Mog35 55

EAE were induced as described previously19 (link). Mice were subcutaneously immunized with 300 μg mouse myelin oligodendrocyte glycoprotein 35–55 peptide (MOG_35–55, MEVGWYRSPFSRVVHLYRNGK) in Freund’s complete adjuvant (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 3 mg/ml of Tuberculosis H37Ra (BD Difco, NJ, USA). In addition, 750 ng Pertussis toxin (Enzo Life Sciences, NY, USA) was injected intraperitoneally on days 0 and 2 p.i. MOG_35–55 was produced in an automatic synthesizer (CL. Bio-Scientific., Xi’an, China). Purity of the peptide was > 95% as determined by HPLC.

C57BL/6J mice were obtained from the Animal Resource Centre (Perth, Australia) and housed under standard conditions with food and water ad libitum. All procedures were approved by the institutional Animal Ethics Committee and performed strictly in accordance with regulations set by the National Health and Medical Research Council of Australia. Only female mice aged 12–16 weeks were used (32 (link)). EAE was performed as previously described (36 (link)). Briefly, mice received 200 µg of peptide 35-55 of myelin oligodendrocyte glycoprotein (MOG_35-55), emulsified in complete Freund’s adjuvant (Sigma-Aldrich, St. Louis, MO) supplemented with 4 mg/ml of heat inactivated Mycobacterium tuberculosis (Becton Dickinson, Franklin Lakes, NJ) and administered subcutaneously. This was immediately followed by an intraperitoneal injection of 350 ng of Bordetella pertussis toxin (Sigma-Aldrich), which was repeated 48 hours later. Control groups included sham where MOG_33–55 was substituted with phosphate buffered saline (PBS, 0.01 M phosphate, 15 mM NaCl, pH 7.4) and normal mice. Clinical progression was followed by daily weighing and visual assessment of ambulatory difficulties, scored as follows: 0 = no symptoms, 1= limp tail, 2 = hind limb weakness, 3 = hind limb paralysis, 4 = ascending paralysis, and 5 = moribund (36 (link)). The experimental design is summarized in Supplementary Figure 1.

Six to eight-week old C57Bl/6 female mice were obtained from Harlan, Italy Srl. The mice were housed in macrocolon cages on a 12 hr light/dark cycle at 23 °C, with ad libitum access to food and water. Adequate measures were taken to minimize pain and discomfort. Mice were immunized subcutaneously in the flanks and at the base of the tail with a total of 200 μg of MOG_35–55 (synthesized by EspiKem Srl, Università di Firenze) per animal emulsified in complete Freund adjuvant (Sigma-Aldrich) supplemented with 4 mg/ml of Mycobacteriun tibercolosis (strain H37Ra; Difco Laboratories). Immediately thereafter, and 48 hr later, the mice received an intraperitoneal injection of 500 ng pertussis toxin (Sigma-Aldrich) in 100 μl of phosphate buffer saline (PBS). The animal were examined daily for weight loss and disability, and were clinically graded by investigators blind to group identity, as follows: zero indicate no signs, 0.5 partial loss of tail tonicity, 1 paralyzed tail, 2 ataxia and difficulty in righting, 3 paralysis of the hind limbs and/or paresis of the forelimbs, 4 tetraparalysis, 5 moribund or death.

EAE was induced by subcutaneous immunization with 200 µg myelin oligodendrocyte glycoprotein peptide 35–55 (MOG_35-55) (Sigma, ST Louis, MO) in CFA emulsion, containing 2.5 mg/ml of heat killed, pulverized Mycobacterium tuberculosis strain H37RA. The mice also received two doses of 0.3 µg Bordetella pertussis toxin (Sigma, St. Louis, MO) on day 0, and day 2 post-immunization by intraperitoneal (i.p.) injection in 100 µl of RPMI 1640 medium containing 0.1% normal mouse serum. The control (cd19^+/CREstat3^+/+) and CD19-STAT3KO (cd19^+/CREstat3^−/−) (n = 6) were euthanized 17 days post-immunization. The mice were monitored and disease severity was assessed daily by a masked observer. Clinical signs of EAE were graded according to the following scale: 0, No clinical symptoms; 1, clumsiness, incontinence or atonic bladder, flaccid tail; 2, mild paraparesis (trouble initiating movement); 3, moderate paraparesis (hind limb weakness); 4, complete front and hind limb paralysis; 5, moribund state^{31 (link)}. Spinal cord and brain were harvested 17 days post-immunization and infiltrated lymphocytes and other immune cells were isolated by collagenase digestion followed by Percoll-gradient centrifugation and subsequent analysis by FACS and intracellular cytokine staining.

The EAE model was performed in the “Salari Institute of Cognitive and Behavioural Disorders (SICBD)”. The animal model was induced by injection of MOG35-55 (MEVGWYRSPFSRVVHLYRNGK) as is previously described^{51 (link)}. Briefly, Under the anaesthesia caused by ketamine hydrochloride (50 mg kg − 1; Alfasan, Woerden-Holland) plus Xylazine (5 mg kg⁻¹; Alfasan, Woerden-Holland), mice were immunized with 300 µg of myelin oligodendrocytes glycoprotein (MOG35-55; Sigma-Aldrich, United States) dissolved in phosphate-buffered saline (PBS) and emulsified with an equal volume of complete Freund adjuvant (CFA; 400 µg of Mycobacterium tuberculosis; Sigma Co, USA). Additionally, 300 ng of pertussis toxin (List Biological Labs, Campbell, CA, USA) was injected intraperitoneally, into all animals, on days 0 and 2.