Igd apc cy7

All flow cytometry used appropriate isotype controls. Gating and compensation were aided by the performing fluorescence minus one controls. Cells isolated from biopsies or blood were stained with blue-fluorescent reactive dye (Life Technologies), CD19-BV785, CD27-APC (BD Biosciences) or PE or BV421, CD10-BV605 or APC, CD38-PerCp-ef710 (eBioscience), CD24-PE/Cy7 or BV605, IgD-APC/Cy7, IgM-V450 (BD Biosciences), IgG-PE/Cy7 and IgA-FITC (Miltenyi Biotec), in 2% FCS staining buffer with 1 mM EDTA for 15 min at 4 °C before analysis on the BD LSRFortessa (BD Biosciences). For high-throughput sequencing analysis, cells were stained with LIVE/DEAD Fixable Aqua (Life Technologies) or DAPI, CD19-BV785, CD27-FITC or APC, IgD-APC/Cy7, CD38-PerCp-ef710 (eBiosciences), CD10-BV605 in 2% FCS staining buffer with 1 mM EDTA for 15 min at 4 °C before sorted on the BD FACSAria (BD Biosciences). Four subsets from PBMC and three subsets from paired biopsy mononuclear cells from Donors 1 to 4. The numbers of cells used to generate sequences for each sample from Donors 1 to 4 are shown in Supplementary Fig. 14d.

PB was collected in heparin-coated tubes (Venosafe plastic tubes, Terumo Europe N.V., Leuven, Belgium) and PB mononuclear cells (PBMC) were isolated using high density centrifugation (Lympholyte; Cedarlane Laboratories, SanBio B.V., Uden, the Netherlands). PBMC (0.5×10⁶ cells) were stained using anti-human CD19 PerCP-Cy5.5 and CD4 APC to discriminate between B and T cells, respectively (BD Biosciences, Erembodegem, Belgium). B cell subpopulations and surface molecules were defined using following anti-human antibodies: IgD APC-Cy7, CD27 PE-Cy7, HLA-DR/DP/DQ (major histocompatibility complex (MHC)-II) FITC, CD80 PE and CD86 PE-CF594 (all from BD Biosciences, Erembodegem, Belgium). Following anti-human monoclonal antibodies were used for T cell analysis: CD45RA APC-H7, CD45RO PE-CF594, CXCR5 Alexa Fluor 488 and PD-1 PE-Cy7 (all from BD Biosciences, Erembodegem, Belgium), CD25 PerCP-Cy5.5 and CD127 PE (eBioscience, San Diego, USA). Following isotype controls were used: mouse IgG1 PErCP-Cy5.5, IgG1 PE, IgG1 PE-Cy7, IgG2aκ PE-CF594, IgG2bκ APC-H7, IgG1 APC, IgG2aκ FITC, IgG1κ PE-CF594, IgG1 PE-Cy7, IgG2aκ APC-H7 and rat IgG2b Alexa Fluor 488 (all from BD Biosciences, Erembodegem, Belgium). All flow cytometric analyses were performed on a FACSAriaII flow cytometer and analyzed with FACS Diva software (BD Biosciences).

For analysis of B cell differentiation, 5 x 10⁴ transduced HSCs were transferred to flasks of OP9 stromal cells and co-cultured in B lymphoid-promoting medium (Alpha MEM supplemented with 20% FBS, 1% penicillin-streptomycin, 50 μM 2-mercaptoethanol, 10 ng/mL SCF, 10 ng/mL Flt3L and 10 ng/mL mIL-7 (PeproTech, Rocky Hill, NJ), and 250 ng/mL amphotericin B). Cells were harvested by trypsinization and resuspended in sorting buffer after 7 days of co-culture. Hardy fraction analysis of B cell differentiation was performed as previously described [35 (link)]. Briefly, cells were stained with 7-AAD (eBioscience) and the following antibodies: B220-Pacific blue, CD43-APC, BP-1-BV605, CD24-PE-Cy7, IgD-APC-Cy7, and IgM-PE-CF594 (BD Biosciences). Single-color stained cells and UltraComp beads (eBioscience) were used for compensation and to establish Hardy fraction gating. The following controls were used to exclude OP9 cells and non-transduced cells from ZsGreen⁺/RFP⁺ analysis: OP9 cells alone, non-transduced HSPCs alone, and OP9 cells with non-transduced HSPCs. Samples were analyzed on an LSR II flow cytometer and FACS plots were generated using FlowJo software.