Induction of EAE and development and treatment with IC-100
Active EAE was induced in 2-months old C57BL/6 female mice with myelin oligodendrocyte glycoprotein 35-55 peptide (MOG35-55, BioSynthesis) as previously described . Briefly, mice received an intraperitoneal (i.p.) injection of pertussis toxin dissolved in PBS (350 ng/mouse; day 0), followed by sub-cutaneous administration of MOG35–55 (300 ng/mouse; day 1) emulsified in Complete Freund’s Adjuvant, and a second i.p. injection of pertussis toxin (350 ng/mouse; day 2). IC100 (IgG4) was developed by humanization of a mouse monoclonal (IgG1) against human ASC (Abzena, Cambridge England). IC100 was cloned into a CHO cell manufacturing cell line (Selexis, Geneva, Switzerland). IC100 was purified from CHO cell supernatants using ProSepA high capacity column chromatography (Antibody Solutions, Santa Clara, CA). Mice were administered vehicle (0.9% saline) or IC100 at three different doses (10, 30 and 45 mg/kg) via i.p. injection every 4 days, starting at day 8 after induction of EAE. Clinical symptoms of EAE were assessed daily by a blind investigator on a scale of 0 to 6 as follows: 0, no clinical signs; 1, loss of tail tone; 2, completely flaccid tail; 3, complete hind limb paralysis; 4, complete forelimb paralysis; 5, moribund; 6, dead. All experiments were performed according to protocols and guidelines approved by the Institutional Animal Care and Use Committee of the University of Miami.
Cell isolation for flow cytometry
Following transcardial perfusion with PBS, spinal cords were harvested and placed in cold Hanks’ Balanced Salt Solution without Mg2+ and Ca2+ (HBSS w/o). Samples were then manually dissociated into single cell suspensions through a 70 µm strainer and washed in HBSS w/o. Spleen samples were spun at 1200 rpm for 10 min at 4˚C, supernatants were removed, and red blood cells (RBCs) lysed in 2 ml RBC lysis buffer (eBioscience) according the manufacturer’s instructions. Spleen cells were then resuspended in PBS. Cells isolated from the spinal cord were resuspended in flow cytometry buffer (FCB, eBioscience), and incubated with Myelin Removal Beads II (Miltenyi). Myelin was depleted using the LS magnetic columns as described in the manufacturer’s protocol (Miltenyi). Similar to the splenocytes, spinal cord cells were resuspended in PBS and stained as described below.
Immunolabeling and flow cytometric analysis
Cells were resuspended in 100 µl FCB, blocked with 0.5 µl TruStainFcX (anti-mouse CD16/32 FcR block, Biolegend) for 5 min at room temperature, and stained for 30 min at 4°C with: FITC-anti-CD45 (1:1000, Biolegend), PE-Cy7-anti-CD4 (1:200, eBioscience), PerCP-Cy5.5-anti-CD8 (1:200, Biolegend), PE-anti-B220 (1:200, Biolegend), APC-anti-MHCII (1:200, eBioscience), and APC-eFluor780-anti-CD11b (1:200, eBioscience). Cells were then fixed in 1% PFA for 1 h. Finally, cells were resuspended in 500 µl FCB, labelled with DAPI to exclude cell debris and analyzed with a CytoFLEX S flow cytometer (Beckman-Coulter) equipped with CytExpert software (Beckman-Coulter).
Quantification of IC100 in Tissues
IC100 was quantified in brain, spinal cord, liver and spleen at 35 days post-induction (dpi) of EAE using a proprietary assay developed by InflamaCORE, LLC using Meso Scale Technology. Protein lysates were obtained as described in . The assay was read using the QuickPlex SQ 120 instrument (Meso Scale Diagnostics, Maryland).
EAE curves were analyzed point-by-point by 2-way repeated measures ANOVA, followed by Tukey test for multiple comparisons. The EAE curves as a whole were also analyzed by non-parametric Mann-Whitney test comparing treated groups to the control group, individually. All other assessments were analyzed by Student’s t test, if the data were normally distributed, or by non-parametric Mann-Whitney test if the data were not normally distributed. Normality was determined by the Pearson & D’Agostino test. Data were expressed as mean ± SEM, and p values equal or less than 0.05 were considered statistically significant. Statistical analyses were carried out with GraphPad Prism software.