Plp139 151

All experimental protocols were approved by the UCLA Animal Protection Committee and carried out in compliance with the ARRIVE guidelines and all relevant guidelines and regulations were followed for the experiment. Nine weeks old female C57BL/6 or SJL mice were obtained from the Jackson Laboratory and housed in a specific pathogen-free facility with free access to food and water. C57BL/6 mice were immunized subcutaneously with MOG_35-55 (200 µg, > 95% purity, GenScript)) in 50% IFA containing Mycobacterium tuberculosis H37R (5 mg/ml, Difco) in multiple sites near the base of their tail on day 1 and injected intraperitoneally with pertussis toxin (200 ng/mouse) on day 0 and 2. Individual SJL mice were immunized with PLP_139-151 (100 µg/mouse, > 95% purity, GenScript) using a similar protocol to that described for C57BL/6 mice. The mice were monitored for EAE onset daily: 0, no disease; 1, limp tail; 2, hind limb weakness; 3, complete hind limb paralysis; 4, quadriplegia; and 5, death. Mice that were in between the clear-cut gradations were scored intermediate in increments of 0.5. When the mice developed EAE with a score of 1 at 10–13 days post-immunization, they were randomized to receive plain water or water containing homotaurine (0.25 mg/ml) or GABA (6 mg/ml).

Peptide encapsulating nanoparticles were fabricated with the double emulsion method⁴⁶ . Briefly, 150 µL of antigen dissolved in PBS (50 mg/mL PLP_139–151 or OVA_323–339 [Genscript]) was added to 2 mL of 20% w/v poly(lactide-co-glycolide) (inherent viscosity: 0.17 dL/g, Lactel). This solution was sonicated for 30 s before the addition of 10 mL of 1% w/v poly(ethylene-alt-maleic anhydride) in water. This was again sonicated to form the double emulsion which was poured into 200 mL of 0.5% w/v poly(ethylene-alt-maleic anhydride) and stirred overnight. Particles were washed extensively before lyophilization in cryoprotectant. The size and zeta potential of the particles were determined by dynamic light scattering (DLS) by mixing 10 mL of a 25 mg/mL particle solution into 990 mL of MilliQ water using a Malvern Zetasizer ZSP.
For treatment studies, mice were implanted with INs and 14 days later adoptively transferred with 30 million T-cells. Two days, post-transfer, mice received a single bolus i.v. injection of 2.5 mg PLP_139–151 or OVA_323–339 loaded nanoparticles. Mice were monitored daily for symptoms of EAE, and INs were removed on day 9.