Regulation of the Alpha and Delta T Cell Receptor Repertoires

The adaptive immune system, comprised of B and T lymphocytes, responds with unique specificity to antigens. Specificity is imparted by the process of V(D)J recombination. During V(D)J recombination, gene segments spread across genomic space are assembled to generate unique antigen receptors by a cut-and-paste reaction in somatic cells. The mechanisms that regulate V(D)J recombination remain unclear.In T cells, two receptor classes are possible. These TCRs, the TCR and TCR, are assembled in part from genes sharing a single genetic locus. This single locus for both the T cell receptor and T cell receptor  (Tcra-Tcrd) chains has a unique nested structure that makes its regulation and recombination particularly interesting. In the Tcra-Tcrd locus, Vsegments are interspersed among the V segments. Rearrangement of Tcrd precedes Tcra recombination, and since these events are on the same locus, Tcrd recombination was hypothesized to diversify the starting point for primary Tcra recombinations, which are the first of a series of sequential recombination events possible for functional TCRchain generation. We generated a novel mouse model genetically modified to be incapable of Tcrd recombination. Using this model, we have determined that Tcrd recombination indeed diversifies the Tcra repertoire. Particularly, primary rearrangements are skewed toward more proximal V segments, while later rounds of rearrangement proceed as expected from the restricted primary V repertoire. We also investigated the specific impact of rearrangement to two relatively distal V segments of particular interest, Trav15d-1-dv6d-1 and Trav15-1-dv6-1, on the Tcra repertoire. To understand the significance of rearrangements to these V segments, we used two lines of mice lacking each of these segments. We found that primary Tcra recombinations utilizing V segments immediately upstream of each deleted gene segment were reduced. In the case of deletion of Trav15d-1-dv6d-1, the most distal V segment, we observed a reduction in primary recombinations using all upstream V segments. In the case of Trav15-1-dv6-1 deletion, primary recombinations upstream of this segment were reduced, but utilization of V segments 5′ of the most proximal upstream V segment was not disrupted. Unexpectedly, we also observed a reduction in secondary recombinations to V segments immediately upstream of either Trav15d-1-dv6-1 or Trav15-1-dv6-1 when either segment was deleted. These gene segments or their surrounding regions appear to be important in promoting diversity of the Tcra repertoire not only by their use as Tcrd recombination substrates, but also by a mechanism that remains to be explored. The structure of the Tcra-Tcrd locus also plays a role in recombination. Early in thymocyte development, the Tcra-Tcrd locus is contracted. During this period, Tcrd recombinations are capable of assembling segments distal in linear space. Later in development, when Tcra recombines, the locus is decontracted, and rearrangements generally proceed using the most proximal available segments. We and others have previously demonstrated that chromatin topology has a major impact on transcription and V(D)J recombination. Control of locus conformation is heavily influenced by contacts between CCCTC binding factor (CTCF)-bound elements. How CTCF regulates recombination at the Tcra-Tcrd locus is only partially understood. Herein, we used a novel mouse model lacking an intergenic CTCF binding element (CBE) in the Tcra-Tcrd locus (‘INT1’) to determine the impact of this specific CBE on Tcrd recombination. We find that loss of INT1 skews Tcrd rearrangements toward utilization of proximal V segments. These results suggest that INT1 serves as an important structural feature facilitating varied Tcrd recombination.