Adoptive Transfer EAE in Mice

Mice were immunized s.c. with 100 µg of the MOG_35–55 peptide (MEVGWYRSP-FSRVVHLYRNGK; Biosynthesis) in CFA (Difco) at four sites over the flanks. For active immunization, mice were also administered 300 ng inactivated Bordetella pertussis toxin i.p. on days 0 and 2. For adoptive transfer EAE, inguinal, axial, and brachial dLNs were harvested from donor mice 10–14 days post-immunization, pooled, homogenized, and passed through a 70 µm strainer (Fisher Scientific). Cells were cultured under Th17-polarizing conditions: MOG_35–55 [50 µg/ml], rmIL-23 [8 ng/ml], rmIL-1α [10 ng/ml], and α-IFNγ [10 µg/ml]. Following 96 hours of culture, CD4⁺ T cells were purified by positive selection using L3T4 magnetic microbeads (Miltenyi Biotec). 3–6×10⁶ CD4⁺ T cells were injected i.p. to naïve recipients. The recipient mice were observed daily for signs of EAE, and rated for degree of disability using a 5 point scale, as previously described (3 (link)).