The Mechanisms Of PD-L1/PD-1 Costimulatory Signal Pathway In The Regulation Of Allogeneic Transplant Immune Responses

Organ transplantation is an effective treatment for several end-stage diseases. Tremendous advances have been made during the last decade in therapeutic strategies to prevent allograft rejection. However, toxicities and complications of current immuno-suppressive drugs make apparent the need for new targets involved in allograft tolerance induction. Manipulation of a ligand central to the initiation of alloimmune responses may previde an opportunity to control the host response to an allograft with enhanced precision.PD-L1, a homolog to B7, has recently been identified as a ligand for PD-1 and is expressed on a variety of tissues and cells including T cells (CD4, CD8, natural killer [NK] T cells), B cells, and antigen-presenting cells (APCs). PD-L1 negatively regulates T-cell function, inhibits T-cell positive selection and development in the thymus, and plays a critical role in the regulation of peripheral tolerance. It has also been shown that PD-1 engagement by PD-L1 immunoglobulin (Ig) fusion protein administration induces long-term islet allograft survival. PD-L1 blockade using anti-PD-L1 monoclonal antibodies accelerates skin allograft rejection and cardiac graft arterial disease. The exact role of the regulation of alloimmune responses by PD-L1 on each cell type remains unclear.Aims:We used PD-L1-/- mice to evaluate the role of the PD-1/PD-L1 signal on allogeneic immune responses in vivo and the underlying mechanisms.Methods:(1) We compared the characteristics of APCs and T cells from the spleens of PD-L1-/- and WT B6 mice by four-color flow cytometry. (2) To determine the functional abilities of peripheral T lymphocytes and APCs from PD-L1-/- mice, a standard mixed lymphocytes reaction assay was performed. (3) To determined the cytokine profiles of PD-L1-/- T cells under allostimulation, or the T cells stimulated by allogeneic SCs from PD-L1-/- mice, ELISPOT assays for cytokine were performed. (4) 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:(1) The data revealed a significant increases in CD11c+ DCs (5.4±2.2 Vs 3.5±1.1, P<0.05) and CDllb+ macrophages (12.7±4.7Vs 5.4±0.4, P<0.05) in the spleens of PD-L1-/- mice compared with WT controls, but a significant decrease in Foxp3+CD4+ Treg from PD-L1-/- mice compared with WT B6 mice (5.7±1.9Vs 7.3±1.5, P<0.05). All of the CD4+CD25+ T cells expresses Foxp3, phenotype Tregs. As expected, these Treg expressed higher PD-L1 on the surface in WT, but not knockout mice. The CD11c+ DCs demonstrated higher levels of PD-L1, but not PD-L2 expression on their surface in the WT B6 mice, whereas CD11c+ DCs from PD-L1-/- mice expressed slightly higher levels of CD80, CD95L, OX40L, PD-L2, and MHC II (IAb). (2) PD-L1-/- T cells demonstrated enhanced proliferation when stimulated with allogeneic SCs. When PD-L1-/- SCs served as stimulators, they demonstrated enhanced allostimulatory ability to C3H spleen T cells compared with their response to WT controls. (3) We performed ELIPOT assays for cytokine IL-2, IFN-y, IL-10, IL-4 production. T cells from PD-L1-/- mice demonstrated enhanced cytokine production in both Thl and Th2 types (vs. WT controls, P<0.05 for IFN-y; P<0.01 for IL-4; P<0.05 for IL-10) compared with the WT B6 mice. However, the T cells stimulated by allogeneic PD-L1-/- SCs produced an increased level of IFN-y and a decreased level of IL-4 (vs. WT controls, P<0.05) compared with the T cells stimulated by WT SCs. IL-10及IL-2 productions were equivocal between groups. (4) We performed a series of allogeneic HTx between two MHC fully mismatched strain combinations, B6 and C3H, and B6 and BALB/c, respectively. PD-L1-/- mice were tested as donors or recipients in both strain combinations. The heart allograft survival in all strain combinations demonstrated accelerated acute rejection in PD-L1-/- donors or recipients when compared with their comparable WT strain crosses. The mean survival time (MST) of PD-L1-/- allografts in C3H recipients was 9.3±1.1 days compared with 36±37 days in WT controls (P<0.01); the MST of C3H allografts in PD-L1-/ recipients was 8.5±0.6 days compared with 13.3±1.3 days in WT B6 recipients (P<0.01). Similarly, in B6 and