Abstract
<jats:p>Highly expanded clonotypes are widely used to enrich tumor-reactive TCRs, yet expansion may not faithfully report functional reactivity. Using direct tumor-cell recognition as the benchmark, we found that in native E0771 and LLC tumors, highly expanded clonotypes did not consistently show the strongest responses, whereas reactive TCRs were also present among low-frequency clones. We next used the antigen-controlled MB49-OVA model to jointly measure TCR functional avidity, peptide–MHC presentation abundance and tumor-cell recognition. In this setting, TCRs with different avidities for the same antigen could converge to similar expansion levels in vivo. Moreover, peptide–MHC presentation abundance was sufficient to reshape the reactivity hierarchy predicted from avidity alone, and distinct avidity–presentation combinations were linked to biased transcriptional states. Thus, clonal expansion is a context-dependent enrichment cue rather than a stable surrogate of tumor reactivity, and low-frequency clonotypes may provide a valuable reservoir for tumor-reactive TCR discovery.</jats:p>