DNAM-1通过IL-2/STAT-5通路调节Ⅰ型调节性T细胞的增殖和功能

DNAM-1 regulates the proliferation and function of T regulatory type 1 cells via the IL-2/STAT5 pathway

目的探讨DNAM-1对Ⅰ型调节性T细胞(Tr1细胞)活化、增殖和功能的影响及相关分子机制。方法利用anti-CD3/CD28激活小鼠T细胞,采用流式细胞术分别检测静息和激活状态下CD4+T细胞和Tr1细胞DNAM-1分子表达变化;分离DNAM-1基因敲除小鼠(KO小鼠)脾脏初始CD4+T细胞并体外诱导Tr1细胞,流式细胞术检测CD25和CD69活化分子表达水平,CFSE标记后检测增殖能力,IL-2刺激前后检测KO小鼠Tr1细胞分泌IL-10和转录激活蛋白(p-STAT5)水平变化。结果流式细胞术结果显示:与静息状态下相比较,激活状态的CD4+T细胞和Tr1细胞表达DNAM-1分子均增高(P<0.05);敲除DNAM-1不影响小鼠脾脏Tr1细胞的数量和比例,但KO小鼠Tr1细胞表达细胞激活分子CD25和CD69均降低,差异有统计学意义(P<0.05);与WT小鼠比较,KO小鼠诱导型Tr1细胞体外增殖能力降低(P<0.05);与WT组Tr1细胞比较,KO小鼠Tr1细胞分泌抑制性细胞因子IL-10水平降低(P<0.05),给予IL-2刺激后仍无法逆转,表达Il-10 mRNA和Gzmb mRNA水平降低(P<0.05);给予不同剂量IL-2刺激Tr1细胞后,KO小鼠Tr1细胞表达p-STAT5水平相比较WT组均降低(P<0.05)。结论DNAM-1参与Tr1细胞的活化和增殖,并通过IL-2/STAT5信号通路影响Tr1细胞抑制功能。

Objective To explore the role of DNAM-1 in the activation,proliferation and function of type I regulatory T cells(Tr1 cells).Methods Anti-CD3/CD28 antibodies were used to stimulate mouse T cells derived from the spleen of wild-type(WT)mice,and the expression level of DNAM-1 in resting and activated Tr1 cells was evaluated with flow cytometry.Naïve CD4^(+)T cells isolated by magnetic cell sorting from the spleens of WT mice and DNAM-1 knockout(KO)mice were cultured in Tr1 polarizing conditions for 3 days,after which CD25 and CD69 expressions were measured using flow cytometry.The induced Tr1 cells were labelled with CFSE and cultured in the presence of anti-CD/CD28 antibodies for 3 days,and their proliferative activity was analyzed.The expressions of IL-10 and p-STAT5 in DNAM-1-deficient Tr1 cells were detected before and after IL-2 stimulation.Results The expression level of DNAM-1 was significantly upregulated in CD4^(+)T cells and Tr1 cells after stimulation with anti-CD3/CD28 antibodies(P<0.05).DNAM-1 knockout did not cause significant changes in the number or proportion of Tr1 cells,but but significantly increased the expression levels of the activation markers CD69 and CD25(P<0.05).Compared with WT Tr1 cells,DNAM-1-deficient Tr1 cells exhibited reduced proliferative activity in vitro(P<0.05)with downregulated IL-10 production(P<0.05)and decreased expressions of Il-10 and Gzmb mRNA(P<0.05).In DNAM-1-deficient Tr1 cells,IL-2 stimulation significantly reduced IL-10 secretion level and the expression of p-STAT5 as compared with WT Tr1 cells.Conclusion DNAM-1 participate in the activation and proliferation of Tr1 cells and affect the biological functions of Tr1 cells through the IL-2/STAT5 pathway.