Tahiro Shin

Expression of Programmed Death 1 Ligands by Murine T Cells and APC

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.

Cooperative B7-1/2 (CD80/CD86) and B7-DC Costimulation of CD4+ T Cells Independent of the PD-1 Receptor

B7-DC is a recently discovered member of the B7 family that binds to PD-1 and is selectively expressed by dendritic cells (DCs). It has been shown to either costimulate or inhibit T cell responses. To assess the role of B7-DC in DC–T cell interactions, DCs from B7-DC knockout (KO) mice were generated and compared with DCs from wild-type (WT) and B7–1/B7–2 double KO mice. B7–1/B7–2–deficient DCs, while strongly diminished in their ability to stimulate naive CD4+ T cells, nonetheless retain partial activity. DCs from B7-DC KO mice are diminished in their ability to activate CD4+ T cells, demonstrating that DC-expressed B7-DC serves a predominantly stimulatory rather than inhibitory function in the initiation of T cell responses. B7-DC costimulates expression of CD40L with faster kinetics than B7–1 and displays potent synergy with B7–1 and B7–2 for T cell proliferation and cytokine production, indicating that these B7 family members work in concert to stimulate T cells. Finally, costimulation with B7-DC alone or in conjunction with B7–1 is PD-1 independent, indicating that B7-DC costimulates T cells via a second receptor.

In vivo costimulatory role of B7-DC in tuning T helper cell 1 and cytotoxic T lymphocyte responses

B7-DC, one of the recently described B7 family members, has the capacity to inhibit T cell responses via engagement of the immunoreceptor tyrosine-based inhibitory motif–containing inhibitory PD-1 receptor as well as enhance responses via an as yet unidentified costimulatory receptor. B7-DC is highly homologous to a coinhibitory B7 family member, B7-H1, which also binds PD-1. It is currently unclear which B7-DC function—costimulation or inhibition—predominates in vivo. To study in vivo functions of B7-DC, we evaluated immune responses in B7-DC knockout (KO) mice. Although not eliminated, interferon-γ (IFN-γ) production by CD4 T cells and IFN-γ–dependent humoral responses were reduced in B7-DC KO mice relative to wild type mice. Antigen-specific CD8 T cell responses and cytotoxic T lymphocyte (CTL) activity were also diminished in B7-DC KO mice. Hepatic tumors grew more quickly in B7-DC KO mice, associated with a decrease in intrahepatic tumor-specific CD8 T cells. These results highlight the contrasting in vivo roles of B7-DC and B7-H1 and indicate that B7-DC functions as a tuning molecule, selectively augmenting T helper 1 and CTL responses.

CD73 has distinct roles in nonhematopoietic and hematopoietic cells to promote tumor growth in mice

CD73 is overexpressed in many types of human and mouse cancers and is implicated in the control of tumor progression. However, the specific contribution from tumor or host CD73 expression to tumor growth remains unknown to date. Here, we show that host CD73 promotes tumor growth in a T cell-dependent manner and that the optimal antitumor effect of CD73 blockade requires inhibiting both tumor and host CD73. Notably, enzymatic activity of CD73 on nonhematopoietic cells limited tumor-infiltrating T cells by controlling antitumor T cell homing to tumors in multiple mouse tumor models. In contrast, CD73 on hematopoietic cells (including CD4⁺CD25⁺ Tregs) inhibited systemic antitumor T cell expansion and effector functions. Thus, CD73 on hematopoietic and nonhematopoietic cells has distinct adenosinergic effects in regulating systemic and local antitumor T cell responses. Importantly, pharmacological blockade of CD73 using its selective inhibitor or an anti-CD73 mAb inhibited tumor growth and completely restored efficacy of adoptive T cell therapy in mice. These findings suggest that both tumor and host CD73 cooperatively protect tumors from incoming antitumor T cells and show the potential of targeting CD73 as a cancer immunotherapy strategy.

Cross-linking the B7 family molecule B7-DC directly activates immune functions of dendritic cells

B7-DC molecules are known to function as ligands on antigen-presenting cells (APCs), enhancing T cell activation. In this study, cross-linking B7-DC with the monoclonal antibody sHIgM12 directly potentiates dendritic cell (DC) function by enhancing DC presentation of major histocompatibility complex–peptide complexes, promoting DC survival; and increasing secretion of interleukin (IL)-12p70, a key T helper cell type 1 promoting cytokine. Furthermore, ex vivo treatment of DCs or systemic treatment of mice with sHIgM12 increases the number of transplanted DCs that reach draining lymph nodes and increases the ability of lymph node APCs to activate naive T cells. Systemic administration of the antibody has an equivalent effect on DCs transferred at a distant site. These findings implicate B7-DC expressed on DCs in bidirectional communication. In addition to the established costimulatory and inhibitory functions associated with B7-DC, this molecule can also function as a conduit for extracellular signals to DCs modifying DC functions.

