不同病程尖锐湿疣患者特异性CTL细胞频率和功能分析

Analysis of the frequency and function of antigen specific CTL in different courses＇ patients with candyloma acuminata

目的利用负载人乳头瘤病毒6型（HPV-6）E7抗原肽的HIA-A$0201四聚体染色结合流式细胞术，分析不同病程HLA—A2阳性HPV-6所致尖锐湿疣（CA）患者外周血中特异性CTL细胞的频率和功能。方法以负载HPV-6E7抗原肽1、2细胞（表达HLA—A2分子）作为刺激细胞，与不同病程CA患者的外周血淋巴细胞（PBL）共培养，获得特异性CTL。体外制备PE标记HLA—A2／HPV-6E7四聚体，利用该四聚体和FITC．CD8单克隆抗体对这些特异性CTL进行双色荧光染色，经流式细胞仪分析双阳性细胞的频率；同时采用乳酸脱氢酶释放法（LDH）测定这些特异性CTL的杀伤活性。结果经混合淋巴细胞培养4周后，正常对照组特异性CTL细胞频率为（0．320±0．076）％，复发期CA患者组特异性CTL细胞频率为（1．100±0．064）％，缓解期cA患者组的特异性CTL为（1．115±O．071）％，均显著高于正常对照组（t值分别为19．23和18．66，P均〈0．001），复发期CA患者组与缓解期CA患者组间差异无统计学意义（t值为0．34，P=0．74）。复发期患者组特异性CTL杀伤活性为（51．18±0．48）％，缓解期CA患者的特异性CTL杀伤活性为（59．33±1．71）％，两组间差异有统计学意义（P〈0．05）。结论缓解期CA患者与复发期CA患者外周血中特异性CTL数量相近，但缓解期CA患者特异性CTL杀伤活性明显强于复发期CA患者，这可能是不同病程CA患者转归差异的根本原因。

Objective To explore the frequency and function of antigen-specific T ceils at different courses of condyloma acuminate （CA） by means of the sHLA-A2/HPV-6 E7 tetramer constructed from HLA-A ＊ 0201 and its restricted peptide HPV-6 E7（22-30）. Methods Peripheral blood lymphocytes （PBL） were col- lected from the HLA-A2 positive individuals without clinical manifestations and those CA cases at different cour- ses. The T2 （ HLA-A2 ~ ） cells born with HPV-6 E7 were used as the priming cells to co-cultivate with the PBL taken from different courses of the patients to generate the peptide-specific CTL through long term co-cultivation. The HLA-A2/HPV-6 E7 tetramer was labeled with PE in vitro and the proliferated specific CTLs after cultivation were stained with fluorescence by making use of sHLA-A2/HPV-6 E7 tetramer and FITC-CD8 McAb. And the killing activities of these specific CTLs were tested by LDH assay. Results The frequency of specific CTLs in control group was （0.320± 0. 076）%. The frequencies of specific CTLs in recurrent CA and during remission were （ 1. 100± 0.064） % and （ 1.115 ± 0. 071 ） % , respectively, both of which were of no significant difference （ t = 0.34, P = 0.74） and higher than that of the control. The killing activities in recurrent CA and during re- mission were （51.18±0.48）% and （59.33 ± 1.71 ）% , respectively. The significant difference existed be- tween these two groups. Conclusion The tetramer can be applied directly to visualize antigen-specific CTLs ef- ficiently and become the critical approach in the assessment of T cell immune responses in CA patients. The difference is not obvious in the frequencies of specific CTLs at different courses of CA. But the difference is prominent in the CTLs killing frequency at different courses of CA. The CTLs can be highly activated and elimi- nate the HPV in remission CA, so the disease can be cured.