Tcrd rearrangement redirects a processive Tcra recombination program to expand the Tcra repertoire

The Tcra-Tcrd locus undergoes Tcrd rearrangement in DN thymocytes followed by primary and secondary Tcra rearrangements in DP thymocytes. Here we reveal the mechanisms underpinning combinatorial diversity in the Tcra repertoire. We show that V-alpha and J-alpha segments on individual Tcra alleles are used in stepwise and coordinated proximal-to-distal progressions during primary and secondary rearrangements, substantially constraining combinatorial diversity. Notably, Tcrd recombination in DN thymocytes diversifies the Tcra repertoire by truncating the V-alpha array to permit otherwise disfavored V-J combinations. Such diversification is important, because Trav1-Traj33+ mucosa-associated invariant T cells are depleted when Tcrd rearrangement is impaired. Our results reveal an important biological advantage conferred by the nested organization of Tcrd and Tcra gene segments. Overall design: High-throughput sequencing was used to analyze Tcra repertoire from transcripts purified from sorted DP thymocytes from groups of 1 to 4 mice bearing either wild-type or genetically modified Tcra alleles. Higher-order chromatin structure of wild-type and HYa Tcra alleles in DP thymocytes was analyzed using 4C-seq from 5 viewpoints.