HLA alleles shape distinct biases in the usage preferences of TCR Vβ segments.

T cells must co-recognize peptide and HLA, yet the extent to which this specificity is shaped by germline-driven TCR-HLA contacts has remained difficult to quantify. Leveraging population-scale TCRβ repertoires linked to donor HLA genotypes inferred using validated imputation models, we construct allele-specific TCR Vβ gene usage profiles and normalize them to repertoire-wide baselines to derive interpretable HLA-Vβ preference vectors. We demonstrate that different HLA alleles shape distinct biases in the usage frequency of Vβ gene segments among the public TCRs that engage those alleles: certain Vβ genes are over-represented for particular HLA alleles. Similarities in HLA amino-acid sequence predict similarities in both their Vβ preferences and peptide-binding motifs. A residue-level analysis disentangles HLA positions primarily associated with Vβ engagement from those primarily associated with peptide motifs; the Vβ-associated HLA positions localize to canonical TCR-facing helices, whereas peptide-associated HLA positions track binding pockets, revealing distinct molecular routes by which HLA polymorphism shapes the TCR and peptide sides of recognition. CMV exposure stratifications confirmed these Vβ preference vectors are not explained by any shift of repertoire composition associated with a dominant infection. Together, these data support that germline-driven constraints set the landscape of TCR-HLA compatibility. These HLA-specific TCR Vβ biases are a biological prior that provide a foundation for better understanding of TCR-pHLA specificity as a whole and should be accounted for in future evaluation of any TCR-pHLA specificity prediction methods.