The Tcra-Tcrd locus undergoes V(D)J recombination in CD4−CD8−double-negative thymocytes and CD4+CD8+ double-positive thymocytes to generate diverse TCRδ and TCRα repertoires, respectively. Here we reveal a Tcra-Tcrd locus chromatin interaction network in double-negative thymocytes that was formed by interactions between CTCF-binding elements. Disruption of a discrete chromatin loop encompassing Tcrd diversity, joining and constant gene segments allowed a single variable gene segment to frequently contact and rearrange to diversity and joining gene segments and dominate the adult TCRδ repertoire. Disruption of this loop also narrowed the TCRα repertoire, which, we believe, follows as a consequence of the restricted TCRδ repertoire. Hence, a single CTCF-mediated chromatin loop directly regulates TCRδ diversity and indirectly regulates TCRα diversity.

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SUMMARY Adaptive immunity depends on diverse T cell receptor repertoires generated by V(D)J recombination. Here, we define the principles by which combinatorial diversity is generated in the murine Tcra repertoire. Tcra and Tcrd gene segments share the Tcra-Tcrd locus, with interspersed Vα and Vδ segments undergoing Vδ-Dδ-Jδ rearrangement in CD4−CD8− thymocytes and then multiple rounds of Vα−Jα rearrangement in CD4+CD8+ thymocytes. We document stepwise, highly coordinated proximal-to-distal progressions of Vα and Vδ use on individual Tcra alleles, limiting combinatorial diversity. This behavior is supported by an extended chromatin conformation in CD4+CD8+ thymocytes, with only nearby Vα and Vδ segments contacting each other. Tcrd rearrangements can use distal Vδ segments due to a contracted Tcra-Tcrd conformation in CD4−CD8− thymocytes. These rearrangements expand the Tcra repertoire by truncating the Vα array to permit otherwise disfavored Vα−Jα combinations. Therefore, recombination events at two developmental stages with distinct chromatin conformations synergize to promote Tcra repertoire diversity.