MOG antigen recall assay

For T cell priming analysis, the spleens and inguinal lymph nodes of immunized mice were removed 10 days after immunization, and single-cell suspensions were prepared. For peak EAE analysis, mice were transcardially perfused with 0.9% NaCl solution, and the spinal cords were removed and digested with collagenase II (Thermo Fisher Scientific) and DNase I (Roche), before being separated on 30/37/70% Percoll gradient. Single-cell suspensions of all organs were in vitro cultured in the presence of MOG peptide (20 μg/ml). After 2 hours, cultures were supplemented with brefeldin A (1 μg/ml; Merck) to block the secretion of cytokines. After additional 4 hours of culture, cells were harvested and stained for flow cytometry analysis. T cell expression of CD154 was assessed as marker for recent activation, thereby serving as surrogate marker for MOG antigen specificity. CD154^high T cells were then further analyzed for cytokine expression.