Germline-enforced enrichment for charged amino acids in TCR beta chain (TCRß) complementarity determining region 3 (CDR-B3) alters T cell development, repertoire content, and antigen recognition.

T cell receptor beta chain (TCRß) diversity (Dß) gene segments are highly conserved across evolution, with trout Dß1 sequence identical to human and mouse Dß1. A key conserved feature is enrichment for glycine in all three Dß reading frames (RFs). Previously, we found that replacement of mouse Dß1 with a typical immunoglobulin DH sequence, which unlike Dß is enriched for tyrosine, leads to an increase in the use of tyrosine in TCRß complementarity determining region 3 (CDR-B3) after thymic selection, altering T cell numbers, CDR-B3 diversity, and T cell function. To test whether the incorporation of charged amino acids into the Dß sequence in place of glycine would also influence T cell biology, we targeted the TCRß locus with a novel glycine-deficient DßDKRQ allele that replaces Dß1 coding sequence with charged amino acids in all three reading frames. Developing T cells using DßDKRQ expressed TCR CDR-B3s depleted of tyrosine and glycine and enriched for germline-encoded lysine, arginine, and glutamine. Total thymocytes declined in number during the process of ß selection that occurs during the transition from the DN3bc to DN4 stage. Conventional thymocyte and T cell numbers remained reduced at all subsequent thymic stages and in the spleen. By contrast, regulatory T cell numbers were increased in Peyer's patches and the large intestine. In terms of functional consequences, T cell reactivity to an ovalbumin immunodominant epitope was reduced. These findings buttress the view that natural selection of Dß sequence is used to shape the pre-immune TCRß repertoire, affecting both conventional and regulatory T cell development and influencing epitope recognition.