The αβT cell receptor (TCR) orchestrates immunity through the recognition of peptides, derived from degraded proteins, presented on major histocompatibility complex (MHC) molecules. The remarkable ability of the receptor to respond to a vast plethora of antigens is driven by V(D)J recombination, a process which generates a highly diverse TCR repertoire by somatic gene rearrangement of coding DNA. TCR diversity is confined to three short hairpin loops on each TCR chain, called the complementarity determining region (CDR), which form the antigen-binding site. The germline-encoded CDR1 and CDR2 loops predominantly contact MHC, whereas the hypervariable CDR3 are non-germline and primarily bind to the MHC-bound peptide.
In this study, we developed a novel in vivo mutagenesis approach which redirects somatic gene rearrangement using V(D)J recombination machinery to diversify and optimise TCR binding. This approach involves embedding a gene recombination cassette into the peptide-binding CDR3β region of established TCRs. A retrogenic system was employed to facilitate the in vivo processes necessary for gene rearrangement and thymic selection. We demonstrate that the recombination cassette can successfully induce gene rearrangement and introduce variation to the targeted CDR3β site. Thymocytes expressing the diversified TCRs can be selected on MHC and develop into functional peripheral T cells. Subsequent exposure to cognate ligands also allowed us to identify optimised and ‘immunodominant’ TCRs.
In addition, we produced a novel chimeric TCR chain which comprises Vα and Cβ domains. This TCR chain forms a heterodimer with endogenous TCRα chains to form a unique Vα-Vα antigen-binding surface. Thymocytes expressing this novel form of αβTCR were able to engage efficiently with both MHC classes and develop normally into functional T cells typical of a conventional repertoire. Collectively, these findings suggest that the germline CDR loops are not essential for mediating MHC recognition during MHC-restricted T cell development and function.