Pentamidine treatment enhances a spinal cord T cell regulatory phenotype. Seventeen days post-EAE induction, spinal cord mononuclear cells were stimulated ex vivo with PMA/ionomycin in the presence of protein secretion inhibitors and the expression of IFN-γ, IL-17A and Foxp3 was evaluated by intracellular flow cytometry. (A) Representative contour plots, (B) percentage of positive cells within parent population and (C) Th/Treg ratio are shown. Sorting strategy is described in Supplementary Fig. 6. At the same timepoint, cells from cervical lymph nodes were stimulated ex vivo with MOG_35–55 and (D) IFN-γ, (E) IL-17A and (F) IL-10 were quantified in the culture supernatants after 72 h by ELISA. Graph bars represent the ratio between stimulated and non-stimulated conditions. Unpaired Mann–Whitney t-test was used for statistical significance (^#P < 0.05, and ^##P < 0.01 versus EAE) with n = 5–7 animals per group.