Inactivation of GSK3b by Ser389 phosphorylation during early thymocyte development contributes to the diversity of the Tcrb and Tcra repertoire.  

During thymocyte development, double negative (DN) thymocytes go through consecutive rounds of Tcr and Tcr gene recombination that involve the generation of DNA double strand breaks (DSB), addition of N-nucleotides between coding ends, and DNA repair with the final goal of enhancing the diversity of the TCR repertoire. How DN thymocytes can survive such a genomic instability during this complex process and generate functional Tcr and Tcr genes remain unclear. Performing scRNAseq analyses of isolated DN3 and DN4 thymocytes we show here a defined trajectory from DN3 through DN4 stages that is delineated by a G1 cell cycle checkpoint, but also by a G2M cell cycle checkpoint to help DNA repair during recombination of Tcr and Tcr genes prior to cell proliferation. We have previously shown that inactivation of GSK3 by phosphorylation on Ser389 occurs selectively in response to DNA DSB to enhance survival during DNA repair. In this study we show that failure to inactivate GSK3 by Ser389-phosphorylation during the transition from DN3 to DN4 stages compromises Tcr and Tcr repertoire diversity. Inactivation of GSK3 during V(D)J recombination is needed to prevent cell death by necroptosis. Thus, cell cycle checkpoints to ensure proper DNA repair during V(D)J recombination as well as the use of unique survival pathways to avoid necroptosis during this process are critical to warrant a large diversity of pre-selection TCR repertoire.