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本研究旨在探讨与抗原提呈细胞相作用的人CD8~+T细胞的特性及其机制.在GM-CSF和IL-4作用下从外周血单个核细胞诱导培养后获得树突状细胞(DC),DC经Mtb感染(MOI=100)过夜,再与去除CD4~+的T淋巴细胞(3×10~6/ml)共孵育并加入10ng/ml的IL-7,第7天用新鲜的Mtb感染的DC再次刺激T细胞,第8天及以后每隔1天均加入0.2ng/ml的IL-2,第9天时对T细胞进行阳性克隆选择,2天后收集细胞进行CD4~+、CD8~+表达分析,并测定IFN-γ的分泌水平.结果显示培养至第12天时96%的T细胞为CD8~+,Mtb感染的DC能触发CD8~+T细胞应答,IFN-γ的分泌依赖于抗原(Mtb)和DC的同时存在.为了证实这种应答具有回忆性或在体外起到触发作用,又用同样的方法对5个PPD~+、6个PPD~-的个体进行观察,结果发现,PPD~+的个体均能产生CD8~+T细胞应答.将CD8~+T细胞经有限稀释进行克隆,结果有两个克隆(23和29)对Mtb有特异性应答.这些CD8~+T细胞与对Mtb感染的靶细胞的识别被抗MHCⅠ类抗体抑制,但与来自所有个体的Mtb感染的DC共培养均可产生IFN-γ,提示两者之间的识别不受MHC-ⅠA、-B、-C及CD1的限制.在Mtb感染DC前1小时,分别将作用于抗原处理不同阶段的
The aim of this study was to investigate the characteristics and mechanisms of human CD8 ~ + T cells acting on antigen presenting cells.Dendritic cells (DCs) were induced from peripheral blood mononuclear cells under the action of GM-CSF and IL-4 DCs were infected with Mtb overnight (MOI = 100), incubated with CD4 (+) T lymphocytes (3 × 10 ~ 6 / ml) and added with 10ng / ml IL-7. Of Mtb-infected DCs, T cells were stimulated again. 0.2ng / ml of IL-2 was added every other day on the 8th day and later. T-cells were selected on the 9th day. After 2 days, the cells were harvested for CD4 ~ +, The results showed that 96% of T cells cultured on day 12 were CD8 ~ +, and Mtb-infected DCs could trigger CD8 + T cell responses and secretion of IFN-γ Dependent on the presence of antigens (Mtb) and DC In order to confirm that this response has recurrent or in vitro triggering effects, 5 individuals with PPD ~ + and 6 PPD ~ were observed in the same way, The results showed that individuals with PPD ~ + could produce CD8 ~ + T cell responses.During cloning of CD8 ~ + T cells by limited dilution, two clones (23 and 29) had specific responses to Mtb.These CD8 ~ + T cells and sense of Mtb Of target cells were inhibited by anti-MHC class I antibodies but co-cultured with Mtb-infected DCs from all individuals produced IFN-γ, suggesting that the recognition between the two is not affected by MHC-I A, -B, -C CD1 in Mtb infected with DC 1 hour before the role of antigen in different stages will be treated