应用E7多肽/HLA-DR8四聚体监测治疗前后肺结核患者外周血多肽特异性CD4+ T细胞的变化

中国防痨杂志 ›› 2011, Vol. 33 ›› Issue (7): 400-406.

应用E7多肽/HLA-DR8四聚体监测治疗前后肺结核患者外周血多肽特异性CD4⁺ T细胞的变化

黎意芬² 黄利荣¹ 刘国标² 方毅敏² 李研¹, 彭毅¹ 赖小敏¹

1广州市胸科医院； 2中山大学中山医学院微生物学教研室热带病防治研究教育部重点实验室海洋微生物功能分子广东省高校重点实验室；广东省重大传染病预防和控制技术研究中心；

出版日期:2011-07-12 发布日期:2012-03-14
基金资助:
国家自然科学基金重点项目(30430660);传染病重大专项(2008ZX10003-007、2008ZX10003-012);广东省科技计划项目(73107)

Tracking of peptide-specific CD4⁺ T cells in peripheral bloods from untreated and treated patients with pulmonary tuberculosis by using E7/HLA-DR8 tetramers

Li Yifen²,Huang Lirong¹,Liu Guobiao²,Fang Yimin²,Li Yan¹,Peng Yi¹,Lai Xiaomin¹

1.Guangzhou Chest Hospital,Guangzhou 510095,China; 2.Department of Microbiology,Ministry of Education Key Laboratory of Tropical Diseases Control,Guangdong Provincial Department of Education Key Laboratory of Functional Molecules from Marine Microorganisms,Guangdong Provincial Research Center for Severe Infectious Disease Prevention and Control Technology,Zhongshan School of Medicine,Sun Yat-sen University,Guangzhou 510080,China

Online:2011-07-12 Published:2012-03-14

摘要/Abstract

摘要： 目的监测治疗前及治疗6个月内各时间点肺结核患者外周血E7多肽特异性CD4+T细胞及总CD4⁺T细胞的变化。方法应用已有的果蝇S2恒定细胞株,分别表达、纯化获得2种生物素化E7/HLA-DR8（E7/HLA-DRB1*08032和E7/HLA-DRB1*0818）复合物单体,进而制备成相应四聚体,分别用2种四聚体与抗人CD4-FITC共染色,流式细胞分析检测治疗前以及规律抗结核治疗15、30、45、60、90、120、150、180 d肺结核患者外周血四聚体阳性（E7多肽特异性）CD4⁺ T细胞及总CD4⁺ T细胞,同时以健康成人对照者、非结核呼吸道感染患者外周血及脐带血作为研究对照。结果多数病例的外周血总CD4⁺ T细胞在治疗前略低于正常水平,随着治疗开始很快升高并在短期内恢复至正常水平;但除治疗前组与脐带血差异有统计学意义（P<0.05）外,治疗前后各组之间,以及治疗前组与健康成人、非结核呼吸道感染者之间的差异无统计学意义（P>0.05）。治疗前以及规律抗结核治疗15、30、456、0 d内肺结核患者组外周血可检出高水平的四聚体阳性CD4+ T细胞,与60 d后各组及3个对照组差异均有统计学意义（P<0.01或P<0.05）,2种四聚体之间的检测结果无统计学意义（P>0.05）;肺结核患者外周血四聚体阳性CD4+ T细胞在治疗前即有较高水平,治疗1560 d后达到最高,一些患者中间有波动,以后开始逐渐降低甚至降至正常水平。结论多肽/HLA-DR四聚体可以用于监测治疗前后结核患者外周血多肽特异性CD4⁺ T细胞的变化,配合总CD4⁺ T细胞的检测,对于治疗期结核患者疾病发展和恢复的监测具有指导意义。

关键词: 结核,肺/免疫学, CD4阳性T淋巴细胞, HLA-DR抗原

Abstract: Objective To track peptide E7-specific CD4⁺ T cells in peripheral bloods(PBL) from pulmonary tuberculosis(PTB) patients during the untreated and 6-months treatment.Methods Two types of E7/HLA-DR8(E7/HLA-DRB1*08032 and E7/HLA-DRB1*0818) tetramers were prepared by using biotinylated monomers expressed and purified from constructed stable Drosophila Schneider 2 cell(S2 cell) lines,and then respectively used to detect tetramer-positive(peptide E7-specific) CD4⁺ T cells and total CD4⁺ T cells in the PBL of PTB patients about at days 0,15,30,45,60,90,120,150 and 180 during their regular anti-tuberculosis(TB) therapy by anti-human CD4-FITC staining and flow cytometric analysis.The PBL of non-TB patients with pulmonary infection and healthy adults,and umbilicalcord blood were used as controls.Results The percentage of total CD4⁺ T cells in the PBL of most untreated PTB patients was slightly lower than normal level;along with treatment,it got to increase rapidly and reach almost normal level in a short period;but statistical difference was only found between the PBL of untreated PTB patients and umbilicalcord blood(P<0.05),and statistical differences were not seen between other groups(P>0.05).High levels of tetramer-positive CD4⁺ T cells in the PBL of PTB patients could be detected about at days 0,15,30,45 and 60 during their regular anti-TB therapy,respectively,and statistical differences were found between the PBL of these time-points and both of subsequent time-points and 3 control groups(P<0.01 or P<0.05).In contrast,there was no statistical difference between 2 tetramer staining(P>0.05).The results suggested that relatively high and the highest levels of tetramer-positive CD4⁺ T cells appeared in the PBL of untreated PTB patients and treated cases at days 15-60,respectively.Subsequently,although some small rise and fall in some patients,the percentages of tetramer-positive CD4⁺ T cells began to decrease,and gradually reached and kept at a relatively low even normal level.Conclusion Peptide/HLA-DR tetramers could be used to track peptide-specific CD4⁺ T cells in the PBL of TB patients during the untreatment and treatment,which combined with the detection of total CD4⁺ T cells is significant for monitoring TB development and recovery.

Key words: Tuberculosis,pulmonary/immunology, CD4-positive T-lymphocytes, HLA-DR antigens

中图分类号:

R521

黎意芬黄利荣刘国标方毅敏李研彭毅赖小敏. 应用E7多肽/HLA-DR8四聚体监测治疗前后肺结核患者外周血多肽特异性CD4⁺ T细胞的变化[J]. 中国防痨杂志, 2011, 33(7): 400-406.

Li Yifen,Huang Lirong,Liu Guobiao,Fang Yimin,Li Yan,Peng Yi,Lai Xiaomin. Tracking of peptide-specific CD4⁺ T cells in peripheral bloods from untreated and treated patients with pulmonary tuberculosis by using E7/HLA-DR8 tetramers[J]. Chinese Journal of Antituberculosis, 2011, 33(7): 400-406.

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