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

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。

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

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. Na?ve 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.