Jes5 16e3

After homogenizing spleens, cells were treated with erythrocyte lysis buffer, meshed through a 35-μm cell strainer, washed with PBS, and analyzed by FACS.
DCs were phenotyped by using fluorochrome-labeled mAbs directed against CD11c (N418; BioLegend), CD80 (16-10A1; BD Pharmingen, Heidelberg, Germany), CD86 (PO3; Thermo Fisher Scientific), PD-1 (J43, Thermo Fisher Scientific), CTLA-4 (UC10-4B9; BioLegend), CD83 (Michel-19; BioLegend), CD40 (3/23; BioLegend), MHCII (2G9; BD Pharmingen) and PD-L1 (MIH6; AbD Serotec, Puchheim, Germany). Dead cells were excluded using the LIVE/DEAD™ Fixable Blue Dead Cell Stain Kit (Thermo Fisher Scientific). Following mAb labeling for 30 min at 4 °C and final washing, cells were analyzed on an LSR II flow cytometer.
To measure intracellular cytokines, cells were stimulated with PMA and ionomycin (1 μg/ml each; Sigma-Aldrich, Taufkirchen, Germany) for 4 h in the presence of 3 μg/ml Brefeldin A (Thermo Fisher Scientific), labeled for CD11c, subjected to fixation and permeabilization and subsequently stained with mAbs against IL-10 (JES5-16E3; BD Pharmingen) and IL-12 (C15.6; BD Pharmingen). IFN-γ in T cells was measured by using fluorochrome-labeled anti-IFN-γ (XMG-1.2, BioLegend) and counterstaining with anti-CD4 (RM4-5, eBioscience, Frankfurt, Germany).

Single-cell suspensions of splenocytes were stimulated with PMA (1 μg/ml) and ionomycin (1 μg/ml) (both from Sigma-Aldrich,) in the presence of GolgiStop™ (BD Biosciences, 1:1,000) at 37°C for 4 h. Cells were incubated with antibody for CD4, and PD-1 at 4°C, then were fixed and permeabilized with Foxp3 Fix/Perm buffer set (eBioscience) and incubated with antibody for IL-2 (BD Biosciences, JES6-5H4), IL-4 (BD Biosciences, 11B11), IL-10 (eBioscience, JES5-16E3), and IFNγ (BD Biosciences, XMG1.2). For IL-21 staining, cells were incubated with IL-21 R/Fc chimera (R&D Systems) for 1 h, washed and stained with PE-labeled affinity-purified F(ab') α-human IgG Fc Region antibody (R&D Systems) for 30 min.

Flow cytometry analysis was performed according to the methods described previously [28 (link)]. Briefly, the cells isolated from the MLN (the MLN cells) were incubated with an Fcγ receptor-blocking mAb (CD16/32; 2.4G2, BD Biosciences) for 15 minutes at 4°C. For surface antigen detection, the cells were labeled with monoclonal antibodies against Gr-1 (RB6-8C5, BD Biosciences), F4/80 (BM8, BD Biosciences), αβTCR (H57-597, BD BioLegend), γδTCR (GL3, BioLegend), NK1.1 (PK136, BioLegend), CD4 (RM4-5, BD Biosciences), CD44 (IM7, BD Biosciences), CD25 (PC61, BioLegend), B220 (RA3-6B2, BioLegend), and CD19 (MB19-1, BioLegend) for 30 min at 4°C.
For intracellular cytokine staining, the cells isolated from the MLN (the MLN cells) were stimulated with ionomycin (1 mg/mL; Sigma-Aldrich) and PMA (25 ng/mL; Sigma-Aldrich) for 5 h at 37°C, with brefeldin A (10 mg/mL; Sigma-Aldrich) added after 1 h. Then, the cells were fixed and permeabilized with fixation/permeabilization working solution for 20 min at 4°C followed by incubation with monoclonal antibodies against IFN-γ (XMG1.2, BD Biosciences), IL-17A (TC11-18H10.1, BD Biosciences), and IL-10 (JES5-16E3, BD Biosciences). The cells were analyzed using a Cytofix/Cytoperm Plus Kit.