Mechanisms That Direct Assembly of the T Cell Receptor Alpha Repertoire

Abstract

For effective adaptive immune responses, V(D)J recombination must produce diverse, clonally distributed B and T cell receptors (BCRs and TCRs) during lymphocyte development. Recombination of the genes encoding the TCR and TCR chains (Tcra and Tcrd) in developing T cells poses distinct regulatory challenges, as the two genes are encoded by a single locus (Tcra-Tcrd) and share an array of V segments. Tcrd undergoes a single round of V-D-J in CD4-CD8 double-negative (DN) thymocytes, while Tcra is assembled through multiple rounds of V-J rearrangement in the CD4-CD8 double-positive (DP) compartment. However, the mechanisms ensuring that diverse repertoires result from rearrangements at Tcra-Tcrd are poorly understood. Here, we demonstrate that the developmentally-dynamic chromatin architecture of Tcra-Tcrd ensures diverse gene segment usage during both Tcrd and Tcra rearrangement.We began by seeking a better understanding of how the Tcra repertoire is assembled through several rounds of V-J rearrangement. To do this, we developed and applied a high-throughput sequencing (HTS)-based strategy to analyze the pre-selection Tcra repertoire in mice carrying wild-type and genetically modified Tcra-Tcrd alleles. We found that Tcra rearrangement in individual thymocytes is intrinsically highly processive and coordinated along the V and J arrays through multiple rounds of rearrangement, imposing severe constraints on combinatorial diversity. Repertoire diversity is nonetheless achieved by mechanisms that impart combinatorial diversity to primary V-J rearrangement, effectively scrambling the starting points for progressions of secondary rearrangements in individual DP thymocytes. Using 4C-seq and 3D DNA-FISH, we determined that the processive usage of V segments is supported by an extended V-V array architecture in DP thymocytes.We next explored how assembly of the Tcrd repertoire is regulated. Using 4C-seq, we identified a stable chromatin loop mediated by CTCF binding that contains the D and J segments used by Tcrd. This loop was essential for diverse V usage, as disruption of one end by gene targeting biased Tcrd rearrangements toward D-J proximal V segments. We demonstrated that V usage during Tcrd rearrangement also has a substantial impact on the Tcra repertoire. V-D rearrangement in DN thymocytes variably truncates the V-V array on Tcra-Tcrd alleles that will later recombine Tcra in the DP compartment, scrambling the starting point for primary V-J rearrangement. We revealed that these variable truncations impart V diversity during primary V-J rearrangements and allow otherwise disfavored V-J rearrangements to occur. Such diversification is functionally significant, as Trav1-Traj33 mucosa-associated invariant T (MAIT) cells are depleted when Tcrd rearrangement is impaired or absent. Therefore, the regulation of Tcrd and Tcra recombination by Tcra-Tcrd chromatin architecture represents an integrated mechanism for boosting TCR diversity in both the  and  T cell lineages.