结核分枝杆菌抗原多肽递呈时HLA-DR等位基因与多肽特异性CD4+ T细胞克隆的限制性分析

Drift binding between Mycobacterium tuberculosis (MTB) peptide/HLA-DR―CD4 TCR was found during analyzing samples from patients with tuberculosis (TB) using peptide/HLA-DR tetramers or CD4+ TCR tetramers, and same or highly similar CD4+ T cell clones could be sorted using 8 kinds of peptide E7/HLA-DR tetramers. This program is intended to detect samples from more same TB patients using flow cytometric analyses with E7-related 8 kinds of peptide E7/HLA-DR tetramers and 3 kinds of CD4+ TCR tetramers and ELISPOT assay with 8 kinds of peptide E7/HLA-DR tetramers, monitor CD4+ T cell clones sorted by 8 kinds of peptide E7/HLA-DR tetramers by using binding-ELISPOT and proliferation stimulus and blocking test, and then sequence and analyze the sequences of CDR3 and V-D-J of T cell clones and the patients' HLA-DR allels. Hope by analyzing matched or unmatched HLA-DR allels between tetramer and patients and uniformity or heterogeneity of CDR3 region and V-D-J in CD4+ T cell clones, explore that HLA-DR allels, and sequences and spatial conformation of CDR3 and V-D-J influence on recognition and binding and their law between peptide/HLA-DR―CD4 TCR.

在应用各种四聚体分析结核标本时发现多肽/HLA-DR/CD4 TCR之间存在漂移结合现象，8种E7/HLA-DR四聚体分选出的细胞为相同或高度相似的CD4+ T细胞克隆。本项目拟以E7多肽相关的8种HLA-DR四聚体和3种CD4+ TCR四聚体-流式细胞以及E7/HLA-DR四聚体-ELISPOT进一步对同一结核患者标本作同步检测，用E7/HLA-DR四聚体分选患者CD4+ T细胞克隆进行结合-ELISPOT、增殖刺激与阻断试验，序列分析各种试验T细胞CDR3区及V-D-J及患者HLA-DR等位基因。期望能通过分析四聚体与结核患者的HLA-DRB等位基因对应与否，以及CD4+ T细胞克隆的TCR CDR3区和V-D-J 均一性与否，探讨HLA-DR等位基因、CD4+ T细胞克隆的TCR CDR3区与V-D-J 序列或空间构象对多肽/ HLA-DR / TCR之间结合识别的影响与规律。

前期在应用各种四聚体分析结核标本时发现多肽/HLA-DR/CD4 TCR 之间存在漂移结合现象。本项目以8种E7/HLA-DR四聚体-IFN-γ-ELISPOT检测分析了36例结核患者及其他对照外周血标本，用8种E7/HLA-DR四聚体和8种CD4+ α/β TCR四聚体分别流式分析了111～223例、31～147例结核患者标本及其他对照标本。结果提示多肽/HLA-DR-TCR之间确实存在等位基因漂移结合现象。结核多肽/HLA-DR四聚体磁珠分选的结合阳性或未结合CD4+ T细胞克隆的TCR序列分析以及增值刺激与阻断试验，分别完成了14、及15例胸水标本的检测分析。不同等位基因的E7/HLA-DR四聚体在同一患者体内可以识别和结合相同TCR CDR3区的CD4+ T细胞，但其Vα和Vβ可呈现不同或寡克隆现象；在不同患者间则可以识别和结合相似结构或功能CDR3区的CD4+ T细胞。这些结合特性主要由多肽特异性决定，同时也存在一定的HLA-DR限制性。E7/HLA-DR四聚体-磁珠分选不能达到100%的分选率，所获得细胞或许存在不均一性，包含四聚体特异结合的优势CD4+ T细胞克隆及其他非优势残留CD4+ T细胞群。研究结果对于深入探讨多肽/HLA-DR与CD4+ TCR之间的结合规律、多肽在抗原递呈细胞（antigen presenting cell, APC）与CD4+ T细胞之间的递呈机制有一定的科学价值；同时在合理、规范使用CD3、CD4抗体与多肽/HLA-DR四聚体联合检测分析CD4+ T细胞方面也有实际的指导意义。