Entry, distribution and function of group 1 and 3 ILCs in the healthy and inflamed CNS.

健康中枢可见浸润性和常驻性ILCs。我们之前的研究表明CNS-ILCs表现出严格的组织区隔性，ILC2s和1组ILCs是脑实质和脉络膜丛内的主要群体。此外，我们证明cns - 1组ILCs包括传统的cNK细胞，ILC1s和一小部分前ilc3。我们的初步数据表明，在围产期的中枢神经系统中发现的rorr γt+ ILC3s似乎参与了硬脑膜淋巴管的发育。有趣的是，成人自身免疫性神经炎症期间，rorγ γt+淋巴组织诱导剂（LTi）细胞增加，这可能暗示三级淋巴组织结构（TLSs）的形成和疾病的慢性。另一方面，ilc1似乎代表了一种组织常驻人群，在生命早期就开始在大脑中播种，并且不会再循环。此外，我们发现在自身免疫性神经炎症和寄生虫感染等病理条件下，ILC1s数量增加并作为细胞因子的来源。因此，我们假设1组ilc可能通过产生关键细胞因子如ifn - γ来调节疾病的发展。为了验证我们的假设，我们将使用Id2CreERT2 x R26REZFP小鼠研究中枢神经系统中ILC的转换，对所有ILC进行谱系追踪，并使用骨髓嵌合小鼠进行外周ILC浸润。此外，我们将通过免疫荧光分析定位沿中枢神经系统区室的ILCs并评估其功能作用。我们将特别关注ILC3s在发育过程中的作用，并将研究它们在硬脑膜淋巴形成和随后转化为前ILC3s中的作用。为此，将通过免疫荧光和PCR分析RorcGFP/GFP、Rag1-/-和RorcGFP/GFPRag1-/-以及淋巴毒素信号敲除（Ltbflox/flox x RORc-Cre和Ltaflox/flox x RORc-Cre）。围产期ilc3将通过scRNAseq进行表征，并在RORgt-FM动物中追踪其可能的可塑性。为了确定CNS-ILC谱及其在EAE期间的变化，我们将对健康和患病大脑的ilc进行CITE-seq，并分析慢性和复发缓解型EAE期间的ilc。此外，在缺乏1组ILCs和ILC3，或ILC3以及前ILC3的小鼠中进行被动EAE，我们将研究这些群体在EAE发展中的作用，再次关注ILC3对TLS形成和疾病慢性的可能影响。此外，在WT、rorcfp /GFP和Id2CreERT2 x Rorcfloxed动物中，DTH-EAE模型会产生突出的脑膜TLS。最后，我们将在一项包括多发性硬化症患者和hc的初步研究中，利用脑样本和pbmc来证明在动物模型中获得的数据的转化价值。

Infiltrated and resident ILCs are found in the healthy CNS. We previously showed that CNS-ILCs display a strict tissue compartmentalization and that ILC2s and group 1 ILCs are the main populations within the brain parenchyma and choroid plexus. Further, we demonstrated that CNS-group 1 ILCs include conventional cNK cells, ILC1s and a fraction of ex-ILC3. Our preliminary data indicate that RORγt+ ILC3s, which are found in the CNS perinatally, appear to be involved in the development of dura lymphatic vessels. Interestingly, RORγt+ lymphoid tissue inducer (LTi) cells increase in adults during autoimmune neuroinflammation, which may suggest an implication in the formation of tertiary lymphoid structures (TLSs) and disease chronicity. On the other hand, ILC1s appear to represent a tissue-resident population that starts seeding the brain during early life and do not re-circulate. Moreover, we showed that ILC1s increase in numbers and act as source of cytokines during pathological conditions such as autoimmune neuroinflammation and parasite infection. We hypothesize therefore that group 1 ILCs may modulate disease development by production of key cytokines such as IFN-gamma. To test our hypotheses, we will investigate the turn-over of ILCs in CNS using Id2CreERT2 x R26REZFP mice, to lineage trace all ILCs, as well as bone-marrow chimeric mice to access peripheral ILC infiltration. Moreover, we will localize the ILCs along CNS compartments by immunofluorescence analysis and assess their functional roles. We will pay special attention on the implication of ILC3s during development and will investigate their implication in dura lymphatic formation and subsequent conversion into ex-ILC3s. To do so, RorcGFP/GFP, Rag1-/- and RorcGFP/GFPRag1-/- together with lymphotoxin signaling knockouts (Ltbflox/flox x RORc-Cre and Ltaflox/flox x RORc-Cre) will be analyzed by immunofluorescence and PCR. Perinatal ILC3s will be characterized by scRNAseq and track for possible plasticity in RORgt-FM animals. To determine the CNS-ILC profile and its changes during EAE, CITE-seq will be conducted on ILCs from healthy and sick brains, and ILCs will be analyzed during chronic and relapsing-remitting EAE. Further, performing passive EAE in mice lacking either group 1 ILCs and ILC3, or ILC3 as well as ex-ILC3 we will investigate the role of these populations in EAE development, focusing again on possible ILC3 implication on TLS formation and disease chronicity. Additionally, the DTH-EAE model, that develops prominent meningeal TLS will be induced in WT, RorcGFP/GFP and Id2CreERT2 x Rorcfloxed animals. Finally, we will prove the translational value of the data obtain in the animal model using brain samples and PBMCs in the context of a pilot study including MS patients and HCs.