BALB/c strain combinations, PD-L1-/- grafts in the BALB/c recipients were rejected at an accelerated rate at day 7 posttransplantation compared with 13±1.9 days in WT grafts (P<0.01). Although BALB/c grafts in the PD-L1-/- recipients were also rejected at an accelerated rate, MSTs were 7.0±0.8 days vs.13±1.6 days in WT recipients (P<0.001). (5) Standard MLR assays were performed on purified spleen T cells of heart allograft recipients at day 7 posttransplantation. The results demonstrated a significant increase in T-cell proliferation in both PD-L1-/- recipients and the recipients of PD-L1-/- grafts compared with the WT controls (P<0.05). (6) We examined the cytotoxic T cell and NK cell activities of recipient splenocytes both in recipients of PD-L1-/- allografts and PD-L1-/- recipients of WT allografts at day 7 posttransplantation. Antidonor CTL activities (against EL-4[H2b] and R1.1[H2k]) of recipients SCs were markedly increased both in recipients of PD-L1-/- grafts (P<0.01) and PD-L1-/- recipients (P<0.05) compared with WT controls. However, the NK cell activity increased moderately only in the PD-L1-/- recipients and not in the recipients of PD-L1-/- grafts compared with the WT controls. (7) The cytokine production of spleen T cells of heart allograft recipients was evaluated by ELISPOT assay in the combination of C3H to PD-L1-/- and PD-L1-/- to C3H mice at day 7 posttransplantation. WT B6 mice were used as controls. PD-L1-/- donors or recipients demonstrated a different pattern of cytokine production. Both Th1-(IL-2, P<0.05 and IFN-γ, P<0.05) and Th2 (IL-4, P<0.05)-cytokine production were increased significantly in the T cells of PD-L1-/- recipients. However, the T cells from the recipients of PD-L1-/-donors recealed a significant increase only in Thl cytokine IFN-y(P<0.05). Moreover, immunohistochemistry straining of frozen sections of the heart allografts and recipient spleens revealed significant elevations of IFN-γsecretion in the PD-L1-/- heart allografts and recipient spleens. However, increased IL-2 production was detected in the heart allografts and spleens of PD-L1-/- recipients compared with WT controls.Conclusions:(1) PD-L1 deficiency altered the balance of peripheral lymphocytes, resulting in a decrease in the number of Foxp3 +CD4+ Treg and an increase in APCs and their co-stimulatory molecule expression in the PD-L1-/- mice. (2) In vitro PD-L1-/-T cells respond more vigorously to allogeneic stimulation than those from WT mice. (3) PD-L1-/- APCs significantly enhance the response of allogeneic T cells compared with those of WT mice. (4) Absence of PD-L1 signal on APCs promotes Thl-cytokine production and suppresses Th2 cytokine production of allogeneic T cells. PD-L1/PD-1 signal induces different immune responses depending on the cell types. (5) Our in vivo data of heart allograft survival clearly support the role of PD-L1 signaling in modulating allogeneic immune responses after HTx. (6) The communication through PD-L1 is bidirectional, because both PD-L1-/- recipients and recipients of PD-L1 allografts demonstrated an augmented antidonor response. (7) The PD-L1 signaling is involved in the cytotoxic T cells, but may not be directly in involved in NK cell funciton. (8) The mechanisms of the PD-L1 signal-mediated immune response are different in recipients of PD-L1-/- allografts and PD-L1-/- recipients of WT allografts. These may be due to PD-L1 expressed on multiple types of cells.Significance:Our data suggest that PD-L1 not only delivers a negative costimulatory signal to activated T cells, regulating peripheral tolerance to self-antigen, but is also critical to the development of allogeneic tolerance. Manipulation of a ligand central to the initiation of alloimmune responses may provide an opportunity to control with enhanced precision the host response to an allograft. PD-L1, the ligand for PD-1, seems to be integral to the modulation of T-cell activation and the APC responses, suggesting that it is such a target. This subject can support a new theoretical foundation by fully understand the mechanisms of PD-1/PD-L1 signal in transplantation immunology; provide a new idea on clinical tolerance induction and maintenance; and have a good clinical application for allografts long-term survival.