RORγt up-regulates RAG gene expression in DP thymocytes to expand the repertoire.

Recombination activating gene (RAG) expression increases as thymocytes transition from the CD4CD8 double-negative (DN) to the CD4CD8 double-positive (DP) stage, but the physiological importance and mechanism of transcriptional up-regulation are unknown. Here, we show that a DP-specific component of the recombination activating genes antisilencer (DPASE) provokes elevated RAG expression in DP thymocytes. Mouse DP thymocytes lacking the DPASE display RAG expression equivalent to that in DN thymocytes, but this supports only a partial repertoire due to inefficient secondary Vα-Jα rearrangement.

E protein binding at the enhancer promotes repertoire diversity.

V(D)J recombination of antigen receptor loci is a highly developmentally regulated process. During T lymphocyte development, recombination of the gene occurs in CD4CD8 double positive (DP) thymocytes and requires the enhancer (Eα). E proteins are known regulators of DP thymocyte development and have three identified binding sites in Eα.

Methods for Study of Mouse T Cell Receptor α and β Gene Rearrangements.

Quantitative real-time PCR and next-generation sequencing (NGS) are invaluable techniques to analyze T cell receptor (Tcr) gene rearrangements in mouse lymphocyte populations. Although these approaches are powerful, they also have limitations that must be accounted for in experimental design and data interpretation. Here, we provide relevant background required for understanding these limitations and then outline established quantitative real-time PCR and NGS methods that can be used for analysis of mouse Tcra and Tcrb gene rearrangements in mice.

Trav15-dv6 family Tcrd rearrangements diversify the Tcra repertoire.

The Tcra repertoire is generated by multiple rounds of Vα-Jα rearrangement. However, Tcrd recombination precedes Tcra recombination within the complex Tcra-Tcrd locus. Here, by ablating Tcrd recombination, we report that Tcrd rearrangement broadens primary Vα use to diversify the Tcra repertoire in mice.

The order and logic of CD4 versus CD8 lineage choice and differentiation in mouse thymus.

CD4 and CD8 mark helper and cytotoxic T cell lineages, respectively, and serve as coreceptors for MHC-restricted TCR recognition. How coreceptor expression is matched with TCR specificity is central to understanding CD4/CD8 lineage choice, but visualising coreceptor gene activity in individual selection intermediates has been technically challenging. It therefore remains unclear whether the sequence of coreceptor gene expression in selection intermediates follows a stereotypic pattern, or is responsive to signaling.