br Experimental Procedures br Author Contributions br Acknow
Acknowledgments We acknowledge Géraldine Le Goff and Mathieu Canales for blood sampling, Cécile Gameiro and Jean-Pierre Aubry for flow cytometry technical assistance, Giuseppe Pantaleo for support, and Britta Engelhardt, Gisella Puga-Young, Nicolo Brembilla, and Matthieu Perreau for scientific discussions. This work was supported by the Swiss National Science Foundation (PP00P3_157476), the Swiss Multiple Sclerosis Society, and the Ernst and Lucie Schmidheiny Foundation. C.P. holds stipendiary professorships of the Swiss National Science Foundation (PP00P3_157476). A.S.C. is supported by the Hirsch Foundation and the Clinical Research Center (Geneva University Hospitals). A.W.S. is employee of Novartis Pharma and holds stock and stock options in his company.
Introduction Epstein-Barr virus-induced G protein-coupled receptor 2 (EBI2, also known as GPR183) was discovered in 1993 in a screen of genes induced by in vitro Epstein-Barr virus (EBV) infection of a Burkitt’s lymphoma cell line (Birkenbach et al., 1993) and identified by sequence similarity as a G protein-coupled receptor (GPCR). EBI2 was shown to have an important role in B cell positioning in the germinal center reaction (Gatto et al., 2009, Pereira et al., 2009), resulting in fewer plasma Nystatin and reduced antibody titers in EBI2-deficient animals. Also, CD4+ conventional dendritic cells (cDCs) are profoundly diminished in the spleen of EBI2 mutant mice (Gatto et al., 2013, Yi and Cyster, 2013). Recently, a role for EBI2 in differentiation of T follicular helper (Tfh) cells was demonstrated. Activated T cells were shown to migrate in an EBI2-dependent manner to the outer T zone to receive inducible T cell co-stimulator (ICOS) stimulation under IL-2 deprived conditions by IL-2 quenching DCs that fosters Tfh differentiation (Li et al., 2016). Furthermore, Suan et al. (2015) showed that Tfh cells in the germinal centers downregulate expression of EBI2. Thus, EBI2 regulates positioning of immune cells in secondary lymphoid organs. Oxysterols were recently identified as natural ligands for EBI2 (Hannedouche et al., 2011, Liu et al., 2011). The most active ligand, 7α,25-dihydroxycholesterol (7α,25-OHC), is generated via sequential hydroxylation of cholesterol by cholesterol-25-hydroxylase (CH25H) and 25-hydroxycholesterol by 7-alpha-hydroxylase (CYP7B1). In naive mice, CH25H is highly expressed in the spleen, whereas CYP7B1 is expressed quite ubiquitously with highest expression in the liver. In addition, both enzymes are expressed in lymphoid stromal cells (Hannedouche et al., 2011, Yi et al., 2012). Contradictory data were published about the role of CH25H in experimental autoimmune encephalomyelitis (EAE) (Chalmin et al., 2015, Reboldi et al., 2014), and no direct contribution of EBI2 in EAE pathogenesis has been described. Expression of EBI2 and its function for migration in vitro in T cells was recently reported (Chalmin et al., 2015, Hannedouche et al., 2011, Liu et al., 2011, Pereira et al., 2009). Pereira et al. (2009) used an EBI2 reporter mouse and found that most CD4+ T cells, but only approximately half of the CD8+ T cells, expressed EBI2. In vitro migration of murine T cell toward 7α,25-OHC was demonstrated previously (Chalmin et al., 2015, Liu et al., 2011). To analyze EBI2 expression and its function in T cells in vivo, we created an EGFP reporter/knockout mouse strain, termed EBI2EGFP. This mouse strain in heterozygous configuration allows for a systematic analysis of the expression of EBI2 in distinct cell types in steady-state and under inflammatory conditions. Among murine and human T cell subsets, EBI2 was highly and uniformly expressed by either naive CD4+ T cells or within the Th17 subset under inflammatory conditions. Its expression was strongly repressed under transforming growth factor β (TGF-β) + interleukin-6 (IL-6)-induced Th17 differentiation conditions but sustained by interleukin-1 beta (IL-1β) and interleukin-23 (IL-23) during T helper cell differentiation. Strikingly, transfer of myelin oligodendrocyte glycoprotein (MOG)-specific EBI2-deficient Th17 cells isolated from homozygous EBI2EGFP/EGFP mice induced EAE with a significantly delayed onset. Moreover, we found that expression of the enzymes CH25H and CYP7B1 changes dramatically during the course of EAE with a strong upregulation detected in the spinal cord (SC) leading to enhanced tissue levels of the EBI2 ligand in the SC in EAE. Additionally, we found that microglia start expressing CH25H early in EAE and that CYP7B1 is expressed in EAE by infiltrating monocytes and lymphocytes. In accordance with our mouse data, EBI2 was highly expressed by human Th17 cells derived from peripheral blood mononuclear cells (PBMCs). Furthermore, macrophages and a subset of T cells in lesions of multiple sclerosis (MS) patients stained positive for EBI2. Therefore, we suggest that EBI2 and its ligand(s) play important roles for efficient and early migration of encephalitogenic CD4+ T cells into the CNS. This mechanism may also be more generally implicated in the infiltration of human lymphocytes into inflamed tissues.