中华皮肤科杂志 ›› 2010, Vol. 43 ›› Issue (5): 342-345.

• 论著 • 上一篇    下一篇

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

钱起丰1,李芳谷2,李青2   

  1. 1. 深圳市福田区梅林1村42幢301
    2. 深圳市皮肤病防治研究所
  • 收稿日期:2009-02-16 修回日期:2009-09-04 出版日期:2010-05-15 发布日期:2012-04-12
  • 通讯作者: 钱起丰 E-mail:qqian2012@sina.com

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

  • Received:2009-02-16 Revised:2009-09-04 Online:2010-05-15 Published:2012-04-12

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摘要：

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HBVcore18－27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。

关键词: T淋巴细胞，细胞毒性

Abstract:

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 16 E711-20 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, HBVcore18-27 （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 non-stimulated 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 E7 peptide 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.