Anti cd38 pe cy7

The staining procedure was the same as described before. In short, after thawing, samples were treated with FcR Blocking Reagent (Miltenyi Biotech, Germany) and stained accordingly with human anti-bodies (mAbs). They were anti-CD8-PerCP-Cy5.5, anti-CD3-APC-Cy7 (SP34–2), anti-CD4-FITC, anti-Vα 7.2-PE, anti-CD161-APC, anti-CD38-PE-Cy7, anti-CD279-PE-Cy7 and all of them were from BD Biosciences (Heidelberg, Germany). Then samples were washed. Acquisition was carried out by six-color flow cytometry using FACSVerse™ flow cytometry (BD Biosciences) with FACSuite software (BD Biosciences). Analyses of the data were made by FlowJo software (Tree Star, Ashland, OR, USA).

B cells enriched cell fractions were stained with the following combination of mAbs: anti-IgD Alexa Vio770 (BioLegend, San Diego, CA, USA); anti-IgM PerCP_Cy5.5, anti-CD27 PE-CF594, anti-CD38 PE-Cy7, anti-CD24 Alexa Fluor 647 and anti-CD5 BV 421, anti-IGκ FITC, anti-IGλ PE, anti-IgA VioGreen (BD). B cell subsets were isolated by FACS sorting (FACSAria, Becton Dickinson, Franklin Lakes, NJ, USA) after depleting IgA⁺ and dead cells with a two-step sorting approach: 1) a four-way pre-sort with yield setting was used to separate enriched B cells into IGκ⁺/CD5⁺, IGκ⁺/CD5^-, IGλ⁺/CD5^+,and IGλ⁺/CD5^-B cells; 2) each of the above cell fractions were then sorted into six main B cell subpopulations (after excluding CD38^highCD24^- plasmablasts): CD24^highCD38^high transitional (TR), IgD^highIgM⁺CD38^-CD27^- naive (N), IgD^low IgM⁺CD38^-CD27⁺ marginal zone-like (MZ), IgM⁺IgD^-CD38^-CD27⁺ IgM-only memory (MO), IgM^-IgD^-CD38^-CD27⁺ switch-memory (SM), and IgM^-IgD^-CD38^-CD27^- double negative (DN) B cells. See also Figure 1 and Supplementary Figure S1 for details.

Serum immunoglobulins (Ig) levels (IgG, IgA and IgM) were measured by immunoturbidimetry using the automatic analyzer Alinity c system (Abbott Laboratories, Chicago, IL, USA). For cellular evaluation, EDTA whole blood samples were collected. Lymphocyte subpopulations (CD4+ T, CD8+ T, B and NK cells) were performed using BD Trucount tubes and the BD Multitest 6 Color BTNK kit (BD Biosciences, San Diego, CA, USA), which included the following mixtures of fluorophore-conjugated monoclonal antibodies (mAb): anti-CD45-PerCP-Cy5.5, anti-CD3-FITC, anti-CD8-APC-Cy7, anti-CD4-PE-Cy7, anti-CD19-APC, and anti-CD16+CD56-PE. B cell phenotype was performed with an eight-color panel of the following mAb: anti-CD45-APC-H7, anti-CD19-V500, anti-CD10-V450, anti-CD38-PE-Cy7, anti-CD21-PE, anti-CD27-PerCP-Cy5, anti-IgD-FITC, and anti-IgM-APC (BD Biosciences, San Diego, CA, USA), following EURO-Class classification. Cells were acquired on a BD FACSCanto II Flow Cytometer (BD Biosciences, San Diego, CA, USA), and the InfinicytTM22.0 software was employed for multiparametric analysis (Cytognos SL, Salamanca, Spain).

Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation through Ficoll-Hypaque of blood samples obtained at the time of allograft biopsy, and stored at—80°C until analyzed. After thawing, 0.5–1 X 10⁶ PBMC were incubated with various antibody combinations for 30 min at 4°C. The fluorochrome-conjugated monoclonal antibodies used were anti-CD19 V500, anti-CD3 V450, anti-CD56 PE, anti-CD14 PE-Cy7, anti-CD45 APC, anti-CD38 PE-Cy7 from BD Biosciences, anti-CD24 APC from Miltenyi Biotec and anti-IgD FITC and anti-CD27 PE from Beckman Coulter. Samples were analyzed with Canto II cytometer (BD Biosciences) and the data were processed using FlowJo software (Tree Star, Ashland, USA).

After 4 h of stimulation, PBMCs were stained with a combination of the following antibodies from BD Bioscience: anti-CD19-PerCP (cat #345–778), anti-CD43-FITC (cat #555–475), anti-CD27-PECy7 (cat #560–609), anti-CD24-FITC (cat #555–427), anti-CD38-PECy7 (cat #335–825), anti-CD25 FITC (cat #555–431), anti-IL-10 APC (cat #554–707) or anti-IL-10-PE (cat #559–330). Anti-CD5 APC was from Dako (cat #C7242, Glostrup, Denmark) and anti-TIM-1 PE was purchased from Biolegend (cat #353–904).
After 48 h of stimulation, IL-10 was detected with the following BD Bioscience Abs: anti-CD19 APC (cat #555–415), anti-CD14 FITC (cat #555–397), anti-CD4 PerCP (cat #345–770), anti-CD8 PECy7 (cat #557–746) and anti-IL-10 PE (cat #559–330). Live/Dead Fixable Near InfraRed staining (cat #L10119; Molecular Probes, Invitrogen, Carlsbad, CA) was included.
The cells were acquired with a FACS Canto (BD Bioscience) flow cytometer with argon laser (488 nm) and Helium-Neon laser (633nm) excitation.
All analyses were carried out using FlowJo V10 (TreeStar, Ashland, OR). Dead cells were excluded based on Live/Dead Fixable Near InfraRed staining and B cells were identified as CD19⁺ events within a morphological lymphocyte gate. Individual IL-10⁺ B cells were identified using the gating strategy demonstrated in S1 Fig.

FACS analysis was performed with a BD LSRII flow cytometer (BD Biosciences, UK). After 1 to 5 weeks of co-culture, nonadherent and adherent cells were harvested through trypsinization. Recovered cells were resuspended and stained in Annexin binding buffer (BD Biosciences) and with anti-SCA-1-PE or anti-CD56-PE or anti-CD31-PE, and anti-CD45-APC-Cy7, anti-CD34-Percp, anti-CD38-PE-Cy7 antibodies, and Lin-FITC (BD Biosciences), as well as with AlexaFluor647-conjugated Annexin-V (Invitrogen) and DAPI (Sigma). Only viable (both DAPI and Annexin-V negative fraction) human hematopoietic cells (CD45-APC-Cy7 positive and Sca-1-PE or CD56-PE or CD31-PE negative) were assessed for all analyses. Cell viability was expressed as the percentage of DAPI- and Annexin-V-negative cells within human hematopoietic cells.