GSK3B in TCR repertoire and immune diseases

摘要 免疫保护高度依赖于T细胞和多种TCR谱系的存在，这些TCR谱系可提供 针对感染性病原体的广泛保护。尽管外围TCR曲目部分由以下因素决定 抗原介导的胸腺TCRαβ的阳性和阴性选择，也是由成功与否决定的 Tcrβ重排。胸腺细胞发育Dn2/Dn3期TcRβ基因座的重排 是一个复杂的过程，涉及两个(先是Dβ-to-Jβ，然后是Vβ-to-DβJβ)，甚至三个(VβDβ1Jβ1 to-J VβDβ2Jβ2)需要染色质构象开关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，事件之间没有细胞分裂。我们 认为在TcRβ重排过程中没有足够的生存信号来保护基因组的不稳定性 可能导致特定Dβ-Jβ或Vβ-DβJβ重排的频率较低，从而在 预选TCRβ曲目。我们已经确定了一种新的通路，它是选择性地触发的 DNADSB并调节细胞存活。Gsk3β是一种通过诱导细胞降解而促进细胞死亡的激酶 支持生存的因素。我们最近已经证明，dsb通过以下方式特异性地灭活Gsk3β的核池 P38MAPK在Ser389上的磷酸化，也定位于细胞核中，对DSB的反应。DSB自然 在胸腺细胞中由V(D)J介导的重组产生的β也通过 丝氨酸389位的磷酸化。使用Gsk3βSer389Ala Ki小鼠，我们已经证明这一途径是细胞所必需的 在对DSB的反应中存活。因此，经历CSR的Gsk3βSer389 Ala Ki B细胞死于死于 以促进GSK3β底物核Mcl-1的降解。胸腺细胞中Gsk3βSer389突变 干扰胸腺细胞中TcRβ的成功重组，从而干扰DN4的产生 胸腺细胞。我们在胸腺细胞中发现了一些TrbV-DβJβ重排的频率较低的βKI。我们 提出有必要通过丝氨酸389磷酸化使核GSK3β失活以保护 胸腺细胞在TcRβ重排过程中由DSB引发的坏死性下垂。此外，我们建议 在ββ重排过程中，未能使GSK3失活会损害这些细胞的适合性，因此 这会导致某些TRBV-DβJβ重排的频率降低，并影响预选 Tcrβ曲目。为了解决这一假设，我们将确定：1)Gsk3β途径在 对VDJ介导的DSB的应答保护胸腺细胞免受TCRDNA重组时的坏死性下垂(目标1)；2) P38MAPK/GSK3β通路对预选择TCRβ谱系的影响(目标2)；3) 由于核GSK3β失活失败而导致的预选TCRβ谱系的改变对 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.