肿瘤患者T细胞受体多样性和克隆性的高通量测序分析_杨黎.pdf

临床研究与应用 肿瘤患者 T 细胞受体多样性和克隆性的高通量测序分析*杨黎张茹黄建敏宋亚东张志新张毅摘要目的：探讨肺癌和食管癌患者外周血 T 细胞受体（T cell receptor，TCR）多样性和克隆分布，以及食管癌外周血和肿瘤浸润淋巴细胞中 T 细胞 TCR 的差异情况。方法：收集自 2019 年 4 月至 2021 年 5 月郑州大学第一附属医院未行放、化疗，并排除严重感染性疾病、自身免疫性疾病以及近期使用免疫抑制剂治疗史的 8 例食管癌、8 例肺癌患者的外周血标本，选取其中 1 例食管癌患者肿瘤组织标本进行 TCR 测序。17 例健康人外周血标本作为对照组。分析肿瘤患者与健康人之间以及早期、晚期肿瘤患者之间TCR 多样性指数（D50）、VJ 基因使用频率以及 CDR3 序列克隆型频率的差异。结果：食管癌、肺癌患者外周血 TCR CDR3 序列的 D50值 分别为（0.0310.028）和（0.0770.034），均低于健康人（0.1450.057），P0.000 1 和 P=0.005 3。食管癌、肺癌外周血 TCR CDR3 最大克隆比例 分别为（10.6407.640）%和（8.3345.575）%，均高于健康人（4.0702.341）%，P=0.003 1和 P=0.011 9。且 TCR CDR3 序列的最大克隆比例与 D50值呈负相关。食管癌和肺癌患者早期外周血 T 细胞 TCR 多样性（D50）分别为（0.0570.028）和（0.1060.007）明显高于晚期患者 分别为（0.0150.014，P=0.028 5）和（0.0590.030，P=0.041 7）。随着疾病进展，肺癌和食管癌患者外周血 TCR CDR3 中 TRBV 和 TRBJ 基因组合使用减少，单个或多个 TCR 克隆型占比明显增高，存在明显的单一性。结论：肺癌和食管癌中外周血 TCR 谱多样性低于健康人，且存在克隆性增殖。在肿瘤发展中，TCR 谱多样性降低，提示 TCR 谱为肿瘤的诊断、免疫评估提供了潜在的生物标志物，具有重要的临床指导意义。关键词T 细胞受体高通量测序多样性克隆性生物标志物doi:10.12354/j.issn.1000-8179.2023.20221143High-throughput sequencing analysis of T cell receptor diversity and clonality in pa-tients with cancerLi Yang1,Ru Zhang1,Jianmin Huang1,Yadong Song2,Zhixin Zhang3,Yi Zhang1Correspondence to:Li Yang;E-mail:1Biotherapy Center,The First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China;2Department of Gynecological Ra-diotherapy,The First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China;3Department of Health Management&In-stitute of Health Management,Sichuan Provincial Peoples Hospital,University of Electronic Science and Technology of China,Cheng-du 610044,ChinaThis work was supported by National Key Research and Development Program of China(No.2021YFE0110600),National Natural Sci-ence Foundation of China(No.82072578，No.91942314，No.U1804281)and Henan Young Health Science and Technology InnovationOutstanding Program(No.YXK2020027)Abstract Objective:To analyze the characteristics of the peripheral blood T cell receptor(TCR)repertoire,including diversity and clonality,in patients with esophageal cancer(EC)and lung cancer(LC),and to compare TCR repertoires between peripheral blood mononuclear cellsand tumor-infiltrating lymphocytes in patients with EC.Methods:From April 2019 to May 2021 in The First Affiliated Hospital of ZhengzhouUniversity,we collected peripheral blood samples from eight patients with EC and eight with LC,without radiotherapy or chemotherapy,andcollected tissue from one patient with EC to perform high-throughput TCR sequencing.Patients with severe infectious diseases,autoim-mune diseases,and recent treatment histories of immunosuppressants were excluded.Peripheral blood samples from 17 healthy individu-als were used as the control group.The difference in TCR diversity index(D50),V and J gene usage frequency,and CDR3 clonotype fre-quency between healthy individuals and cancer patients,as well as between patients with early and advanced cancer,were analyzed,re-spectively.Results:The D50 value of TCR CDR3 transcripts in patients with EC and LC(0.0310.028)and(0.0770.034)was significantlylower compared to healthy individuals(0.1450.057),P0.000 1 and P=0.005 3.The frequency of the largest dominant clone in patientswith EC and LC(10.647.640)%and(8.3345.575)%was significantly higher than in healthy individuals(4.0702.341)%,P=0.003 1 andP=0.011 9.In addition,there was a negative correlation between the D50 value of the TCR repertoire and the frequency of the largest dom-inant clone(P=0.000 6).The peripheral blood TCR repertoire diversity(D50)in patients with early EC(0.057 0.028)and LC(0.1060.007)作者单位：郑州大学第一附属医院生物细胞治疗中心（郑州市450052）；郑州大学第一附属医院妇科放疗科；健康管理中心/健康管理研究所，电子科技大学附属医院四川省人民医院*本文课题受国家重点研发计划项目（编号：2021YFE0110600）、国家自然科学基金项目（编号：82072578、91942314、U1804281）和河南省中青年卫生健康科技创新杰出青年人才项目（编号：YXK2020027）资助通信作者：杨黎 中国肿瘤临床 2023 年第 50 卷第 2 期Chin J Clin Oncol 2023.Vol.50.N55was also significantly higher than in patients with advanced EC(0.0150.014,P=0.028 5)and LC(0.0590.030,P=0.041 7).With the progres-sion of disease,the recombination between TRBV and TRBJ of the peripheral blood TCR repertoire in patients with EC and LC decreased,andTCR clonotypes conferred a growth advantage with significantly monoclonality.Conclusions:The peripheral blood TCR repertoire diversity inpatients with EC and LC is lower than in healthy individuals,and patients with cancer showed a higher degree of expanded clones.Addition-ally,the circulating TCR repertoire diversity gradually decreases with cancer progression.TCR repertoire can reflect human immune functionand also provide a potential biomarker for the diagnosis of tumors.Keywords:T cell receptor(TCR),high-throughput sequencing,diversity,clonality,biomarker 根据 2019 年世界卫生组织（WHO）统计，癌症作为目前全球主要死因之一，肺癌、食管癌是发病率和死亡率均较高的主要恶性肿瘤1。继常规治疗（手术、放疗、化疗）、靶向治疗之后，免疫疗法已成为癌症治疗的第四大支柱2，且具有效果好、不良反应小和防止复发等优点。T 细胞在病原体清除和肿瘤监测中起着核心作用，且已被证实具有杀伤恶性细胞的能力3。因此，目前肿瘤免疫治疗以 T 细胞为中心，主要集中于增强 T 细胞特异性反应，增强抗肿瘤免疫反应4。T 细胞是获得性免疫系统的主要组成部分。T 细胞表达细胞表面的抗原受体，即 T 细胞受体（TCR），识别抗原提呈细胞提呈的主要组织相容性复合体（major histocompatibility complex，MHC）分子结合的多肽。克隆型 TCR 为 链和 链组成的异二聚体5。链和 链由恒定区（C 区）