尖锐湿疣患者外周血及经体外诱生的HPV16抗原特异性CD8^+细胞毒性T细胞检测

Quantification of peripheral blood HPV16 antigen-specific CD8^+ cytotoxic T cells in patients with condyloma acuminatum

目的研究尖锐湿疣患者外周血HPV16抗原特异性CD8^+细胞毒性T细胞(CTL)表型变化及正常人T细胞对病毒抗原肽体外刺激的细胞免疫应答反应在尖锐湿疣发病机制中的作用。方法采用免疫荧光标染重组MHCⅠ类分子-多肽五聚体技术,流式细胞仪定量检测10例尖锐湿疣患者外周血HPV16E7_(11-20)(HLA-A*0201/YMLDLQPETF)抗原特异性CTL(Pent^+CD8^+)、活化CTL(Pent^+CD8^+CD69^+)及记忆性CTL(Pent^+D8^+CD45RO^+)数量。同时,用HPV16E7_(11-20)合成多肽、重组人白介素7、白介素2体外刺激13例正常人外周血单一核细胞产生抗原特异性CTL,并对其进行表型分析,以无关肽HBVcore_(18-27)作为阴性对照。结果尖锐湿疣患者外周血CD8^+T细胞中HPV16抗原特异性CTL、活化CTL及记忆性CTL百分数分别为0．76％±0．43％．0．36％±0．20％及0．33％±0．15％,对照组分别为0．24％±0．07％,0．17％±0．05％及0．15％±0．06％。两组比较,P值分别＜0．01,＜0．05及＜0．01。病毒多肽体外刺激T细胞7d后,抗原特异性CTL、活化CTL及记忆性CTL百分数分别为0．75％±0．16％,0．35％±0．15％及0．33％±0．18％,均比非刺激组的0．24％±0．06％,0．16％±0．03％及0．13％±0．04％明显增多,P值均＜0．01。结论HPV感染诱导CD8^+T细胞克隆性增殖,产生抗原特异性CD8^+CTL,高效、特异性直接杀伤病毒感染的靶细胞,在抗病毒T细胞免疫应答过程中发挥作用。

Objective To study the roles of phenotype switch of HPV16 antigen-specific CD8^＋ cytotoxic T cells （ CTLs ） in peripheral blood as well as the cellular immune response of human T lymphocytes to viral peptides in the pathogenesis of condyloma acuminatum. Methods HPV16E711-22 （ HLA-A＊0201/YMLDLQPETT ） antigen-specific CTLs were stained with fluorescent-labeled recombinant MHC class Ⅰ -peptide pentamer complex, and co-stained with CD69 （ activated CTL ） and CD45RO （ memory CTL ） surface markers, in peripheral blood fi＇om 10 patients with condyloma acuminatum and 15 normal controls. The CTLs were quantitated by flow cytometric analysis. Meanwhile, peripheral blood mononuclear cells of 13 healthy donors were incubated for 7 days with HPV16E711-20 peptide, recombinant human interleukin 7 and interleukin 2 to yield antigen-specific CD8^＋ CTLs and their other surface markers. An irrelevant peptide, HBVcore18-27 （ FLPSDFFPSV ）, was used as an isotype control. Results The percentages of peripheral blood HPV16E7 antigen-specific CD8^＋CTLs, activated CTLs and memory CTLs of total CD8^＋ T cells were increased significantly in patients with condyloma acuminatum （ 0.76% ±0.43 %, 0.36%±0.20%, and 0.33%±0.15%, respectively） than those in normal controls （0.24%±0.07%, 0.17%±0.05% and 0.15% ±0.06%, respectively ） （P 〈 0.01, P 〈 0.05, and P 〈 0.01, respectively ）. For T cells in vitro stimulated with viral peptide for 7 days, the percentages of peptide-specific CD8＋ CTLs, activated CTLs and memory CTLs were significantly higher （ 0.75% ±0.16%, 0.35% ±0.15% and 0.33% ±0.18%, respectively ） in comparison with those in non-stimulated group （ 0.24% ±0.06%, 0.16% ±0.03% and 0.13%± 0.04%, respectively ） （ P 〈 0.001, P 〈 0.01 and P 〈 0.01, respectively ）. Conclusions These results, in vivo and in vitro, demonstrate that the proliferation of CD8^＋ T cell clones induced by HPV infection could generate antigen-specific CD8^＋ CTLs, causing a highly efficient and specific killing of viral-infected target cells directly, which may play an important role in antiviral T cell-mediated immune response.