PDL1 Is Required for Peripheral Transplantation Tolerance and Protection from Chronic Allograft Rejection

The PD-1:PDL pathway plays an important role in regulating alloimmune responses but its role in transplantation tolerance is unknown. We investigated the role of PD-1:PDL costimulatory pathway in peripheral and a well established model of central transplantation tolerance. Early as well as delayed blockade of PDL1 but not PDL2 abrogated tolerance induced by CTLA4Ig in a fully MHC-mismatched cardiac allograft model. Accelerated rejection was associated with a significant increase in the frequency of IFN-γ-producing alloreactive T cells and expansion of effector CD8+ T cells in the periphery, and a decline in the percentage of Foxp3+ graft infiltrating cells. Similarly, studies using PDL1/L2-deficient recipients confirmed the results with Ab blockade. Interestingly, while PDL1-deficient donor allografts were accepted by wild-type recipients treated with CTLA4Ig, the grafts developed severe chronic rejection and vasculopathy when compared with wild-type grafts. Finally, in a model of central tolerance induced by mixed allogeneic chimerism, engraftment was not abrogated by PDL1/L2 blockade. These novel data demonstrate the critical role of PDL1 for induction and maintenance of peripheral transplantation tolerance by its ability to alter the balance between pathogenic and regulatory T cells. Expression of PDL1 in donor tissue is critical for prevention of in situ graft pathology and chronic rejection.

Naturally Occurring Human IgM Antibody That Binds B7-DC and Potentiates T Cell Stimulation by Dendritic Cells

A human IgM Ab, serum-derived human IgM 12 (sHIgM12), is identified that binds mouse and human dendritic cells (DC), inducing dramatic immunopotentiation following treatment of the mouse DC in vitro. Competition, transfection, and knockout studies identified the ligand on mouse DC as the costimulatory molecule family member B7-DC. Potent T cell responses are stimulated by Ag-pulsed DC treated with the sHIgM12 Ab in vitro and upon adoptive transfer of Ab-treated Ag-pulsed DC into animals. The multivalent structure of pentameric IgM provides the potential for cross-linking cell surface targets, endowing the soluble Abs with biological potential not normally associated with immune function. The ability of the sHIgM12 Ab to potentiate the immune response is dependent on the multimeric structure of IgM, as bivalent monomers do not retain this property. Furthermore, pretreatment of DC with IgM monomers blocks subsequent potentiation by intact IgM pentamers, an indication that cross-linking of B7-DC on the cell surface is critical for potentiation of Ag presentation. These findings imply that, in addition to known costimulatory roles, B7-DC can function as a receptor for signals delivered by cells expressing B7-DC ligands.

PD-1 ligands expressed on myeloid-derived APC in the CNS regulate T-cell responses in EAE

Disease progression in experimental autoimmune encephalomyelitis (EAE) is regulated by programmed death receptor 1 (PD‐1) and its ligands, B7‐H1 (programmed death ligand 1 (PD‐L1)) and B7‐DC (PD‐L2). B7‐H1 and B7‐DC have negative regulatory effects upon binding PD‐1 on activated T cells and B7‐H1 deficiency increases severity of both diabetes and EAE. However, the role of PD‐L expression on different APC in the CNS in regulating local T‐cell function during relapsing EAE has not been examined. Our data show that the majority of CNS CD4+ T cells isolated during acute EAE are PD‐1+, and T cells specific for relapse‐associated epitopes express PD‐1 upon antigen stimulation in the CNS. B7‐H1 and B7‐DC are differentially expressed on discrete APC populations in the inflamed CNS. B7‐H1 and PD‐1 have mainly inhibitory functions on CNS T cells. B7‐H1 negatively regulates the stimulation of activated PD‐1+ TH cells, in co‐cultures with microglia and different CNS‐infiltrating APC presenting endogenously processed peptides. The preponderance of IFN‐γ+ versus IL‐17+ T cells in the CNS of B7‐H1−/− mice suggests that B7‐H1 more selectively suppresses TH‐1 than TH‐17 responses in vivo. In contrast, blockade of B7‐DC has less pronounced regulatory effects. Overall, the results demonstrate that B7‐H1 expressed by CNS myeloid APC negatively regulates T‐cell activation during acute relapsing EAE.

Crucial roles of B7-H1 and B7-DC expressed on mesenteric lymph node dendritic cells in the generation of antigen-specific CD4+Foxp3+ regulatory T cells in the establishment of oral tolerance.

Oral tolerance is a key feature of intestinal immunity, generating systemic tolerance to fed antigens. However, the molecular mechanism mediating oral tolerance remains unclear. In this study, we examined the role of the B7 family members of costimulatory molecules in the establishment of oral tolerance. Deficiencies of B7-H1 and B7-DC abrogated the oral tolerance, accompanied by enhanced antigen-specific CD4(+) T-cell response and IgG(1) production. Mesenteric lymph node (MLN) dendritic cells (DCs) displayed higher levels of B7-H1 and B7-DC than systemic DCs, whereas they showed similar levels of CD80, CD86, and B7-H2. MLN DCs enhanced the antigen-specific generation of CD4(+)Foxp3(+) inducible regulatory T cells (iT(regs)) from CD4(+)Foxp3(-) T cells rather than CD4(+) effector T cells (T(eff)) relative to systemic DCs, owing to the dominant expression of B7-H1 and B7-DC. Furthermore, the antigen-specific conversion of CD4(+)Foxp3(-) T cells into CD4(+)Foxp3(+) iT(regs) occurred in MLNs greater than in peripheral organs during oral tolerance under steady-state conditions, and such conversion required B7-H1 and B7-DC more than other B7 family members, whereas it was severely impaired under inflammatory conditions. In conclusion, our findings suggest that B7-H1 and B7-DC expressed on MLN DCs are essential for establishing oral tolerance through the de novo generation of antigen-specific CD4(+)Foxp3(+) iT(regs).

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.