Pd l1 neutralizing antibody

To investigate neutralizing the suppressor effect, purified anti-PD-1 antibody (0.5 and 1 μg/mL; Biolegend) was added in the same culture conditions described for the suppression assay above, using the corresponding isotype-matched mAb as control. Purified PD-L1 neutralizing antibody (Biolegend) was used at 5 μg/mL. To test involvement of CTLA-4 added together with anti-PD-1 in the suppression mechanism, 5 μg/mL of anti-CTLA-4 (BD Biosciences) was used.

C57BL/6 mice were intraperitoneally immunized with 100 µg of NP-CGG (LGC, Middlesex, UK) in 200 µL Imject Alum (Thermo Scientific) 7 days prior to sacrifice, at which point spleens were collected. Then, CD4⁺ CD19⁻ PD-1⁺ CXCR5⁺ T_FH and CD19⁺ B220⁺ B cells were sorted with Beckman Coulter MoFlo XDP cell sorter. Later, 10⁵ cells of each B and T_FH cells were co-cultured in R-10 medium and treated either with PBS, 10 mM ethanol, 0.5 mM acetate, or 20 µg mL⁻¹ of PD-L1 neutralizing antibody (Biolegend, clone 10 F.9G2). NP-CGG was added to a final concentration of 20 µg mL⁻¹ at the beginning of the experiment. 48 h later, cells were stained for FACS analysis as described before.