GSK3B in TCR repertoire and immune diseases

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SUMMARY Immune protection is highly dependent on T cells and the presence of a diverse TCR repertoire that provides broad protection against infectious agents. Although the peripheral TCR repertoire is determined partially by antigen-mediated positive and negative selection of TCRαβ in the thymus, it is also determined by the success of the Tcrβ rearrangement. Rearrangement of the Tcrβ locus at the DN2/DN3 stage of thymocyte development is a convoluted process that involves two (Dβ-to-Jβ first followed by Vβ-to-DβJβ) or even three (VβDβ1Jβ1-to- VβDβ2Jβ2) sequential recombination events of the same locus requiring chromatin conformation switches, generation of DNA double strand breaks (DSB) and DNA repair, without cell division between the events. We propose that insufficient survival signals to protect from the genomic instability during Tcrβ rearrangement could result in lower frequency of specific Dβ-Jβ or Vβ-DβJβ rearrangements and, thereby, in a compromised pre-selection TCRβ repertoire. We have identified a novel pathway that is triggered selectively in response to DNA DSB and regulates cell survival. GSK3β is a kinase that promotes cell death by inducing degradation of pro-survival factors. We have recently shown that DSB inactivate specifically the nuclear pool of GSK3β by phosphorylation on Ser389 by p38 MAPK, also localized in the nucleus in response to DSB. DSB naturally generated by V(D)J-mediated recombination in thymocytes also inactivate nuclear GSK3β through phosphorylation at Ser389. Using GSK3β Ser389Ala KI mice we have shown that this pathway is essential for cell survival in response to DSB. Thus, GSK3β Ser389Ala KI B cells undergoing CSR die through necroptosis due to enhanced degradation of nuclear Mcl-1, a GSK3β substrate. In thymocytes, GSK3β Ser389 mutation interferes with a successful recombination of Tcrβ in DN3 thymocytes and, therefore, the generation of DN4 thymocytes. We found lower frequency of some Trbv-DβJβ rearrangements in GSK3β KI thymocytes. We propose that inactivation of nuclear GSK3β by Ser389 phosphorylation is necessary to protect thymocytes from necroptosis triggered by DSB during Tcrβ rearrangement. Further, we propose that failure to inactivate GSK3β impairs fitness of these cells during Tcrβ rearrangement and, consequently it results in reduced frequency of some Trbv-DβJβ rearrangements and a compromised pre-selection TCRβ repertoire. To address this hypothesis we will determine: 1) whether inactivation of GSK3β pathway in response to VDJ-mediated DSB protect thymocytes from necroptosis during TCRβ recombination (Aim 1); 2) impact that the p38 MAPK/GSK3β pathway has on the pre-selection TCRβ repertoire (Aim 2); 3) whether the altered pre-selection TCRβ repertoire caused by failure to inactivate nuclear GSK3β has an impact on the immune response.