Isotype control mab

FITC-conjugated mAbs to CD86 [clone 2331 (FUN-1)], CD274 (clone MIH1), PE-conjugated mAbs to pSTAT3 (clone 4/P-STAT3), CD80 (clone L307.4), PD-L2 (clone 2D3/B7-H2), antigen-presenting cell (APC)-conjugated mAbs to HLA-DR (clone G46-6), PD-1 (clone MIH4), Alexa 700-conjugated mAb to CD4 (clone RPA-T4), and BV421-conjugated mAb to CD4 were from BD Biosciences (Le Pont de Claix, France). PE-conjugated mAbs to IL-17A (clone eBio64CAP17), human–mouse RORγt (AFKJS-9), APC-conjugated mAb to FoxP3 (clone 236A/E7), and Fixable Vibility Dye eFluor^® 506 were from eBioscience (Paris, France). PE-conjugated mAb to CD40 (clone MAB89) was from Beckman Coulter (Villepinte, France). Blocking mAb to human PD-L1 (clone MIH1) and isotype control mAb were from eBioscience. Alexa-488 conjugated mAb to IL-10 (clone JES59D7) and blocking mAb to PD-1 (clone EH12.2H7) were from Biolegend (London, UK).

BMNC isolated as described²⁸ were stained with anti-CD3, anti-CD4, anti-CD8, anti-CD11b, anti-CD11c, anti-CD45, anti-F4/80, anti-Gr-1 or appropriate isotype control mAb (all from eBiosciences). CFSE (0.7 mg in 0.3 ml PBS-DMSO 7%; Santa Cruz Biotechnologies) or vehicle were injected i.v. on day 5 post-infection as reported^{3 (link)}. BMNC were then collected and stained with anti-CD11b, anti-CD11c or isotype control mAb (eBiosciences) and examined for expression of CFSE. Splenocytes were incubated with or without anti-CD3 mAb and Brefeldin A (10 μg/ml; eBiosciences). Cells were stained with anti-CD3, anti-CD4 and anti-CD8 (eBiosciences), permeabilized, stained with anti-IFN-γ or isotype control mAb (eBiosciences) and analyzed in an LSR II flow cytometer (BD Biosciences).