外周CD8αα＋TCRαβ＋调节性T细胞表型与功能初步研究

Study on the phenotype and the function of CD8αα＋TCRαβ＋regulatory T cells

目的：研究外周CD8αα＋TCRαβ＋调节性T细胞的表型和免疫调节功能。方法采用流式细胞术分析CD8αα＋TCRαβ＋T细胞在 C57BL／6J小鼠体内的分布情况，并对其进行了相关表型分析，随后采用anti-CD3抗体刺激，流式微球分析细胞因子分泌格局；采用流式细胞术分选和CFSE标记方法，在体外研究CD8αα＋TCRαβ＋T细胞调节抑制功能，并采用细胞过继免疫实验观察CD8αα＋TCRαβ＋T细胞对实验性变态反应性脑脊髓炎（ EAE）小鼠模型的保护作用。结果在C57BL／6J小鼠肝脏、脾脏和外周血中存在一群 CD8αα＋TCRαβ＋T 细胞与CD8αβ＋TCRαβ＋T细胞相比具有CD25＋CD122highCD44highCD62LlowCD69highNK1．1＋DX5＋记忆效应表型，激活后能迅速产生IL-2，随后产生IFN-γ、TNF-α、IL-4和IL-17A以及微量IL-6和IL-10。体外功能实验表明CD8αα＋TCRαβ＋T细胞专一性抑制活化CD4＋T细胞的分化（P＜0．01），体内实验证明该群细胞具有抑制自身活化CD4＋T细胞介导的EAE，延缓疾病发展和保护的效果（ P＜0．01）。结论 CD8αα＋TCRαβ＋T细胞是体内一群固有的具有免疫调节功能的CD8＋调节性T细胞，有望成为细胞治疗新靶点用于自身免疫性等各类疾病。

Objective To investigate the phenotype and the immunoregulatory function of CD8αα＋TCRαβ＋regulatory T cells in peripheral blood samples from mice.Methods The distribution profile and the phenotype of CD8αα＋TCRαβ＋regulatory T cells in C57BL/6 mice were detected by flow cytometry.The cytokines released by CD8αα＋TCRαβ＋regulatory T cells upon the stimulation with anti-CD3 antibody were analyzed by cytometric bead array.The in vitro immunosuppressive activity of CD8αα＋TCRαβ＋regulatory T cells on activated CD4＋T cells was analyzed by using flow cytometry and carboxyfluorescein succinimidyl ester （ CFSE ） .An adoptive cell transfer assay was set up to evaluate the immunoprotective effects of CD8αα＋TCRαβ＋ regulatory T cells in a mouse model of experimental autoimmune encephalomyelitis （ EAE） .Results CD8αα＋TCRαβ＋regulatory T cells were detected in liver, spleen and peripheral blood samples collected from na？ve C57BL/6 mice.Compared with CD8αβ＋TCRαβ＋regulatory T cells, CD8αα＋TCRαβ＋regulatory T cells showed a memory-activated phenotype of CD25＋CD122high CD44high CD62Llow CD69high NK1.1＋DX5＋.CD8αα＋TCRαβ＋regulatory T cells could produce IL-2 after 24 hours stimulation with anti-CD3 antibody, followed by producing IFN-γ, TNF-α, IL-4, IL-17A and traces of IL-6 and IL-10. In vitro, CD8αα＋TCRαβ＋regulatory T cells specifically suppressed the proliferation of activated CD4＋T cells （ P〈0.01 ）.Moreover, they could delay the onset of EAE in mice and reduce clinical score （P〈0.01）.Conclusion CD8αα＋TCRαβ＋regulatory T cells were a unique population with immunoregula-tory function, which could be used as a potential therapeutic target in the treatment of autoimmune disease.