Infection history and TCR sequence features shape the breadth of TCR cross-reactivity

The T cell receptor (TCR) repertoire enables recognition of a vast array of pathogens, despite containing far fewer unique TCRs than the diversity of encountered epitopes. This potential contradiction is resolved by TCR cross-reactivity, whereby individual TCRs recognize multiple distinct epitopes. However, the features defining broadly cross-reactive TCRs remain poorly understood. Here we systematically analyze TCRs from mice sequentially infected with influenza viruses containing naturally occurring single amino acid variants of the D^bNP₃₆₆ epitope. We identify 967 unique paired TCR sequences across 18 infection histories and test 22 representative TCRs against 172 epitope variants, yielding 3,784 TCR-epitope interaction measurements. These TCRs exhibit a continuum of cross-reactivity, with infection history influencing both TCR sequence similarity and reactivity profiles. We find specific TCR sequence features associated with broader cross-reactivity, highlighting the interplay between epitope exposure, TCR diversity, and function, and providing insights into CD8⁺ T cell adaptability, immune memory, and vaccine design.