BMMCs, Tregs, and Th17 cells colocalize in EAE priming and effector sites. EAE was induced in C57BL/6 mice by MOG_35-55 immunization. Animals were killed after 10 days during the acute phase. Lymph nodes draining the MOG_35-55 injection site (DLN) and spinal cords (SC) were analyzed by flow cytometry. (A) Representative dot plots showing IL-6–producing cells among FcϵRI⁺c-kit⁺ MCs (top panels) and IL-17 versus Foxp3 expression in gated CD4+ T cells (bottom panels) in DLN of immunized mice. Histogram shows TGF-β expression in gated CD4⁺Foxp3⁺ Tregs (solid line) overlaid to isotype staining (filled area). (B) Same analysis as in panel A, performed on SC samples. (C) Formalin-fixed, paraffin-embedded sections obtained from DLN and SC were evaluated by immunohistochemistry for IL-6 (left panels) and IL-17 (right panels) expression (original magnification ×200). (D-E) In DLN, expression of Foxp3 (brown) versus IL-6 (red, D) or versus IL-17 (red, E) was analyzed. Insets show (at higher magnification) the proximity of Tregs with Th17 cells or IL-6⁺ cells (original magnification ×200). (F) Double fluorescence confocal microscopy for IL-6 (green) and FcϵRI (red) reveals a few IL6⁺ FcϵRI⁺ MCs (white arrows) scattered among IL-6⁺ FcϵRI⁻ lymphocytes. The inset shows (at higher magnification) the interaction between MCs and IL-6–producing T cells (original magnification ×200). (G) Double immunohistochemistry for OX40 (blue) and IL-17 (red) shows IL-17⁺ cells (cytoplasmatic reactivity) intermingling with small clusters of OX40⁺ lymphocytes (membrane reactivity). IL-17–expressing cells also showed some degree of reactivity to anti-OX40 on the cell surface (arrows) Original magnification ×200. Data are from 2 independent immunization experiments, each including at least 7 mice.