Comprehensive analysis of αβT-cell receptor repertoires reveals signatures of thymic selection

Abstract Thymic selection is a multi-stage process that establishes a T-cell immunity that is efficient in fighting diverse foreign pathogens, while not being self-reactive. During the process, T-cell receptor (TCR)α and β chains are rearranged to form a highly diverse set of heterodimers that are selected based on their affinity to peptides presented by major histocompatibility molecules (MHC) in the thymus. Here we employ high-throughput TCR sequencing data, theoretical model of TCR rearrangement and dedicated statistical methods to infer how the selection process affects human TCR repertoire on different scales. On a global scale, our results indicate differences in V(D)J gene usage, complementarity determining region 3 (CDR3) amino acid composition, CDR3 physicochemical properties, k-mer composition and differences in loop structure induced by the selection process. On a local scale, we were able to determine enriched TCR motifs and “holes” in repertoire induced by positive and negative selection and characterize their features. Finally, we demonstrated how TCR sequence composition affects lineage commitment via thymic selection and highlighted the effect of individual MHC haplotype. Our results can aid in identification of potentially self-reactive TCRs in donor repertoires in autoimmunity and immunotherapy studies. Graphical abstract (Figure 0) Study overview. A. Datasets used in the study and the way TCR sequences were analyzed. B. Main directions in which thymic selection shapes TCR repertoire. Selection results in TCR-CDR3 losing positively charged and large amino-acids while increasing its flexibility. Unlike CDR3 of the TCRα chain, CDR3β hydrophobicity is also increased by selection. CDR3s carrying Cysteines and glycosylation sites are unlikely to pass the selection. In contrast, CDR3 carrying poly-Glycine regions are more likely to be selected for both chains. T-cells committed to CD8+ lineage were more likely to feature bulged CDR3s compared to CD4+. C. CDR3s enriched after thymic selection guide lineage commitment according to single-cell RNA sequencing data analysis, e.g. the MAIT cells and CD8+ phenotypes. D. Enrichment and depletion of certain TCR motifs pinpoint fine-structure of post-selection repertoires, as inferred by sequence neighborhood analysis. E. Comparative analysis of enriched and depleted TCR clusters in monozygotic twins revealed that the selection process is shaped by HLA haplotype. Competing Interest Statement The authors have declared no competing interest. Footnotes Typos and minor inaccuracies were fixed in the following revision. Figure 3 and Supplementary Note 2 were updated. Grant information updated.