HPV16E7多肽体外诱生抗原特异性细胞毒性T细胞表型及功能的研究

Study on phenotype and function of antigen-specific CD8＋ cytotoxic T cells induced by HPV 16 E7 peptide in vitro

目的 探讨高致癌性HPV16E7病毒抗原肽体外诱生HLA-A2阳性正常人外周血抗原特异性CD8＋细胞毒性T细胞（CTL）的表型及功能状态.方法 以HPV16E711-20合成多肽、重组人IL-7、IL-2体外刺激26例HLA-A2阳性正常人外周血T细胞,用HLA-A＊0201限制性表位肽/YMLDLQPETT五聚体技术,结合流式细胞仪对抗原特异性CTL进行频率、表型[CD45RA＋CD27-效应性CTL、CD45RA-CD27-效应性记忆CTL（TEM）、CD45RA-CD27＋中枢性记忆CTL（TCM）、CD45＋CD27＋初始性CTL]及功能（穿孔素、颗粒酶B、FasL）分析,并以无关肽HBVcoxe18-27作为阴性对照.结果 病毒多肽体外刺激T细胞7 d后,抗原特异性CD8＋CTL数为（0.73±0.33）%,比未刺激组的（0.02±0.03）%明显增多（P＜0.01）.进一步分析,在抗原特异性CTL中,效应性CTL、TEM、TCM及初始性CTL百分比分别为（26.07±13.46）%、（7.97±7.11）%、（33.25±19.68）%及（32.73±13.89）%,均比未刺激组的（0.02±0.03）%、（0.02±0.03）%、（0.02±0.03）%及（0.02±0.05）%明显增多,P值均＜0.01.HPV16E711-20表位特异性CTL分泌穿孔素、颗粒酶B、Fas-L的水平分别为（47.01±18.69）%、（80.53±13.32）%及（26.48±7.81）%,均比未刺激组的（0.38±0.55）%、（0.34±0.22）%及（0.16±0.16）%明显增多,P值均＜0.01.结论 HPV16E711-20多肽诱导CD8＋T细胞克隆扩增,产生抗原特异性CD8＋CTL,分泌毒性细胞因子,通过不同机制特异性杀伤病毒感染的靶细胞,在抗病毒免疫应答中发挥作用.

Objective To investigate the phenotype and function of antigen-specific CD8＋ cytotoxic T cells （CTL） from HLA-A2＋ healthy human donors induced by high-carcinogenic HPV 16 E7 peptide in vitro.Methods Peripheral blood T cells from 26 HLA-A2＋ healthy human donors were incubated with HPV 16E711-20 peptide （HLA-A＊0201/YMLDLQPETT）, recombinant human interleukin 7 （IL-7） and IL-2 for 7 days to yield antigen-specific CD8＋ CTL. Then, four-color flow cytometric analysis was performed to detect the percentage of antigen-specific CD8＋ CTL expressing different surface markers including CD45 and CD27. The expression of three intracellular cytokines including perforin, granzyme-B and FasL in the antigen-specific CD8＋ CTL was also measured by intracellular flow cytometry. A wrong peptide, HBVcoxe18-27 （FLPSDFFPSV）,was used as an isotype control. Results A significant increase was observed in the percentage of antigen-specific CD8＋ CTL in peripheral T cells stimulated with HPV 16 E7-peptide compared with the non-stimulated T cells （0.73% ± 0.33% vs 0.02% ± 0.03%, P 〈 0.01）. The percentage of CD45RA＋CD27- effector T cells,CD45RA-CD27- effector memory T （TEM） cells, CD45RA-CD27＋ central memory T （TCM） cells, and CD45RA＋CD27＋ naive T cells in antigen-specific CD8＋ CTL was 26.07% ± 13.46%, 7.97% ± 7.11%, 33.25% ± 19.68%and 32.73% ± 13.89%, respectively in HPV 16 E7-stimulated group, significantly higher than that in nonstimulated group （0.02% ± 0.03%, 0.02% ± 0.03%, 0.02% ± 0.03% and 0.02% ± 0.05%, all P 〈 0.01 ）. Elevated proportions of perforin-, granzyme-B- and FasL-expressing antigen specific CTL were observed in HPV 16 E7-stimulated group compared with non-stimulated group （47.01% ± 18.69% vs 0.38% ± 0.55%, 80.53% ±13.32% vs 0.34% ± 0.22%, 26.48% ± 7.81% vs 0.16% ± 0.16%, all P 〈 0.01 ）. Conclusions HPV 16 E7peptide could induce the clonal proliferation of CD8＋ T cells, generation of antigen-specific CD8＋ CTL and secretion of toxic cytokines, finally lead to a highly efficient and specific killing of virus-infected target cells through different mechanisms, hence, it might play a crucial role in antiviral immune responses.