多发性硬化抗原特异性CD4^+CD25^+调节性T细胞的体外扩增及其功能效应

Expansion and function analysis of antigen-specific CD4^+CD25^(high) regulatory T cell in patients with

目的针对多发性硬化(MS)患者CD4+CD25+调节性T细胞(Treg)的免疫功能缺陷,尝试利用体外培养法获取功能正常的Treg。方法密度梯度离心法获得MS患者(n=5)和健康对照者(n=5)外周血单个核细胞(PBMC),免疫磁珠法分选B细胞和CD4+CD25-初始T细胞,前者进一步经可溶性CD40配体(sCD40L)刺激活化后作为抗原提呈细胞(APC),与后者混合体外共培养,并以髓鞘碱性蛋白(MBP)85-99等刺激后收获细胞;经流式细胞仪分选获得CD4+CD25highCD127low Treg。通过增殖分析鉴定所获Treg的免疫抑制功能。结果磁珠法分选B细胞、初始T细胞的纯化率分别达到90%和80.62%。体外诱导扩增所获的MBP反应性Treg比例为3.5%~6%,MS组与健康对照组Treg比较抑制功能活性无明显差异。结论经此方法可扩增获得免疫功能正常的Treg细胞,进而为今后开展MS的特异性免疫调节治疗提供客观的依据。

Objective To obtain normal regulatory T cells（Treg） through in vitro culture in patients with multiple sclerosis（MS） demonstrating immune defect of CD4^＋CD25^＋Treg. Methods Peripheral blood mononuclear cells（PBMC） of MS patients and healthy control was isolated by density gradient centrifugation, and CD4^＋CD25^- naive T cells and allogeneic B cells were further sorted with immunomagnetic beads. Allogeneic B cell, after activation with sCD40L, was co-cultured with CD4^＋CD25^- naive T cells in the presence of myelin basic protein（MBP）85-99 peptide, Rapamycin and 4-1BBL for 10 days. CD4^＋ CD25^highCD127^low Treg were sorted with flow cytometry, and its immunosuppressive function was identified by proliferation assay. Results Purification rate of CD4＋CD25- naive T cells and allogeneic B cells through immnomagnetic beads were 90% and 80.62% as well as for MBP-reactive Treg 3.5%-6% through in vitro culture. The Treg immunoactivities demonstrated no significant difference compared with that of healthy control. Conclusion Treg with normal immunoactivities can be obtained through this coculture method in vitro, and will provide reliable evidence for further antigen-specific immunomodulatory therapy.