a) A representative mass spectra of S1P_{1 342–352} peptide depicting mass/charge (m/z) of identified amino-terminal (b, red) and carboxyl-terminal (y, blue) fragmented ions. Phosphorylation of S351 was assigned by the presence or loss of phosphate group among identified ions. Four unique S1P_{1 342–352} peptides were identified from two independent mass spectrometric analyses. Immunohistochemistry of formalin-fixed, paraffin-embedded sections from a MS brain lesion labeled with antibodies against S1P₁ (anti-S1P₁)(b and d), CD3 (anti-CD3)(c) and glia fibrillary acidic protein (GFAP)(anti-GFAP) (e). Perivascular immune cells (black arrow), endothelial cells (green triangle) and astrocytes (green arrowheads) expressed S1P₁. Scale bar = 50 μm. Representative data from analysis of three MS brain samples.

(a) Mean clinical score (± S. E. M.) of MOG_35–55–immunized S1P₁(S5A)(mice carrying phosphorylation defective S1pr1 gene) and WT (C57BL/6J) EAE mice (females, 8–9 weeks old). Immune cell proliferation (measured by ³H[thymidine] incorporation) (b) and cytokine expression (measured by ELISA) (c) of ex vivo recall assay from MOG_35–55-immunized S1P₁(S5A)and WT EAE splenocytes (day 8 post-immunization). MOG; myelin oligodendrocyte glycoprotein, c.p.m.; counts per minute. (d) Photomicrograph (Luxol Fast Blue, Hematoxylin stain) and (e) quantification of CNS inflammation in WT (top) and S1P₁ (S5A) (bottom) EAE mice from (a). *p<0.05, **p<0.01, Mann-Whitney U-test (a) and Student’s t-test (b, c and e). n=9–10/arm (a) and 3–5/arm (b–e). These experiments were repeated 3 times.

(a) Mean clinical scores ((±S. E. M.) of Rag1^−/− adoptive transfer recipients of encephalitogenic cells from MOG_35–55-immunized S1P₁(S5A)and WT (C57BL/6J) EAE mice. Donors, n=10, females, 8–9 weeks old; recipients, n=5, females, 5–6 weeks old. (b) Quantification of total CNS infiltrating immune cells, (c) CD4⁺ and (d, e) CD4⁺IL-17⁺ cells from CNS of S1P₁(S5A)and WT EAE mice (at peak disease, days 13–15). Immunoblot depicting p-STAT3 (f), p-p38, p38 and IκBα (h) expression in splenocytes of S1Ps₁(S5A) and WT EAE mice (day8, post-immunization). (g) Quantification of relative p-STAT3 expression [form (f)]. (b-g) n=3–5 mice/arm. *p<0.05, Mann-Whitney U-test (a) and Student’s t-test (b–d). These experiments were repeated twice (a) and at least 3 times (b–g).

(a) Immunoblot depicting p-STAT3 expression in WT and S5A [S1P₁(S5A)] EAE splenocytes (day 8 post-immunization) following in vitro activation with S1P (100nM) or W146 (S1P₁ antagonist) (20nM). (b) Quantification of normalized p-STAT3 expression from (a). (c) Cytokine expression in splenocyte cultures of MOG_35–55-immunized WT EAE mice treated in vivo with S1P lyase inhibitor, THI (6.25mg/kg, daily intraperitoneal injections). IL-17 expression in CD3⁺ cells (activated with anti-CD3/anti-CD28) from (d) S1P₁(S5A) naive mice treated in vitro with W146 (0–10nM), (e) WT naïve mice, treated in vitro with scrambled control (Ctrl) or S1pr1-specific (S1pr1) siRNA, or (h) naïve S1pr1^+/+(WT) or S1pr1^−/− (S1pr1^f/fRosa26-CreER^T2) mice. (f and g) Flow cytometric analysis of S1P₁ expression following treatment with S1pr1 or Ctrl siRNA treatment. *p<0.05, **p<0.01, Student’s t-test. c–e and h; analyzed by ELISA. (a, b, d–g) were performed 3–5 times, (c and h) twice. These experiments were performed with n=3–5 mice/arm. S1P; sphingosine-1-phosphate, W146; S1P₁-specific antagonist, THI; 2-Acetyl-5-tetrahydroxybutyl Imidazole.

(a) IL-17 expression in culture supernatants of mixed lymphocyte cultures [CD3⁺ cells from MOG_35–55-immunized S1P₁(S5A) (S5A T) or C57BL/6J WT (WT T) EAE mice co-cultured with naïve, irradiated antigen presenting cells (APCs)from S1P₁(S5A) (S5A APC) or WT (WT APC) in the presence of MOG_35–55 peptide] measured by ELISA. (b) IL-6 expression in an ex vivo recall assay of splenocyte cultures from MOG_35–55-imunized S5A [S1P₁(S5A)]and WT EAE mice (day 8 post-immunization). (c) Immunoblot depicting p-STAT3 expression in splenocytes from WT and S1P₁(S5A) EAE mice (day 8 post-immunization) treated in vitro with recombinant IL-6 (IL-6) or anti-IL-6 (α-IL-6) (20ng/ml, respectively). (d) Quantification of normalized p-STAT3 expression from (c). IL-17 expression in the culture supernatants of splenocyte cultures from MOG_35–55-immunized S1P₁(S5A) mice treated in vitro with Stattic (STAT3 inhibitor) (0–9 μM) (e) or Jak inhibitor (0–5nM)(f). **p<0.01, *p<0.05 by Students t-test. Experiments were performed 3–5 times with n=3–5 mice.