Induction of experimental autoimmune encephalomyelitis (EAE)

Active EAE in C57BL/6 J mice was induced, as described before⁵⁵, by subcutaneous four point immunization with 200 µg MOG_35-55 peptide, which was either from Hook Kit EK 2210 according to manufacturer’s instructions or self-prepared. Additionally, 800 mg H37RA (Difco) emulsified in Complete Freund’s Adjuvant was given, followed by two applications of 200 ng pertussis toxin in PBS injected intraperitoneally at the time of immunization. Active EAE in SJL/J mice was induced similarly, using 250 µg PLP_139-151 peptide (Hooke Laboratories) instead of the MOG peptide. For passive EAE, 5 × 10⁶ B6.2D2 Tfh-skewed cells were injected intravenously into B6.Rag1^−/− mice. Clinical signs of EAE were monitored daily and converted into clinical scores as described before⁵⁶: 0: no detectable signs of EAE; 0.5: tail weakness; 1: complete tail paralysis; 2: partial hind limb paralysis; 2.5: unilateral complete hind limb paralysis; 3: complete bilateral hind limb paralysis; 3.5: complete hind limb paralysis and partial forelimb paralysis; 4: total paralysis of forelimbs and hind limbs; and 5: death.

Mice were anesthetized with 1.5% ketamine solution. The spleen was separated before perfusion and placed in RPMI media. In order to eliminate blood cells from the organs of interest, perfusion was performed by cutting the atrium and the vascular circulation was rinsed with a minimum of 20 ml PBS. Brain and spinal cord (CNS) were isolated in IMDM. The upper part of the skull was removed and placed in 5 ml PBS with 2% FCS. The small intestine was isolated, then the Peyer’s patches were cut off and collected in 5 ml washing media (RPMI media).