Intravital imaging of T cell priming in the gut-associated lymphatic tissue (GALT)

多发性硬化症(MS)是一种常见的自身免疫性疾病，其特征是免疫细胞渗入中枢神经系统(CNS)。在我之前的工作中，我通过活体成像显示，中枢神经系统中自身反应性的CD4+T细胞的渗透和激活是启动中枢神经系统炎症的关键。然而，为什么脑源性T细胞开始向中枢神经系统渗透，目前仍不清楚。虽然这些细胞正常存在于健康的免疫系统中，但它们只有在被触发时才开始渗透到中枢神经系统并诱导局部炎症损害。由于肠道微生物区系可以触发实验性自身免疫性脑脊髓炎(EAE)的发病，这是一种MS的动物模型，我假设脑源性T细胞为中枢神经系统在肠道中的渗透做好了准备。为了直接验证这一假设，我建立了一种活体成像，允许通过双光子显微镜观察肠道相关淋巴组织(GalT)中T细胞何时何地受到刺激。为了做到这一点，我将表达我之前在脑源性T细胞中开发的两个激活传感器。钙感应蛋白Twitch检测细胞内钙水平的变化，从而对T细胞的刺激进行分级评估，而NFAT-GFP融合蛋白的核转位选择性地检测抗原依赖的T细胞激活，这只由饱和钙信号诱导。为了研究T细胞在GALT中启动的后果，将从传出淋巴管中回收GALT向外迁移的细胞，并将用下一代测序(NGS)分析其表型。基于高尔基转移细胞的分子图谱，我将定义“脑源性开关，这种开关可以作为治疗靶点，防止随后诱发的中枢神经系统炎症。

Multiple sclerosis (MS), a common autoimmune disease, is characterized by infiltration of immune cells into the central nervous system (CNS). In my previous work I showed by intravital imaging that the infiltration and activation of autoreactive CD4 positive T cells in the CNS is critical for the initiation of CNS inflammation. However, it is still largely unknown why encephalitogenic T cells start infiltration into the CNS. Although these cells exist normally in healthy immune repertoire, they only start infiltrating into the CNS and induce local inflammatory lesions if triggered to do. Since it was shown that gut microbiota can trigger the onset of experimental autoimmune encephalomyelitis (EAE), an animal model of MS, I hypothesize that the encephalitogenic T cells get primed for CNS infiltration in the gut. To directly test this hypothesis, I have established an intravital imaging that allows to visualize by two-photon microscopy when and where T cells are stimulated in the gut-associated lymphatic tissue (GALT). To do so I will express two activation sensors that I have previously developed in encephalitogenic T cells. Whereas the calcium sensing protein, Twitch, detects changes of the intracellular calcium level that allow a graded assessment of T cell stimulation, the nuclear translocation of the NFAT-GFP fusion protein selectively detects antigen-dependent T cell activation, which is only induced by saturated calcium signaling. To study the consequences of T cell priming in the GALT, the GALT-emigrating cells will be recovered from efferent lymphatics and their phenotype will be analyzed by next-generation sequencing (NGS). Based on the molecular profiling of the GALT-emigrating cells I will define "encephalitogenic switches that can be therapeutically targeted to prevent the subsequent induction of CNS inflammation.