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

• Applicant: Professorin Dr. Carmen Infante Duarte
• Subject Area: Molecular and Cellular Neurology and Neuropathology ; Immunology ; Clinical Immunology and Allergology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 534335032

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.

• DFG Programme: Research Grants