Induction of Adoptive Transfer Experimental Autoimmune Encephalitis (EAE)

Adoptive transfer EAE was induced as described by Miller and Karpus (13). Briefly, myelin oligodendrocyte glycoprotein (MOG) reactive T cells were primed in donor C57BL/6 mice by two sub-cutaneous injections of MOG_35–55-peptide emulsified in complete Freud’s Adjuvant (Sigma). Intraperitoneal injection of 200 ng Pertussis Toxin (Sigma) was used to boost lymphocyte priming. In order to study the effect of tolerogenic molecules, MOG_35–55 peptide-pulsed DCs were intravenously injected 1 day before and 1 day after the immunization. Twelve days post immunization, inguinal, brachial, and axillary lymph nodes were isolated by digestion in 1 mg/ml Collagenase D (Roche) for 20 min at 37°C. Single cells suspension was prepared using a 40-µm cell strainer (Falcon). Lymph node cells were expanded in vitro for 3 days in the presence of MOG_35–55-pulsed DCs and 0.5 ng/ml recombinant IL-12p70 (eBioscience). Then, 5 × 10⁶ MOG-specific T cells were then transferred to C57BL/6 recipient mice by i.p. injection. Mice were i.v. injected with MOG_35–55-pulsed DCs 1 day after T cell transfer. Clinical EAE symptoms were scored daily in a blinded manner: 0—no obvious changes in motor functions; 1—decreased tail tonus; 2—abnormal gait (ataxia and/or impaired righting reflex); 3—partial hind limb paralysis; 4—complete hind limb paralysis; and 5—complete hind and fore limb paralysis.