Iga pe

Lymphocytes were isolated as described and stained with anti-mouse IgD PerCP-Cy5.5 (1:400, 405710), LPAM (integrin α4β7) PE (1:100, 120605; (Biolegend, San Diego, CA), B220 V500 (1:400, 561227), CD19 APC-H7 (1:200, 560143), CD80 PE (1:500, 553769), CD273 APC (1:200, 560086), CD138 PE (1:200, 553714), IgM PE-Cy7 (1:200, 552867; BD Biosciences), CCR9 PE-Cy7 (1:100, 25-1991), CD73 PE-Cy7 (1:50, 25-0731), IgA PE (1:50, 12-4204), GL7 eFluor 450 (1:100, 48-5902), CD38 Alexa700 (1:800, 56-0381), CD21/35 Pacific Blue (1:800, 57-0212; eBiosciences) or CCR10 APC (1:100, FAB2815A; R and D systems. Minneapolis, MN) and were analysed using LSR II (BD Biosciences) or Navios (Beckman Coulter, Brea, CA) flow cytometers. For sorting, cells were labelled with anti-mouse CD138 PE (1:200, 553714), CD19 PE-Cy7 (1:200, 552854), CD80 APC (1:200, 560016) and GL7 FITC (1:100, 553666) before sorting using a FACSAria (BD Biosciences). Cells were sorted into tubes that had been coated with 2% BSA/PBS overnight, and pelleted by centrifugation at 600 g before being resuspended in PBS and injected into recipient mice or used for gene sequence analysis.

Peyer’s patches (PP) were isolated from the small intestine and cells were extracted as previously described^{16 (link)}. Briefly, PP were finely cut and incubated for 40 min at room temperature with collagenase/DNase. Cells were then filtered through a 70 µm cell strainer and pelleted by centrifuging at 300×g, 4 °C during 5 min and then resuspended in FACS buffer (PBS, 2% FCS, 5 mM EDTA) and counted before antibody staining. For flow cytometry, cells were first incubated on ice in FACS buffer containing an anti-CD16/CD32 antibody to block the Fc receptor for 10 min and then incubated 30 min on ice in the dark with antibodies against the following surface markers : CD45-PE-eF610 (eBiosciences, clone 30-F11), CD19-PerCp-Cy5.5 (Biolegend, clone 6D5), CD3-PeCy7 (eBiosciences, clone 145-2C11), B220-AF647 (BD Biosciences, clone RA3-6B2), IgD-BV421 (BD Biosciences, clone 11-26c.2a), IgM-FITC (BD Bioscience, clone II/41) and IgA-PE (eBiosciences, clone mA-6E1). Cell viability was evaluated using Fixable Viability Dye eFluor 506 (eBiosciences). Cell acquisition was performed using a CytoFlex (Beckman Coulter) and data were analyzed with the CytoExpert software (Beckman Coulter).