Cd38 percp cy5

Frozen PBMC were thawed and stained ex vivo with HLA*0201 or HLA*0101 PE-labelled pentamers (Proimmune Oxford; 20 min in PBS, RT), stained with LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (Thermo Fisher; 20 min RT), fixed (1% formaldehyde in PBS, 20 mins RT) then stained with: CD3-PO (Pacific Orange; Invitrogen, UCHT1), CD8–PB (Pacific Blue; BD biosciences, RPA-T8), and either stained with CCR7-PeCy7 (CD197; BD biosciences, 3D12), CD45RA-FITC (BD biosciences, HI100), CD38-PerCP-Cy5.5 (Biolegend, HIT2), HLA-DR-AlexaFlour700 (BD biosciences, G46-6) for 30 min RT; or permeabilised (10x permeabilisation buffer; ebiosciences) and stained with PD1-PeCy7 (BD biosciences, EH12.1), Perforin-FITC (BE biosciences, dG9), Granzyme B- Alexa fluor700 (BD biosciences, GB11) and Granzyme A-PerCpCy5.5 (Biolegend, CB9) 30 min RT. For intranuclear staining, PBMC were stained as above for pentamers and Live/Dead dye, and then surface stained with: CD3-Pacific-orange, CD8-PB (30 min RT). PBMC were then fixed (1 h RT) and permeabilised using the Foxp3/Transcription Factor Staining Buffer Set (ebiosciences; 45 min RT) and stained with eomes-eFluor660 (eBiosciences, WD1928) and Tbet-BV605 (Biolegend, 4B10).

Peripheral blood mononuclear cells were separated from whole blood using Ficoll-Pague solution (Sigma Aldrich, Germany), plated at a density of 1 × 10⁶ cells/ml in complete RPMI 1640 medium supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, penicillin, streptomycin, 4 mM L-glutamine, and 0.1% 2-mercaptoethanol (all from Gibco) in the presence of phorbol myristate acetate (50 ng/ml; Sigma-Aldrich), ionomycin (1 μg/ml; Sigma-Aldrich), and brefeldin A (BioLegend) in 12-well plates for 18 h, at 37°C. For analysis of different Breg subsets, cells were surface stained with the following anti-human mAb (all from BioLegend): CD19 APC, CD24 FITC, CD38 PerCP-Cy5.5, and CD27 PE. For intracellular IL10 staining, cells were fixed and permeabilized using the Fixation/Permeabilization kit (BD Biosciences) and stained with anti-human IL10 PE-Cy7. Stained cells were acquired by FACS Canto II flow cytometer (BD Bioscience), and data were analyzed using FlowJo software 7.6.1 (Tree Star Inc., USA). The gating strategy used for B cell phenotyping is shown in Figure 1. The following B cell subsets were identified: CD19+ (total B cells), B10 cells (CD24^hiCD27⁺), immature transitional B cells (CD24hiCD38hi), and plasmablasts (CD38^hiCD27⁺).

For external staining, cells from cell lines or PBMCs in PBS were incubated with anti-surface antibodies at room temperature (RT) for 20 min. Live/dead staining was performed using LIVE/DEAD® Fixable Near-IR Dead Cell Stain Kit(Invitrogen), at 633 or 635 nm excitation.
For internal staining, cells were fixed with 2% formaldehyde (Sigma Aldrich) in PBS for 10 min and permeabilized with IX permeabilization buffer (eBioscience) in water.
The following antibodies were used: CD3-FITC (BioLegend, Catalog No. 300406, clone UCHT1, Mouse IgG1, k), CD8-PerCP-Cy5.5 (BioLegend, Catalog No. 344710, clone SK1, Mouse IgG1, k), CD38-PerCP-Cy5.5 (BioLegend, Catalog No. 303522, clone HIT2, Mouse IgG1, k), CD56-APC (Biolegend, Catalog No. 318310, clone HCD56, Mouse IgG1, k); CD19-BV421 (BD Bioscience, Catalog No. 562441, clone HIB19, mouse IgG1, k); CD4-VioGreen (Miltenyi Biotec, Catalog No. 130-106-712, clone M-T466, Mouse IgG1, k), CD161-PE (Miltenyi Biotec, Catalog No. 130-092-677, clone 191B8, Mouse IgG2a), IgG2A isotype control (R&D Systems, Catalog No. MAB003, mouse); and 2H7 mAb. When non-conjugated primary antibodies were used, a secondary rat anti-mouse IgG2A-PE (R&D Systems, Catalog No. F0129, clone 344701, IgG1) was used.
FACS analysis was performed on Miltenyi Biotec MACSQuant cytometer and analyzed with FlowJo Version 9.6.2 software (TreeStar).