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.
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