Pertussis toxin

Primary progressive EAE model for C57BL/6 mice was established following the published protocol [22 (link)]. The mice were immunized subcutaneously on the back with 0.2 mL of myelin oligodendrocyte glycoprotein (MOG) 35–55 peptide (MEVGWYRSPFSRVVHLYRNGK, HPLC-purity: >95%) (ChinaPeptides, Shanghai, China) emulsified in CFA (Chondrex, Redmond, WA, USA) containing 4 mg/mL Mycobacterium tuberculosis H37Ra. These injections were distributed over the following three sites: one along the midline of the back between the shoulders and two on either side of the midline on the lower back. The final dose of MOG 35–55 and Mycobacterium tuberculosis H37Ra was 200 μg and 400 μg per mouse. Each mouse received an additional 400 ng of pertussis toxin (R&D Systems, MN, USA) by intraperitoneal injection of 200 μL PBS on day 0 and day 2 postimmunization. Clinical scores were calculated blindly by two researchers daily according to a 0–5 scale as follows [23 (link)]: 1, limp tail or waddling gait with tail tonicity; 2, waddling gait with limp tail (ataxia); 2.5, ataxia with partial limb paralysis; 3, full paralysis of 1 limb; 3.5, full paralysis of one limb with partial paralysis of the second limb; 4, full paralysis of two limbs; 4.5, moribund; and 5, death.

For removing sialic acid residues, cell-sorted subsets of CD8⁺ T cells were treated with 0.1 units of sialidase (from Vibrio cholera, Sigma-Aldrich) in 1 ml RPMI 1640 (Life Technologies) containing 10% FBS (Gemini Bio-Products) and 2 mM L-glutamine for 2.5 hr at 37°C. For inhibiting G_i/o proteins, CD8⁺ T cells were pre-incubated with pertussis toxin (1 μg/ml (R and D Systems) in RPMI 1640 medium containing 10% FBS and 10 mM HEPES for 3 hr at 37°C. For blocking CCR2 and CCR5, pre-incubation was with BMS CCR2 22 (2 μM (Tocris, Minneapolis, MN) or Maraviroc (10 μM (Tocris), respectively, for 30 min at 37°C and inhibitors were left in the medium throughout the assay. For neutralizing CCL20, HUVEC monolayers in flow chambers were pre-treated for 2 hr at 37°C with 20 μg/ml anti-human CCL20/MIP-3α antibody (c67310; R and D Systems), and antibody was maintained at 10 ng/ml throughout the assay.

Primary melanocytes were extracted from fresh discarded human foreskin and surgical specimens as described previously described with some modifications detailed as follows. After overnight incubation in Dispase, the epidermis was separated from the dermis and treated with trypsin for 10 min. Cells were pelleted and plated on selective MC Medium 254 (Invitrogen, Carlsbad, CA) with Human Melanocyte Growth Supplement, and 1% penicillin and streptomycin. Lightly pigmented primary melanocytes were utilized for experiments assaying estrogen and GPER agonist effects, and heavily pigmented primary melanocytes were utilized for experiments assaying progesterone and PAQR7 agonist effects in melanin production. Female iPS-derived human melanocytes were a gift from Meenhard Herlyn (Wistar Institute, Philadelphia, PA, USA). Progesterone (P8783), 17β-Estradiol (E8875), and αMSH (M4135) were purchased from Sigma-Aldrich (St. Louis, MO). G-1 (10008933), G-15 (14673) and G-36 (14397) were purchased from Cayman Chemical (Ann Arbor, MI). CH2P4 (2085) was purchased from Axon Medchem (Groningen, Netherlands). Pertussis toxin was purchased from R&D systems (Minneapolis, MN). These compounds were diluted to working stock solutions in Medium 254.