Role of NK cell subsets in an animal model of relapsing-remittingmultiple sclerosis - PROJECT SUMMARY/ABSTRACT The goal of our lab is to investigate immune regulation for the treatment and resolution/repair of central nervous system (CNS) inflammatory diseases. During neuroinflammation the concerted actions of the CNS and the immune system affect the pathological responses mediated by infiltrating and resident immune cells. In particular, during multiple sclerosis (MS), myeloid and lymphoid cells are found in abundance in the CNS. While anti-CD20 treatment opened the mind of scientists in targeting non-T cells in MS, other immune cells remain severely understudied for their contribution in MS pathology. Recently, innate cells have gained a lot attention in MS, and in particular the natural killer (NK) cells. NK cells are known for their anti-viral/tumor activity, as well as their regulation of adaptive immunity. NK cells are typically divided into immature NK cells (iNK), denoted as CD56bright and mature NK cells (mNK), denoted as CD56dim. In MS, the role of NK cells is inconclusive. NK cells are found in high number in the cerebrospinal fluid (CSF) of MS patients, and depletion of NK cells at peak of disease in the animal model of MS, experimental autoimmune encephalomyelitis (EAE), leads to attenuated disease severity. These data suggest that NK cells are contributing of MS pathology. However, other studies have shown potential beneficial roles of NK cells in MS. Indeed, deletion of NK cells before EAE disease induction leads to a worsen of disease. Moreover, MS patients treated with Daclizumab (IL-2Rα blocker) showed a decrease of brain lesions which correlated with an increase of CD56bright iNK in blood. Together, these data suggest that different NK subsets might play different roles in MS. However, an important hurdle to study NK cell subsets in EAE is the absence of CD56 in mice. Recently, multiple groups were able to overcome this handicap, and identify markers to distinguish mNKs (NKp46+ CD49b+ CD27- CD11b+) from iNKs (NKp46+ CD49b+ CD27+ CD11b-). Preliminary, we are showing that both subsets exist in the spleen and CNS in the relapsing-remitting model of EAE (RR-EAE). We are also showing robust expression of GM-CSF and IL-17 by mNK in the CNS of EAE mice, while iNKs are showing minimal expression of both cytokines. Based on published data as well as our preliminary data, we hypothesized that the mNK subset contributes to EAE pathogenesis while the iNK subset ameliorates it. In our first aim, we plan to characterize NK cell subpopulations in the CNS of RR- EAE mice at different stages of the disease using single cell RNA sequencing. Using publicly available human MS scRNAseq datasets on blood, CSF and RRMS lesions, we plan to overlay our murine scRNAseq to evaluate if NK subsets found in EAE are also present in MS. In the second aim of our study, we will evaluate the functions of iNKs and mNKs in the CNS by using an in vitro co-culture assay between CNS slice culture and NK subsets, as well as carrying out in vivo experiments by transferring CFSE-labelled NK subsets into EAE mice. We believe that understanding which NK subsets are present at different points of disease, along with whether these cells are regulatory or pathogenic, will gain more insight on potential targets that could benefit future therapeutics.
