Genetic determinants of distinct CD8+ α/β-TCR repertoires in the genus Mus

The adaptive immune system's efficacy relies on the diversity of T cell receptors and the ability to distinguish between self and foreign antigens. Analysis of the paired heterodimeric αβ-TCR chains of individual T cells requires single-cell resolution, but existing single-cell approaches offer limited coverage of the vast TCR repertoire diversity. Here we introduce CITR-seq, a novel, instrument-free, high-throughput method for single-cell TCR sequencing with >88% αβ-TCR pairing precision. We analyzed the TCR repertoires of CD8+ T cells originated from 32 inbred mice using CITR-seq, comprising four evolutionary divergent sister species and their F1 hybrids. Overall, we identified more than 5 million confidently paired TCRs. We found that V(D)J gene usage patterns are highly specific to the genotype and that Vβ-gene usage is strongly impacted by thymic selection. Using F1 hybrids, we show that differences in gene segment usage across species are likely caused by cis-acting factors prior to thymic selection, which imposed strong allelic biases. At the greatest divergence, this led to increased rates of TCR depletion through rejection of particular Vβ-genes. TCR repertoire overlap analysis across all mice revealed that sharing of identical paired CDR3 amino acid motifs is four times more frequent than predicted by random pairing of TCRα and TCRβ chains, with significantly increased sharing rates among related individuals. Collectively, we show that beyond the stochastic nature of TCR repertoire generation, genetic factors contribute significantly to the shape of an individual's repertoire.