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 TCR?? in the thymus, it is also determined by the success of the Tcr? rearrangement. Rearrangement of the Tcr? locus at the DN2/DN3 stage of thymocyte development is a convoluted process that involves two (D?-to-J? first followed by V?-to-D?J?) or even three (V?D?1J?1-to- V?D?2J?2) 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 Tcr? rearrangement could result in lower frequency of specific D?-J? or V?-D?J? rearrangements and, thereby, in a compromised pre-selection TCR? repertoire. We have identified a novel pathway that is triggered selectively in response to DNA DSB and regulates cell survival. GSK3? 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 GSK3? 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 GSK3? through phosphorylation at Ser389. Using GSK3? Ser389Ala KI mice we have shown that this pathway is essential for cell survival in response to DSB. Thus, GSK3? Ser389Ala KI B cells undergoing CSR die through necroptosis due to enhanced degradation of nuclear Mcl-1, a GSK3? substrate. In thymocytes, GSK3? Ser389 mutation interferes with a successful recombination of Tcr? in DN3 thymocytes and, therefore, the generation of DN4 thymocytes. We found lower frequency of some Trbv-D?J? rearrangements in GSK3? KI thymocytes. We propose that inactivation of nuclear GSK3? by Ser389 phosphorylation is necessary to protect thymocytes from necroptosis triggered by DSB during Tcr? rearrangement. Further, we propose that failure to inactivate GSK3? impairs fitness of these cells during Tcr? rearrangement and, consequently it results in reduced frequency of some Trbv-D?J? rearrangements and a compromised pre-selection TCR? repertoire. To address this hypothesis we will determine: 1) whether inactivation of GSK3? pathway in response to VDJ-mediated DSB protect thymocytes from necroptosis during TCR? recombination (Aim 1); 2) impact that the p38 MAPK/GSK3? pathway has on the pre-selection TCR? repertoire (Aim 2); 3) whether the altered pre-selection TCR? repertoire caused by failure to inactivate nuclear GSK3? has an impact on the immune response.
It is clear that the immune response to the same antigen or infectious agent is highly heterogeneous among individuals even among those who are very close genetically. The development of an inappropriate immune response to self-antigens causing tissue destruction is also heterogeneous. T cells are a key component of the immune system, have the capacity to recognize a very large spectrum of antigens through their highly diverse T cell receptor (TCR) repertoire, and are essential for protection against infections. However, they are also frequently mediating autoimmune diseases. The high diversity of the TCR that allows recognition of millions of antigens is achieved by DNA recombination of TCR? and TCR? genes, a process that confers genomic instability to the immature T cells. It is generally believed that the TCR cell repertoire is determined by an educational training in the thymus based on their recognition of antigens in the context of the MCH molecules (positive and negative selection). We propose here that the TCR repertoire is first determined by a process independent of antigen recognition, but stochastically dependent on survival to the genomic instability generated while the immature T cells undergoing recombination of the TCR? gene. We have recently identified a novel signaling pathway (inactivation of GSK3? by p38 MAP kinase) that provides survival to these immature T cells while undergoing the DNA recombination process. The goal of this proposal is to determine whether this pathway affects the shape of TCR? repertoire, and how a slim TCR? repertoire can have an impact in protection against infectious agents.
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