Mechanisms Of Age-related Changes in Transcriptional Regulation, TCR Repertoire, and Cytokine Expression

多样化的T细胞受体(TCR)谱系对于控制病毒感染至关重要。然而，关于人类TCR谱系和已定义的病毒抗原的信息是有限的。我们利用高通量测序(HTS)和单细胞配对TCR分析，对两个主要病毒表位：巨细胞病毒的pp65495503(NLV)和甲型流感病毒的M15866(Gil)的CD8+TCR谱进行了全面的分析。我们对CD4+和CD8+T细胞的TCR谱进行了比较分析。在每个研究对象中，CD4+T细胞的TCR多样性从1.8-8.2x105不等，平均比CD8+T细胞的TCR多样性高3-4倍。此外，CD4+T细胞(0.3%)和CD8+T细胞(1.3%)之间的TCR序列几乎没有重叠。进一步的分析表明，CD4+和CD8+T细胞对CDR3中的某些氨基酸表现出不同的偏好，这一点得到了支持向量机分类器的进一步证实，这表明CD4+和CD8+T细胞中的TCR CDR3存在明显和可识别的差异。最后，我们鉴定了6-12%具有相同CDR3和不同V基因的独特TCR。总之，我们的发现揭示了CD4+和CD8+T细胞之间TCR谱系的明显特征，并可能被用于评估T细胞免疫能力。 对于抗原特异的CD8+TCR谱系，我们鉴定了数千个新的NLV和GIL特异的TCR和TCR序列，以及数十个不同的CDR3和CDR3共识基序。这种多样性大大高于之前描述的T细胞对单个病毒表位的反应，无论是私人的还是公共的TCR克隆型，并显示出高度的个体差异(每个受试者12028,000个克隆型)。然而，多样性受到优先的V-J组合、CDR3长度和CDR3/CDR3配对的有效限制。我们进一步发现，GIL特异的而非NLV特异的TCR表现出令人惊讶的广泛的结合亲和力，解离常数(KDS)从2到200M。最后，我们确定了两个无关的GIL特异TCR与GILHL-A2的络合物和两个TCR-NLV-HLA-A2复合体的晶体结构。这些结构解释了为什么GIL特异性谱系的多样性低于NLV特异性谱系。值得注意的是，这两个抗病毒TCR谱占总CD8+TCR谱的0.220%，确保了T细胞对单一表位的广泛和强大的反应。我们对两种不同的抗病毒T细胞反应的基因、生化和结构的综合描述可能有助于未来在个人水平上开发免疫或疾病的预测指标。

A diverse T cell receptor (TCR) repertoire is essential for controlling viral infections. However, information on human TCR repertoires in general and to defined viral antigens is limited. We performed a comprehensive analysis of TCR repertoires of CD4+ and CD8+, and CD8+ TCR repertoires specific for two dominant viral epitopes: pp65495503 (NLV) of cytomegalovirus and M15866 (GIL) of influenza A virus using the high-throughput sequencing (HTS) and single-cell paired TCR analysis. We provided a comparative analysis of the TCR repertoires of CD4+ and CD8+ T cells. TCR diversity of CD4+ T cells ranges from 1.8-8.2 x105 and is 3-4 times greater on average than that of CD8+ T cells in each study subject. Furthermore, there was little overlap in TCR sequences between CD4+ (0.3%) and CD8+ (1.3%) T cells. Further analysis showed that CD4+ and CD8+ T cells exhibited distinct preferences for certain amino acids in the CDR3, and this was further confirmed by a support vector machine classifier, suggesting there are distinct and discernible differences between TCR CDR3 in CD4+ and CD8+ T cells. Finally, we identified 6-12% of the unique TCRs that share an identical CDR3 with different V genes. Together, our findings reveal the distinct features of the TCR repertoire between CD4+ and CD8+ T cells and could potentially be used to evaluate the competency of T cell immunity. For antigen-specific CD8+ TCR repertoires, we identified thousands of new NLV- and GIL-specific TCR and TCR sequences, as well as dozens of distinct CDR3 and CDR3 consensus motifs. This diversity is substantially greater than previously described for T cell responses to single viral epitopes, both for private and public TCR clonotypes, and exhibited a high degree of individual variations (12028,000 clonotypes per subject). However, diversity is effectively restricted by preferential V-J combinations, CDR3 lengths, and CDR3/CDR3 pairings. We further found that GIL-specific, but not NLV-specific, TCRs exhibited a surprisingly wide range of binding affinities, with dissociation constants (KDs) from 2 to 200 M. Finally, we determined the crystal structures of two unrelated GIL-specific TCRs in complex with GILHLA-A2 and two TCR-NLV- HLA-A2 complexes. These structures provide an explanation of the lower diversity of GIL-specific than NLV-specific repertoires. Remarkably, these two anti-viral TCR repertoires occupied 0.220% of the total CD8+ TCR repertoire, ensuring broad and robust T cell responses to single epitopes. Our comprehensive genetic, biochemical, and structural portrait of two different anti-viral T cell responses may contribute to the future development of predictors of immunity or disease at the personal level.