Phosphorylation of GSK3β at Ser389 prevents thymocyte necroptosis and influences the diversity of the Tcr repertoire 4584

Abstract Description The recombination of Tcrb and Tcra genes requires several cycles of programmed DNA double-strand breaks (DSBs) in double-negative (DN) thymocytes, along with effective repair mechanisms. However, the regulatory processes governing cell cycle checkpoints and survival pathways during this repair process are still elusive. Here, we report high-resolution single-cell RNA sequencing (scRNA-seq) analyses of individually sorted DN3 and DN4 thymocytes. We show the presence of a G2/M cell cycle checkpoint as well as the known G1 checkpoint during Tcrb and Tcra recombination. We demonstrate that the inactivation of GSK3β through phosphorylation at Ser389 is crucial for the survival of DN3 and DN4 thymocytes while they are stalled at the G1 and G2/M checkpoints. GSK3β promotes cell death by necroptosis, rather than by apoptosis, during V(D)J recombination of DN3 and DN4 thymocytes. If GSK3β is not inactivated in DN3 thymocytes, it can alter the Tcrb gene repertoire primarily through changes in Trbv segment utilization. GSK3b alters the nucleotide composition of the N2 region of Tcrb by suppressing the expression of the Apobec3 deaminase during Tcrb V(D)J recombination. Additionally, the preferential recombination of proximal V segments in Tcra relies on the inactivation of GSK3β, and this change is induced by necroptosis. Our study identifies a unique survival pathway in thymocytes that helps them navigate cell cycle checkpoints for DNA repair during Tcr gene V(D)J recombination. Funding Sources NIH R01 AI051454 Topic Categories Hematopoiesis and Immune System Development (HEM)