Mog35 55 peptide

Animal handling, animal care, and experimental work involving them were performed according to the Institutional Animal Care and Use Committee (IACUC) guidelines of the, University of Maryland School of Medicine, Baltimore, MD, USA via protocol # 0616004 approved in June 2016. EAE was induced in C57BL/6 mice (JAX, Bar Harbor, ME), 6–8 week old male, following the method described elsewhere [63 (link),64 (link)]. Briefly, mice were immunized subcutaneously with MOG_35–55 peptide (AnaSpec Inc., Fremont, CA, USA) (200 µg per mouse) in complete Freund’s adjuvant (CFA) followed by concomitant intraperitoneal (i.p.) administration of Pertussis toxin (EMD Millipore Corp., Billerica, MA, USA) on day 0 and day 2. Thereafter, these mice were observed regularly for the development of disease. The severity of EAE was graded on a scale of 0 to 5 as described in detail elsewhere (Figure S1) [64 (link)]: grade 1 = partial or total flaccid paralysis of tail; 2 = hind limb weakness/disrupted righting reflex; 3 = flaccid paralysis in one hind limb; 4 = flaccid paralysis in both hind limbs; and 5 = moribund/dead. The severity of disease was further confirmed by histological examination of CNS, particularly spinal cord after staining of tissue sections by hematoxylin and eosin (H&E) as well as Luxol fast blue staining (data not shown.)

All animal experiments were approved by the St. Luke’s Roosevelt Hospital Center IACUC. Heterozygous fetuin-A knockout mice have been described previously [1 (link)]. Wild-type C57BL/6J mice were purchased from Jackson Laboratory (Bar Harbor, ME). EAE induction with MOG35-55 peptide and 0–13 EAE scale have been described previously [9 (link)]. The adoptive transfer EAE model in C57BL/6J mice was performed as previously described [10 (link)]. Briefly, EAE mice were sacrificed at day 11 and splenocytes were restimulated in culture in RPMI media with 10% FBS, 1% sodium pyruvate, 1% non-essential amino acids, Penicillin/Streptomycin, 20 μg/ml MOG 35–55 peptide (Anaspec), along with 20 ng/ml IL-12 (R&D Systems) and 10 μg/mL anti-IFN-γ (BD Pharmingen) to promote Th17 cell expansion. After 3 days, restimulated splenocytes were washed with PBS and the same number of viable cells (10–30 million cells/mouse) were injected i.p. into naïve recipient mice. Splenocytes were also analyzed by flow cytometry or for gene expression as described below. Levels of IL-17 in the supernatants were measured by ELISA (R&D Systems).

EAE was induced in DBA/1j and C57BL/6 mice with either 50 μg of recombinant rat MOG_1–125,^{41 (link)} emulsified in incomplete Freund's adjuvant (Sigma-Aldrich) supplemented with 3 mg ml⁻¹ of heat-inactivated Mycobacterium tuberculosis H37RA (Difco Laboratories, Oxford, UK), or in C57BL/6 mice with 100 μg of MOG_35–55 peptide (AnaSpec, Fremont, CA, USA) emulsified in incomplete Freund's adjuvant (Sigma-Aldrich) supplemented with 4 mg ml⁻¹ of heat-inactivated Mycobacterium tuberculosis H37RA (Difco Laboratories). The emulsion was injected intradermally at the base of the tail. In some experiments (as indicated in the figure legends), animals were treated with 200 ng of pertussis toxin (Enzo Life Sciences, Farmingdale, NY, USA) via intraperitoneal injection on days 0 and 2. Clinical assessment was performed daily by an observer ‘blinded' to mouse genotype and according to the following criteria: 0, no disease; 1, decreased tail tone; 2, abnormal gait (ataxia) and/or impaired righting reflex (hind limb weakness or partial paralysis); 3, partial hind limb paralysis; 4, complete hind limb paralysis; 5, hind limb paralysis with partial forelimb paralysis; and 6, moribund or dead.

The EAE model was produced following the procedure described previously [16 (link)]. Briefly, 1:1 emulsion of MOG_35–55 peptide (1 mg/ml in PBS) (Anaspec) and complete Freund’s adjuvant (CFA, Sigma) was injected subcutaneously (50 μl) into each flank. In addition, pertussis toxin (PTX, 20 ng) (Biological Laboratories Inc.) was given intravenously (200 μl in PBS) at the time of immunization and again 2 days later. The EAE mice were scored daily based on the disease symptom severity scale (from day 0 to day 20 or 25), as we described previously [21 (link)].

Mice (12- to 16-week old) were immunized s.c. with 50 μg of MOG_35–55 peptide (MEVGWYRSPFSRVVHLYRNGK, AnaSpec) and 500 μg of Mycobacterium tuberculosis (Difco) emulsified in incomplete Freund's adjuvant (IFA) (Difco). In addition, the animals received 200 ng of pertussis toxin (List Biological Laboratories) i.p. on days 0 and 2. The severity of EAE was monitored and graded on a clinical score of 0 to 5: 0=no clinical signs; 1=limp tail; 2=paraparesis (weakness, incomplete paralysis of one or two hind limbs); 3=paraplegia (complete paralysis of two hind limbs); 4=paraplegia with forelimb weakness or paralysis; 5=moribund or death. The statistical significance was analysed by nonparametric Mann–Whitney's U-test.

Naive CD4⁺ T cell isolation kit was purchased from Miltenyi Biotec (Cologne, Germany). Anti-CD3/CD28 coated beads were purchased from Gibco (New York, USA). RhIL-2, rhTGF-β, rmIL-6, and rmIL-12 were purchased from R&D Systems (Minneapolis, MN). Anti-TNFR1 PE, anti-TNFR2 PE, anti-Thy1.1 PercP/Cy5.5, anti-LAP APC, anti-PD-1 APC, anti-Nrp-1APC anti-GITR PE, anti- Ki-67 PE, anti-CD4 PercP/Cy5.5, anti-IFN-γ PE, anti-IL-17A APC, rmTNFα, anti-IL-4 antibody, anti-IFN-γ antibody, and CFSE were purchased from Biolegend (San Diego, CA). PMA, ionomycin, and cyclosporin A (CsA) were purchased from Calbiochem-EMD Millipore (Dormstadt, Germany). CFA was purchased from Sigma-Aldrich. Incomplete Freund's Adjuvant (IFA, Difco, MI, USA), killed Mycobacterium tuberculosis (strain H37Ra; Difco), and myelin oligodendrocyte glycoprotein (MOG, 35–55 peptide, (AnaSpec Inc, Fremont, CA) were also purchased.