The Role And Mechanism Of The Recombinant PD-L1 Regulatory Effect T Cell Function In Murine AGVHD

Objective:To explore the effect of recombinant PD-L1 on T cell function and its role and mechanism in murine a GVHD.Methods:1.We separated CD4~+ T cells and CD4~+ CD25-cells in vitro,and cultured cells under three modes that Anti-CD3 、 Anti-CD3 ~+ Anti-CD28 and MLR.We tested cell viability through the CD69、CD44、CTLA-4、Lag-3 and Tim-3,detected cell proliferation and apoptosis by CFSE and Annexin V-FITC/PI,expression of cytokines by ELISA and intracellular cytokine staining,flow cytometry to detect the influence of PD-L1 on Treg cells differentiation.To explore the influence of soluble PD-L1 proteins in cell viability,proliferation,apoptosis and secretion of cytokines of T cells under different conditions,as well as the contaction between Treg cells.2.We constructed PD-L1 plasmid by a method of PAS,and PCDN3.1 was used to connect PD-L1 gene segment.The PCDN3.1 plasmid was injected through the tail vein in the treatment group,and PD-L1 plasmid was injected in the experimental group on one day before transplantation.Three a GVHD models were constructed in vivo:(1)BALB/c(H2Kd)recipient mice were lethally irradiated with 650 c Gy total body irradiation(TBI)and infused with 1×107 C57BL/6(H2Kb)bone marrow(BM)cells and 5×106 splenocytes 4hours later.(2)C57BL/6(H2Kb)recipient mice were lethally irradiated with 800 c Gy total body irradiation(TBI)and infused with 1×107C57BL/6(H2Kb)bone marrow(BM)cells and 5×106 splenocytes 4 hours later.(3)BALB/c(H2Kd)recipient mice were lethally irradiated with 650 c Gy total body irradiation(TBI)and infused with 1×107C57BL/6(H2Kb)bone marrow(BM)cells and 5×106 splenocytes without Treg cells 4 hours later.To observe the survival and weight changes of mice under three models,the a GVHD scores was recorded.FACS analysis was performed to examine the T cells subsets and theexpression of cytokines in spleen on day 7 or 14 after allogeneic hematopoietic stem cell transplantation.Results:1.PD-L1 inhibits the proliferation,activity and cytokine expression of CD4~+T cells and promotes apoptosis in vitro Anti-CD3 stimulation;However,there was no influence on the proliferation and differentiation of Treg.PD-L1 inhibits the proliferation,activity and cytokine expression of CD4~+ T cells,promotes its apoptosis and can induce the production of Treg cells in the case of Anti-CD3 ~+ Anti-CD28 stimulation.2.PD-L1 inhibits the proliferation and differentiation of Treg cells in the presence of IL-2 and TGF-β.3.Overexpression of PD-L1 can alleviate the symptoms of a GVHD significantly and prolong survival of mice.4.PD-L1 can reduce the pathological damage of all organs in mice after transplantation.5.PD-L1 inhibits the proliferation of T cells without changing the proliferation and differentiation of Treg cells in the mixed lymphocyte reaction.6.PD-L1 inhibits mice a GVHD is not dependent on Treg cells,and PD-L1 still plays a role in inhibiting a GVHD under different models.Conclusion:1.PD-L1 can inhibit the activity,appreciation and cytokines secretion of T cells directly.2.PD-L1 alleviates the symptoms of a GVHD in mice by inhibiting the expression and function of T cells.3.PD-L1 inhibits mice a GVHD is not dependent on Treg cells.