Clone ebio1d3

For FACS analysis, single-cell suspensions were stained with the following antibodies: anti-CD4 (BioLegend, clone GK1.5, #100402, 1:400), anti-CD4 (BD Biosciences, clone RM4-5, #560468, 1:500), anti-CD19 (Thermo Fisher Scientific, clone eBio1D3, #45-0193-82, 1:400), and anti-CD8 (BD Biosciences, clone 53-6.7, #560776, 1:500). For intracellular staining, T cells were restimulated with 50 ng/ml PMA and 750 ng/ml ionomycin in the presence of 10 μg/ml brefeldin A (all from Sigma-Aldrich) for 4 h. After fixation and permeabilization, the cells were stained with anti-IL-17A (Thermo Fisher Scientific, clone eBio17B7, #17-7177-81, 1:200) and anti-IFN-γ (Thermo Fisher Scientific, clone XMG1.2, #17-7311-82, 1:500). For detection of apoptotic cells, the cultured Bregs were harvested, washed with HBSS buffer and resuspended in a HBSS solution containing FITC-labeled Annexin V (Thermo Fisher Scientific, #88-8005-72, 1:100). To measure the cell proliferation, the staining with dye carboxyfluorescein succinimidyl ester (5 µM CFSE, Sigma-Aldrich) was performed. The cells were analyzed using FACSCalibur cytometer or BD FACSAria III cell sorter (both BD Biosciences). Data were analyzed with FlowJo analysis software (TreeStar). All FACS sequential gating/sorting strategies for the analysis of T and B cells are provided in Supplementary Fig. 7.

Before cell surface staining, samples were incubated with Fc‐block™ (BD Biosciences). Staining was performed in PBS with 2% FCS and 5 mM EDTA for 45 min on ice. A combination of fluorochrome‐conjugated monoclonal antibodies was used, specific to mouse CD19 (PE‐Cy7 – clone eBio1D3), CD3 (AF700 – clone 17A2) or TCRβ (PE‐Cy7 – clone H57‐597), CD11b (BV711 – clone M1/70), CD11c (BV786 – clone HL3), CD45 (BV605 – clone 30‐F11), CD103 (BV421 – clone M290), CD64 (AF647 – clone X54‐5/7.1), Ly6C (BV605 – clone AL21), and MHCII (PerCP‐Cy5.5 – clone M5/114.15.2) and were purchased either from BD Biosciences, BioLegend, or eBioscience. To determine cell viability, fixable viability dye eFluor 780 (eBioscience) was added to samples. To detect intracellular STM, viable cells were stained for the surface markers, fixed, and permeabilized using the eBioscience™ Foxp3/Transcription Factor Staining Buffer Set (eBioscience) according to the manufacturer's protocol and incubated with a PE‐labeled anti‐Salmonella‐LPS monoclonal antibody (clone 1E6; Abcam). In fixed cells, EGFP was counterstained with an AF488 labeled polyclonal anti‐GFP antibody (Abcam). Data was collected using a BD™ LSR II flow cytometer or a BD LSRFortessa™ cell analyzer. Data analysis was done using the FlowJo™ software.