Deep exploration of the TCR CDR3ß repertoire specific for viral CD4 T cell epitopes inside the circulating T cell repertoire

This study provides an in-depth analysis of the diversity of the CD4 TCR CDR3ß repertoire specific to Influenza A (HA) and Epstein-Barr virus (EBNA) epitopes. Epitope-specific CD4 T cells from 13 healthy donors were enriched using a short-term culture step, isolated based on activation markers, and sequenced for their TCR CDR3ß region using high-throughput sequencing. The frequency of each clonotype were then identified within the complete circulating CD4 T-cell CDR3ß repertoire. For both epitopes, the clonotype distribution was markedly skewed, with a small number of highly expanded clones comprising approximately 60% of the repertoire, alongside numerous low-frequency clonotypes. VJ gene usage and motif preferences differed between the two peptides, highlighting epitope-specific TCR selectivity. The response was predominantly composed of private T-cell clonotypes. The proportion of public clonotypes can increase among donors sharing HLA class II molecules and reveals in HLA-unrelated donors the level of TCR promiscuity. Overall, our data demonstrate that CD4 T-cell responses to these viral epitopes are polyclonal and highly personalized. The modest overlap of clonotypes between donors, coupled with a long tail of low-frequency clones, suggests that the full diversity of the epitope-specific T-cell repertoire is likely broader than previously estimated. Overall design: TCRseq of total and specific CD4 T cells

本研究针对甲型流感病毒（Influenza A，HA）表位与EB病毒（Epstein-Barr virus，EBNA）表位特异性CD4+ T细胞受体（CD4 TCR）CDR3β库的多样性展开深入分析。研究人员从13名健康供体内分离表位特异性CD4+ T细胞，通过短期培养步骤富集此类细胞，并基于活化标志物完成分选，随后采用高通量测序技术对其TCR CDR3β区域进行测序，进而在完整循环CD4+ T细胞CDR3β库中鉴定每种克隆型（clonotype）的频率。
针对两种表位，克隆型分布均呈现显著偏倚：少量高度扩增的克隆约占整个库的60%，同时伴随大量低频克隆型。两种表位的VJ基因使用偏好与基序偏好存在差异，凸显了表位特异性的TCR选择特性。此类免疫应答主要由个体特异性（private）克隆型构成。共享II类人类白细胞抗原（HLA class II）的供体群体中，公共（public）克隆型的占比会有所升高；而在HLA不相关的供体中，该占比可反映TCR的多特异性水平。
综上，本研究数据表明，针对上述病毒表位的CD4+ T细胞应答呈现多克隆性且具有高度个体化特征。不同供体间克隆型的重叠度较低，加之存在大量低频克隆构成的长尾分布，提示表位特异性T细胞库的完整多样性可能比此前预估的更为广泛。
实验整体设计：总CD4+ T细胞与表位特异性CD4+ T细胞的T细胞受体测序（TCRseq）