不同等位基因E7/HLA-DR四聚体结合的结核患者CD4＋T细胞CDR3区序列分析  

Analysis of TCR CDR3 sequences of E7/HLA-DR tetramers-bound CD4＋ T cells from tuberculosis patients by using tetramers constructed with different HLA-DRB1 alleles

作　　者：甘一川 王聪[1] 方毅敏[2] 姚亚男[1] 涂晓欣 申雁鸣[2] 赖小敏[1] Fang Yimin Yao Yanan Tu Xiaoxin Shen Yanming Lai Xiaomin(Department of Microbiology, Zhongshan School of Medicine, Sun Yat-sen University, Key Laboratory of Tropi- cal Diseases Control, Ministry of Education, Institute of Tuberculosis, Key Laboratory of Functional Molecules from Marine Microorganisms of Colleges and Universities in Guangdong Province, Guangdong Provincial Research Center for Severe Infectious Disease Prevention and Control Technology, Guangzhou 510080, China Guangzhou Chest Hospital, State Key Laboratory of Respiratory Disease, Guangzhou 510095, China)

机构地区：[1]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,结核病研究所,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中心,广州510080 [2]国家呼吸疾病重点实验室,广州市胸科医院,510095

出　　处：《中华微生物学和免疫学杂志》2017年第2期91-96,共6页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金（81271779）;十二·五传染病重大专项分任务（2012ZX10004903-004-002）;广东省自然科学基金项目（2014A030313725）;广州市科技计划项目（155700012,201604020019）

摘　　要：目的 探讨机体抗结核免疫过程中CD4＋T细胞TCR与多肽/MHC之间的识别和结合规律.方法 从9例结核患者胸水中用等位基因不同的结核多肽E7/HLA-DR四聚体磁珠分选出E7结合阳性CD4＋T细胞,提取其RNA逆转录成cDNA作为模板扩增TCRα链和β链CDR3区核酸片段,并比较其长度和序列特征.结果 等位基因不同的E7/HLA-DR四聚体能够识别同一患者CDR3区序列相同或结构功能相似的CD4＋T细胞克隆.结论 结核患者体内CD4＋T细胞识别和结合抗原多肽时有一定的HLA-DR限制性,但是主要以多肽特异性为主,这为进一步探讨机体抗结核免疫中CD4＋T细胞和MHC之间的抗原提呈机制提供了数据.Objective To investigate the recognition and binding mechanism between TCR of CD4＋ T cells and peptide/MHC （ major histocompatibility complex） in immune responses against Mycobacte-rium tuberculosis （MTB）. Methods Nine patients with tuberculosis （TB） were recruited in the present study. Peptide E7-bound CD4＋T cells were sorted by using E7/HLA-DR tetramers constructed with different HLA-DRB1 alleles from pleural fluid （ PLF） of TB patients. Total RNA was extracted and reversely tran-scribed into cDNA, which was used as the template to amplify complementarity determining region 3 （CDR3） fragments of T cell receptor （TCR） α and β chains. Amino acid sequences, spectratypes and lengths of the amplified CDR3 fragments were analyzed. Results The CDR3 amino acid sequences of E7-bound CD4＋ T cells were identical completely or partially in a single individual. E7/HLA-DR tetramers with different HLA-DRB1 alleles were capable of recognizing and binding CDR3 fragments with identical sequence or similar structure and function in one single individual. Conclusion The TCRαandβchains CDR3 line-age of CD4＋ T cells of TB patients apparently drifts, and the predominant CDR3 sequences of TCRαandβchains that recognize MTB antigen exhibit peptide specificity with some certain HLA-DR restriction. This study illustrates the possible causes and mechanisms of peptide-specific CD4＋T cells related antigen presen-tation against MTB.

关 键 词：结核 E7 HLA-DR四聚体 CDR3区 CD4+T细胞 E7 CDR3 

分 类 号：R52[医药卫生—内科学]