C9b7w

For in vivo neutralization of LAG-3, 200 μg of anti-LAG-3 mAb (C9B7W, BioLegend) or purified rat IgG (Invitrogen) in PBS were injected intraperitoneally on day −1 and +1 of EAE induction.

For phenotypic analysis of DCs, splenic lymphocyte suspensions were stained with fluorochrome conjugated antibodies specific for the following surface markers: CD11c (N418), CD80 (16–10 A1), CD86 (GL1), CD40 (39164), MHCII (10.2.16), and PDL1 (10F.9G2), all of which were purchased from eBioscience (eBioscience, San Diego, CA, USA). Cell suspensions of thymus, spleen, and pancreatic lymph nodes (PLNs) were stained and analyzed with antibodies against CD4 (RM4-5), CD8α (53-6.7), CD25 (PC61.5), Foxp3 (FJK-16s), IFN-γ (XMG1.2), IL-4 (11B11), IL-2 (JES6-5H4), programmed cell death protein-1 (PD-1) (J43), T-bet (4B10), Eomesodermin (Eomes) (Dan11mag), CD160 (CNX46-3), CD244 (C9.1), CD272 (8F4), KLRG1, or killer cell lectin-like receptor G1 (2F1), all purchased from eBioscience. Antibodies against TNF-α (MP6-XT22), LAG3 or lymphocyte-activation gene 3 (C9B7W), and TIM3 or T cell immunoglobulin and mucin domain 3 (RMT3-23) were purchased from BioLegend (San Diego, CA, USA). For intracellular cytokine staining, splenocytes were stimulated with phorbol 12-myristate 13-acetate/PMA, ionomycin, and monensin (Sigma-Aldrich, St. Louis, MO, USA) for 6 h, followed by staining with antibodies. All analyses were performed on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed using CellQuest software (BD Biosciences).

Anti-mouse: α-CD45 (30-F11, BD, 4 μg ml⁻¹), α-CD3 (145-2C11, Biolegend, 5 μg ml⁻¹), α-CD4 (GK1.5, Biolegend, 5 μg ml⁻¹), α-CD8 (53-6.7, BD, 5 μg ml⁻¹), α-CTLA-4 (1B8, abcam, 30 μg ml⁻¹), α-TIM3 (B8.2C12, Biolegend, 2.5 μg ml⁻¹), α-OX40 (OX-86, Biolegend, 10 μg ml⁻¹), α-CD62L (MEL-14, BD, 5 μg ml⁻¹), α-CD44 (IM7, Biolegend, 2.5 μg ml⁻¹), α-LAG-3 (C9B7W, Biolegend, 5 μg ml⁻¹), α-IFN-γ (XMG1.2, Biolegend, 2.5 μg ml⁻¹), α-TNF-α (MP6-XT22, Biolegend, 2.5 μg ml⁻¹), α-Granzyme B (NGZB, ThermoFisher, 1.25 μg ml⁻¹), α-FOXP3 (MF-14, Biolegend, 10 μg ml⁻¹), α-Ki67 (SolA15, ThermoFisher, 0.6 μg ml⁻¹). Anti-human: α-CD4 (A161A1, Biolegend, 2.5 μg ml⁻¹), α-T-bet (4B10, Biolegend, 2.5 μg ml⁻¹), α-Phospho-Akt (Ser473) (D9E, Cell Signaling, 0.5 μg ml⁻¹), α-Phospho-Akt (Thr308) (D25E6, Cell Signaling, 0.5 μg ml⁻¹), α-Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E, Cell Signaling, 0.5 μg ml⁻¹).