-type recipients. However, this expansion was significantly enhanced when OT-1 CD8+ T cells were adoptively transferred into PD-L1−/− recipients. To extend these results to a tumor model, we used the B16 melanoma system. PD-L1−/− mice given dendritic cells loaded with antigen and α-GalCer had a significant reduction in tumor growth and this was associated with increased trafficking of antigen-presenting cells and CD8+ T cells to the tumors. These data demonstrate that abrogating PDL1:PD-1 interactions during the activation of iNKT cells amplifies an anti-tumor response when coupled with DC vaccination.

PD-L1/PD-1 signal deficiency promotes allogeneic immune responses and accelerates heart allograft rejection.

Background. PD-L1, a ligand for programmed death 1 (PD-1), delivers a negative costimulatory signal to T cells and plays a critical role in the regulation of peripheral tolerance. Methods. We used PD-L1−/− mice to evaluate the role of the PD-L1 signal on allogeneic immune responses in vivo and the underlying mechanisms. Heart transplantation was performed from PD-L1−/− donors or recipients in major histocompatibility complex fully mismatched mouse combinations. The immunologic function of allograft recipients was evaluated ex vivo by enzyme-linked immunospot, mixed lymphocytes reaction, cytotoxic T lymphocyte, and flow cytometry. Results. Our results demonstrated that PD-L1−/− T cells proliferated vigorously under alloantigen stimulation, and also that the antigen-presenting cells (APCs) from PD-L1−/− mice exhibited a stronger allostimulatory activity compared with that in wild-type mice. Heart allografts were rejected at an accelerated rate in both PD-L1−/− donors and recipients. This was associated with significantly augmented donor specific T–cell proliferation and antidonor cytotoxic T lymphocyte activities, and enhanced Th1- or Th2-type immune responses of heart allograft recipients. Conclusions. Absence of PD-L1 input triggers a stimulatory signal to effector T cells and APCs, accelerating heart allograft rejection. Engagement of the PD-L1 signal on T cells or APCs may be necessary to induce transplant tolerance.

The SLAM family member CD48 (Slamf2) protects lupus-prone mice from autoimmune nephritis

Polymorphisms in the SLAM family of leukocyte cell surface regulatory molecules have been associated with lupus-like phenotypes in both humans and mice. The murine Slamf gene cluster lies within the lupus-associated Sle1b region of mouse chromosome 1. Non-autoreactive C57BL/6 (B6) mice that have had this region replaced by syntenic segments from other mouse strains (i.e. 129, NZB and NZW) are B6 congenic strains that spontaneously produce non-nephritogenic lupus-like autoantibodies. We have recently reported that genetic ablation of the SLAM family member CD48 (Slamf2) drives full-blown autoimmune disease with severe proliferative glomerulonephritis (CD48GN) in B6 mice carrying 129 sequences of the Sle1b region (B6.129CD48(-/-)). We also discovered that BALB/c mice with the same 129-derived CD48-null allele (BALB.129CD48(-/-)) have neither nephritis nor anti-DNA autoantibodies, indicating that strain specific background genes modulate the effects of CD48 deficiency. Here we further examine this novel model of lupus nephritis in which CD48 deficiency transforms benign autoreactivity into fatal nephritis. CD48GN is characterized by glomerular hypertrophy with mesangial expansion, proliferation and leukocytic infiltration. Immune complexes deposit in mesangium and in sub-endothelial, sub-epithelial and intramembranous sites along the glomerular basement membrane. Afflicted mice have low-grade proteinuria, intermittent hematuria and their progressive renal injury manifests with elevated urine NGAL levels and with uremia. In contrast to the lupus-like B6.129CD48(-/-) animals, neither BALB.129CD48(-/-) mice nor B6 × BALB/c F1.129CD48(-/-) progeny have autoimmune traits, indicating that B6-specific background genes modulate the effect of CD48 on lupus nephritis in a recessive manner.