PD-L1阻断改善酵母多糖致伤小鼠脾脏耐受性 DC对T淋巴细胞活性的影响

摘要

目的：探讨酵母多糖致伤小鼠脓毒症病程发展过程中脾脏耐受性树突状细胞( dendritic cell, DC)的形成对脾脏T淋巴细胞免疫活性的影响,并通过程序性死亡受体-1(programmed death-1,PD-L1)抗体阻断PD-L1/PD-1途径改善耐受性DC对T淋巴细胞活性的抑制作用。方法采用酵母多糖腹腔注射的方法复制小鼠脓毒症模型。用磁珠法分离致伤小鼠脾脏DC和T淋巴细胞,测定脾脏组织中DC上PD-1、PD-L1、PIR-B的表达水平和分泌IL-12、IL-10的能力；检测脾脏T淋巴细胞增殖活性和IL-2的分泌水平。进一步将致伤组小鼠脾脏DC与正常小鼠脾脏T淋巴细胞混合培养,并加入PD-L1抗体,检测T淋巴细胞增殖活性及混合培养上清中IL-2、IL-12和IL-10含量。结果酵母多糖致伤后5天和12天组小鼠脾脏DC上PD-1、PD-L1及PIR-B的表达大幅上调,IL-12p70分泌减少,IL-12p40和IL-10分泌增加；脾脏T淋巴细胞增殖活性降低、IL-2分泌减少。在致伤组DC与正常T淋巴细胞混合培养体系中加入PD-L1抗体可减轻致伤组DC对T淋巴细胞增殖活性和分泌IL-2的抑制作用,并改善DC上IL-12p70、IL-12p40和IL-10的分泌能力。结论酵母多糖诱导小鼠脓毒症的病程晚期,脾脏耐受性DC的形成导致T淋巴细胞活性降低；PD-L1抗体通过干预PD-1/PD-L1途径改善T淋巴细胞和DC的免疫活性。%Purpose To investigate the effect of tolerogenic dendritic cells ( DC) on T lymphocytes in the spleen during the develop-ment of zymosan-induced sepsis in mice, and to explore whether PD-L1 blockade could alleviate the immunosuppressive effect of tolero-genic DC on T lymphocytes. Methods Mice sepsis model was established by intraperitoneal injection of zymosan. Splenic DC and T lymphocytes were isolated respectively by using anti-CD11c and anti-CD3 magnetic beads. The expressions of PD-L1, PD-1 and PIR-B on splenic DC were measured, and IL-12 and IL-10 secreted from DC were determined. Mitogen-induced T lymphocyte proliferation and IL-2 secretion were assessed. Anti- PD-L1 antibody was added into mixed culture of tolerogenic DCs with normal Tcells. T cell proliferation and IL-2, IL-12 and IL-10 concentrations in the supernatant of mixed culture were determined. Results At 5 days and 12 days after zymosan injection, the expressions of PD-L1, PD-1 and PIR-B on splenic DC increased greatly, secretion of IL-12p70 re-duced and that of IL-12p40 and IL-10 augmented in DC, which were associated with decrease of T cells proliferation and IL-2 secre-tion. Administrating anti-PD-L1 antibody into the mixed culture of tolerogenic DC and Tcell could alleviate the suppression of DC on T lymphocyte proliferation and secretion of IL-2, and ameliorate the ability of DC secreting IL-12 and IL-10 as well. Conclusions At late stage of zymosan-induced sepsis, the formation of splenic tolerogenic DC resulted in immunosuppression of T lymphocytes. Anti-PD-L1 antibody could improve the immunoactivity of DC and T lymphocyte through intervening PD-L1/PD-1 pathway.