Figure 5: CD39 signalling is essential for PSA-mediated immunoregulation during murine EAE.

WT and CD39^KO C57BL/6 mice were treated with PSA as in Fig. 1a and induced sub-optimal active EAE. (a) EAE was monitored till day 25. Mean±s.e.m. scores typical of two independent experiments are shown (n=8, per group). *P<0.05; **P<0.01; ***P<0.001. (b) Mean±s.e.m. cumulative scores were calculated from (a). (c) H&E and Luxol fast blue (LFB) staining was performed same as in Fig. 1c. Depicted are typical pictures (magnification × 40) from EAE-induced PSA-treated WT or CD39^KO mice. All samples were collected (n=4 sections per mouse, n=3 mice per group) at the peak of disease. Scale bars, 100 μm. (d) Tissues were collected at the onset of EAE and flow cytometric analysis of cells was performed. Mean±s.e.m. frequencies of activated subsets within total CD4 T cells were compared for the indicated sites (n=4). (e,f) Total splenocytes were obtained from WT or CD39^KO mice treated as in a on day 14 of EAE induction and cultured in vitro with PSA, MOG_35–55 peptide, or a combination of both at escalating doses (10, 100 or 200 μg ml⁻¹ of each). Supernatants were collected 48 h after culture. IL-10 (e) and IL-17A (f) were measured by ELISA. Mean±s.e.m. results are combined from two experiments (n=3-4). (g) Rag^KO recipient mice were reconstituted with splenic CD4 T cells from day 14 EAE-induced WT and CD39^KO mice treated with PSA as in a or PBS. Two weeks later, optimal active EAE was introduced into recipients. EAE was monitored till day 21. Mean±s.e.m. scores representative of two experiments are shown (n=6–8, per group). *P<0.05; **P<0.01. (h) Mean±s.e.m. cumulative scores were derived from g. P-value was calculated by Mann–Whitney U-test in a, b and h, by two-tailed Student’s t-test in d, e and f, and by Kruskal–Wallis test in g.