Pertussis toxin

The EAE model was constructed as previously reported
¹⁹ (link): MOG_35‐55 (GL Biochem Ltd., Shanghai, China) was dissolved in PBS and then mixed with complete Freund's adjuvant (8 mg/mL) (strain H37RA; Difco, USA) at a 1:1 volume ratio. Female mice were injected with the emulsified solution at four sites. Two intraperitoneal injections of pertussis toxin (200 ng/mouse) (Millipore, Billerica, MA, USA) were given at 0 and 2 days after immunization. Mice were clinically scored by two researchers following the double‐blind principle and recorded as follows: tail weakness, 0.5; tail paralysis, 1; faltering gait with hind limb weakness, 2; bilateral hind limb paralysis, 3; forelimb weakness, 4; and near death, 5. Mice were administered an anti‐CD22 monoclonal antibody (mAb) (CY34.1, BioXcell) or an IgG control via the tail vein at a dose of 400 μg/20 g from Day 4 every 2 days.

On day 0, 10- to 12-week-old mice were immunized subcutaneously with 200 µg of MOG 33–35 peptide (CHINESE PEPTIDE) emulsified with CFA (Sigma-Aldrich) containing 5 mg/ml heat-killed Mycobacterium tuberculosis (BD Bioscience). The mice then received an intraperitoneal injection of 200 ng of pertussis toxin (Millipore) 2 and 26 h after immunization. The mice were monitored daily and scored for clinical signs of disease according to the following criteria: 0 = no clinical symptoms; 1 = limp tail; 2 = weakness in hind limbs; 3 = complete paralysis of hind limbs; 4 = complete hind limb and partial front limb paralysis; and 5 = moribund state^{45 (link)}.

The littermate control, Lck-Cre × Perp^fl/+ and Lck-Cre × Perp^fl/fl mice at 10 weeks of age were immunized with 200 µg MOG_35–55 (Chinese Peptide, Shanghai, China) emulsified in 50% complete Freud’s adjuvant (CFA) containing 4 mg/ml heat-killed Mycobacterium tuberculosis (Chondrex, Redmond, WA, USA) at their dorsal flanks. Individual mice were injected intraperitoneally with pertussis toxin (200 ng/mouse, Millipore) on day 0 and 2. The development and severity of EAE in individual mice were scored daily using the following score system: 0, healthy; 1, tail paralyzed; 2, no coordinated movement; hind limb paresis; 3, both hind limbs paralyzed; 4, forelimbs paralyzed; and 5, moribund state.

Mice were randomly divided into NC, NC+BMSCs, EAE and EAE+BMSCs groups, with 15 mice in each group. Mice of EAE and EAE+BMSCs groups performed EAE modeling. In short, Mice were immunized subcutaneously with 200 μg MOG_35-55 (peptide sequence: Met-Glu-Val-Gly-Trp-Tyr-Arg-Ser-Pro-Phe-Ser-ArgVal-Val-His-Leu-Tyr-Arg-Asn-Gly-Lys; MedChem Express, US) peptide emulsified in complete Freund’s adjuvant (Sigma, USA) containing Mycobacterium tuberculosis H37Ra (BD Biosciences, USA) on 0 day. Then mice were injected intravenously with 300 ng Pertussis Toxin (Millipore, USA) both immediately after immunization and 2 days later. In 8 days post-EAE induction, BMSCs were injected into the lateral ventricle (Start with the dura mater; before and after the halogen: 0.6 mm; opening: 1.5 mm; depth: 1.7 mm) of mice of NC+BMSCs and EAE+BMSCs groups by brain stereotactic technology.

EAE induction was performed as described previously (Arima et al., 2012 (link), 2015 (link); Ogura et al., 2008 (link)). Briefly, C57BL/6 mice were injected with a MOG(35-55) peptide (Sigma-Aldrich, Tokyo) in complete Freund's adjuvant (Sigma-Aldrich) at the base of the tail on day 0 followed by intravenous injection of pertussis toxin (Sigma-Aldrich) on days 0, 2, and 7. On day 9, CD4⁺ T cells from the resulting mice were sorted using anti-CD4 microbeads (Miltenyi Biotec, Tokyo). The resulting CD4⁺ T cell-enriched population (4 × 10⁶ cells) was cocultured with rIL-23 (10 ng/ml; R&D Systems, Minneapolis, MN) in the presence of MOG peptide-pulsed irradiated splenocytes (1 × 10⁷ cells) for 2 days. anti-CD4 microbeads were used to enrich CD4+ T cells. These pathogenic CD4+ T cells (1.5 × 10⁷ cells) were then injected intravenously into wild type mice. Clinical scores were measured as described previously (Arima et al., 2012 (link), 2015 (link); Ogura et al., 2008 (link)). As examples of non-CNS antigens, OVA(323-339) peptide (Sigma-Aldrich, Tokyo) and human IRBP (1-20) peptide (Sigma-Aldrich, Tokyo) were used. Except for the peptides, OVA- and IRBP-specific CD4+ T cells were generated and transferred in the same way as MOG-pathogenic CD4+ T cells.