Cd40 hm40 3

On day 50 (peak of the disease) after induction of EAMG, rats were euthanized under deepening anesthesia. Spleens and inguinal lymph nodes were removed and the weights were measured. Then the spleens and inguinal lymph nodes were minced through a 70 μm cell strainer to prepare single-cell suspensions of mononuclear cells (MNCs) under aseptic conditions. Red blood cells in spleen MNCs were lysed with a lysis buffer (Biolegend) for 5 min. All specimens were coded to facilitate blind testing. The spleen or inguinal lymph nodes MNCs suspension with a final volume of 100 μl were labeled with corresponding monoclonal antibodies and incubated for 30 min at 4 °C in the dark. The following antibodies were used in the assay: CD3 (1F4; Biolegend), CD4 (OX35; eBioscience), B220 (HIS24; eBioscience), CD20 (SP32; Abcam), CD161 (10/78; BD Pharmingen), MHC II (HIS19; Biolegend), CD80 (3H5; Biolegend), CD86 (24F; Biolegend), CD40 (HM40-3; Biolegend), CD27 (LG.7F9; eBioscience), ICOS (C398.4A; Biolegend), CXCR5 (ERP8837; Abcam). Unconjugated primary antibodies were detected with Alexa Fluro 488-conjugated anti-rabbit IgG (Abcam). Samples were analyzed using an Aria II flow cytometer (BD).

After animal sacrifice and perfusion with ice cold PBS, half of the brain of mice was collected and homogenized at defined times p.i. and leukocytes enriched on a Percoll gradient as previously described (Blanc et al., 2014 (link)). Cells were stained using the following antibodies: CD4 (GK1.5, Biolegend), CD3 (145–2 C11, Biolegend), IFN-γ (XMG1.2, Biolegend), FoxP3 (FJK-16s, eBioscience), IL-10 (JES5-16E3, Biolegend), CD8 (53-6.7, Biolegend), CD45 (30-F11, Biolegend), F4/80 (BM8, Biolegend), CD11b (M1/70, Biolegend), CD40 (HM40-3, Biolegend), CD86 (GL-1, Biolegend), MHCI (M1/42, Biolegend), and MHCII (M5/114.15.2, Biolegend). Virus-specific T cells were determined via flow cytometric analysis through either intracellular staining for IFN-γ or defined tetramers specific for immunodominant CD4 +and CD8+T cell-specific viral epitopes (Chen et al., 2014 (link); Stiles et al., 2006 (link)). Samples were analyzed using BD LSRFortessa and FACSDiva software and data was measured using FlowJo. 

Mitomycin C was purchased from Wako. Anti-mouse CD3ε (145-2C11), CD28 (37.51), and CD40 (HM40-3) were obtained from Biolegend (San Diego, CA, USA). Recombinant mouse IL-4 was purchased from R&D Systems (Minneapolis, MN, USA). Ovalbumin (OVA), N-acetylcysteine (NAC), and lipopolysaccharide (LPS; Escherichia coli O55:B5) were purchased from Sigma–Aldrich (St. Louis, MO, USA). NP₄₉-AECM-Ficoll (NP₄₉-Ficoll; 4-hydroxy-3-nitrophenylacetic hapten conjugated to amino-ethyl-carboxy-methyl-Ficoll) and NP₃₀-BSA were obtained from Biosearch Technologies (Novato, CA, USA).