Cd138 percpcy5

Directly conjugated antibodies were purchased from BD Biosciences (San Jose, CA, USA): CCR7-PECy7 (3D12), CD3-PerCPCy5.5 and CD3-V500 (UCHT1), Foxp3-AF488 (259D/C7), CD25-AF700 (M-A251), CD4-APCH7 (RPA-T4), CXCR5-AF647 (RF8.B2), CD45RA-PECF594 (HI100), CD19-APCCy7 (SJ25C1), IgM-FITC (G20-127), IgD-PECF594 (IA6-2), CD38-V450 (HB7), CD138-PerCPCy5.5 (MI15), CD21-APC (B-LY4), CD27-PE (M-T271), IgG-BV605 (G18-145) or BioLegend (San Diego, CA, USA): PD1-PE (EH12.2H7), CD19-BV650 (HIB19), CD57-PB (HCD57), ICOS-FITC and ICOS-PerCPCy5.5 (C398.4A), OX40-PECy7 (BER-ACT35), CD40L-BV605 (24–31).

Spleen and mesenteric lymph node (MLN) were collected and mashed in 70-µm cell strainers with C10 media (RPMI-1640 containing L-glutamine, 10% fetal bovine serum, 100 U/ml penicillin-streptomycin, 10 mM HEPES, 1 mM sodium pyruvate, 1% 100X MEM non-essential amino acids, 55 µM 2-mercaptoethanol, all from Life Technologies). For splenocytes, red blood cells were lysed with RBC lysis buffer (eBioscience). For FACS staining, cells were blocked by anti-mouse CD16/32 (eBioscience), stained with fluorochrome-conjugated antibodies, and analyzed with BD FACSAria Fusion flow cytometer (BD Biosciences). Anti-mouse antibodies used in this study include: CD3-FITC and B220-FITC (eBioscience), CD4-PerCP/Cy5.5, CD8-PE/Dazzle, PD-1-APC/Cy7, CXCR5-BV605, CD25-BV421, FOXP3-PE, CD44-APC, CD62L-APC/Cy7, CD138-PerCP/Cy5.5, CD19-BV421, CD273-PE/Dazzle, CD38-PE/Cy7 and GL7-APC (Biolegend). For intracellular staining, Foxp3/Transcription Factor Staining Kit was used to fix the cells (Biolegend). For intracellular staining of IL-10 producing cells, splenocytes or MLN cells were pre-stimulated for 4 hours with 500X Cell Stimulation Cocktail (Invitrogen) plus protein transport inhibitor (eBioscience) containing phorbol 12-myristate 13-acetate (PMA), ionomycin, brefeldin A and monensin; then stained with CD19-AF700, CD138-BV711, Foxp3-AF647 and IL-10-PE. FlowJo was used to analyze data.