人CD8+CD28-抑制性T淋巴细胞的体外扩增及其抗原特异性调节作用

目的 探讨在体外大量扩增CD8+CD28-抑制性T淋巴细胞(Ts细胞)的方法,并检验其免疫调节作用.方法 分离健康志愿者全血中CD8+T淋巴细胞,在含不同细胞因子和异体抗原提呈细胞(APC)的培养条件下进行体外扩增.应用流式细胞术对扩增过程中CD28 -细胞亚群的比例进行监测.分为3组进行混合淋巴细胞培养,反应细胞均为CD4+T淋巴细胞:B-APc组以来源于原致敏供者的APC作为刺激细胞,I-APC组以HLA-A、B、DR全错配的无关供者的APC作为刺激细胞,Dynabeads组以包被有抗CD3和CD28单克隆抗体的免疫微球Dynabeads作为刺激细胞；扩增后的Ts细胞作为第三方调节细胞加入混合淋巴细胞培养中,测定其对CD4+T淋巴细胞增殖的抑制作用.结果 在含白细胞介素2(IL-2)+ IL-7+ IL-15的培养条件下,CD8+T淋巴细胞培养后CD8+CD28-T淋巴细胞亚群的比例最高(P＜0.05).B-APC组不加入Ts细胞时,增殖的CD4+T淋巴细胞比例为66.7％,当加入的Ts细胞与反应细胞的比例分别为0.5∶1、0.1∶1和0.02∶1时,增殖的CD4+T淋巴细胞比例分别为16.5％、34.1％和62.6％.Ts细胞对CD4+T淋巴细胞的增殖有明显抑制作用,而在I-APC组和Dynabeads组中,此抑制作用不明显.结论 应用含IL-2+ IL-7+ IL-15的培养条件联合异体APC刺激可以在体外大量扩增Ts细胞,扩增所得Ts细胞在体外对供者CD4+T淋巴细胞的增殖有明显抗原特异性抑制作用.

In vitro expansion of human CD8+ CD28- suppressor T cells and their immunological regulatory effect with antigen specificity

Objective To study the In vitro expansion of human CD8+ CD28- suppressor T cells and their immunological regulatory effect.Methods Human CD8 + CD28- suppressor T cells were expanded in vitro driven by the combination of cytokines and allogeneic antigen presenting cells (APCs).Flow cytometry was used to assess the development of CD28- subpopulation.Expanded CD8+ CD28- T cells were isolated by immunomagnetic microspheres and then added as third part modulators into mixed lymphocyte culture to assess their immunological regulatory characteristics.Results The combination of cytokines included IL-2,IL-7 and IL-15 and allogeneic APCs could increase the portion of CD8 + CD28- T cell subtype,and expansion fold of CD8+ CD28- T cell subtype was significantly increased as compared with others (P＜0.05).Expanded CD8+ CD28- T cells could suppress the proliferation of CD4+ T cells stimulated by allogeneic APCs.Moreover,this suppression had antigen specificity.Conclusion Human CD8 + CD28- suppressor T cells can be in vitro expanded in large amounts driven by the combination of cytokines and allogeneic APCs.Expanded CD8 + CD28- T cells in this study have antigen specific regulatory characteristics.