Cd45rb pe

Untouched CD4 T-cells were isolated using the Dynal negative isolation kit (Life Technologies Carlsbad, CA) according to manufacturer’s instructions, then stained using CD4-APC (Ebioscience, San Diego, CA), CD45RB-PE (BD Biosciences, San Jose, CA), CD25-PE/Cy7 (Ebioscience, San Diego, CA). Following staining, cells were sorted into CD4+CD45RB+CD25−(Tresp) or FOXP3/GFP+(Treg) populations using a FacsAria instrument (BD, San Diego, CA). 1E6 Treg and 4E6 Tresp Cells were transferred into lymphopenic (RAG1 KO) recipients at 1∶4 ratio of Treg:Tresp. On day 7–8 post-transfer, recipient animals were harvested, and single-cell suspensions generated from spleens and lymph nodes as above. For analysis, cells were counted by trypan blue exclusion, and stained for CD3-APC (Ebioscience, San Diego, CA), CD4-PacOrange (Life Technologies Carlsbad, CA) or CD4-PE (Ebioscience), CD25-FITC (Ebioscience, San Diego, CA), followed by analysis using an LSR2 instrument (BD Biosciences, San Jose, CA).

Cryopreserved PBMCs were thawed at 37°C, washed in RPMI/60% and RPMI/20% of fetal bovine serum (FBS), and incubated for 1 h at 37°C in RPMI/10%FBS. PBMCs were then stained with the Fixable Viability Stain-FVS780r (APC-H7 detect, BD Biosciences) for 15 mins. After PBMCs washing in PBS/1%FBS, cells were incubated in a U-bottom 96-well plate at a density of 1.5 million/well and stained with selected 14-color panel including: anti-CD19-AF700 (Clone HIB19), anti-IgD-Pe-Cy7 (Clone IA6-2), anti-IgM-BB515 (Clone G-20-127), polyclonal goat anti-IgA-Dylight^®649 (from Jackson Immunoresearch), CD10-BV650 (Clone H10a), CD21-PE-CF594 (Clone B-LY4), CD27-BV510 (Clone L128), CD38-BV786 (Clone HIT2), CD45-RB-PE (Clone MT4), CD86-PerCP-Cy5.5 (Clone 2331), CD279 (PD-1)-BV421 (Clone EH12.1), all from BD Biosciences unless indicated, for 15 mins. After washing twice in PBS/1% FBS, cells were fixed in PBS/1% formaldehyde, acquired in a BD LSRFortessa (BD Biosciences) cytometer using a plate HTS loader (BD Biosciences) and analyzed with FlowJo software (Tree Star). Gating strategy is described in Supplementary Figure 1. Lymphocyte gate was defined manually by morphological parameters excluding non-viable cells. B cells were identified as CD19+ CD21+/− cells and gated according to the expression of different markers to identify B-cell maturation stages as described in Supplementary Figure 1.