GSK3B in TCR repertoire and immune diseases

总结 免疫保护高度依赖于T细胞和提供免疫保护的多种TCR库的存在。 广泛保护免受传染性病原体。尽管外周TCR库部分由 抗原介导的TCRαβ在胸腺中的阳性和阴性选择，也是由成功的 Tcrβ重排。胸腺细胞发育DN 2/DN 3期Tcrβ基因重排 是一个复杂的过程，涉及两个（Dβ到J β，然后是Vβ到D βJβ）甚至三个（VβDβ 1 J β1到 VβDβ 2 J β2）需要染色质构象转换的相同基因座的顺序重组事件， DNA双链断裂（DSB）和DNA修复的产生，在事件之间没有细胞分裂。我们 提出在Tcrβ重排过程中没有足够的存活信号来保护基因组不稳定性， 可能导致特异性Dβ-Jβ或Vβ-DβJβ重排的频率降低，从而损害 预选择TCRβ库。我们已经确定了一种新的途径，它是选择性地响应于 DNA DSB和调节细胞存活。GSK 3 β是一种激酶，通过诱导细胞凋亡来促进细胞死亡。 促生存因素。我们最近的研究表明，DSB特异性地抑制GSK 3 β的核库， P38 MAPK在Ser 389上的磷酸化，也定位于细胞核中响应DSB。DSB自然 在胸腺细胞中由V（D）J介导的重组产生的细胞也通过 Ser 389的磷酸化。使用GSK 3 β Ser 389 Ala KI小鼠，我们已经证明该途径对于细胞增殖和分化是必需的。 DSB的生存率。因此，经历CSR的GSK 3 β Ser 389 Ala KI B细胞通过坏死性凋亡死亡， GSK 3 β底物核Mcl-1的降解增强。在胸腺细胞中，GSK 3 β Ser 389突变 干扰Tcrβ在DN 3胸腺细胞中的成功重组，从而干扰DN 4胸腺细胞的产生。 胸腺细胞我们发现在GSK 3 β KI胸腺细胞中Trbv-DβJβ重排频率较低。我们 提出Ser 389磷酸化导致的细胞核GSK 3 β失活是保护 Tcrβ重排过程中DSB引发的胸腺细胞坏死性凋亡。此外，我们建议， 在Tcrβ重排过程中，不能抑制GSK 3 β会损害这些细胞的适应性， 它导致一些Trbv-DβJβ重排的频率降低和预选择性受损 TCRβ库。为了解决这一假设，我们将确定：1）是否失活的GSK 3 β途径， 对VDJ介导的DSB的应答保护胸腺细胞在TCRβ重组期间免于坏死性凋亡（目的1）; 2） p38 MAPK/GSK 3 β通路对预选择TCRβ库的影响（目的2）; 3） 由于不能抑制细胞核GSK 3 β而引起的预选TCRβ库的改变， 免疫反应

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.