Cd4 apc clone rpa t4

CD4⁺ T cells were magnetically separated from PBMCs (CD4-MicroBeads, Miltenyi) and maintained in IL2-containing (20 ng/ml, SCI-Store) full RPMI-1640 medium until the start of the experiment. Then they were labeled with CTV (cat No. C34557, Thermo Fisher Scientific) and co-cultured (10⁵ cells) with autologous previously differentially activated for 5 days and non-activated B cells (10⁵ cells). After 5 days of co-incubation, supernatants were collected separately for determination of IL10 level by ELISA (cat No. 88-7106-88, Thermo Fisher Scientific). Cells were stained with antibodies to the main phenotypic molecular markers of regulatory T cells: CD4-APC (clone RPA-T4, lot No. B307926, BioLegend), CD25-PE (clone BC96, lot No. 2173867, eBioscience), and CD127-FITC (clone A019D5, lot No. E-AB-F1152L, Elabscience, USA). To determine cell viability, cells were also stained with Viability Dye-eFluor780 (Thermo Fisher Scientific). Cells were then analyzed by flow cytometry using FACS Canto II (BD Biosciences), FlowJo Software version 10 (TreeStar) was used for analysis.

PBMC were washed and stained with 5 μl Live/Dead Violet (Invitrogen) followed by surface staining with the following titrated antibodies: 0.5 μl CD3-AF700 (clone UCHT1, Ebioscience), 2 μl CD4-APC (clone RPA-T4, BioLegend), 2 μl CD8-Efluor605 (clone RPA-T8, Ebioscience), 2 μl CD14-PE/Cy7 (clone HCD14, BioLegend), 2 μl CD16-AF488 (clone 3G8, BioLegend), 1 μl CD19-PE/Cy5 (clone HIB19, BioLegend), 2 μl CD25-APC/Cy7 (clone BC96, BioLegend), 2 μl CD127-NC650 (clone eBioRDR5, Ebioscience), 15 μl HLA-DR-PE (clone L243 BioLegend). Fluorescence minus one controls were used to set gates for CD25, CD127 and HLA-DR. Samples were acquired on a BD LSR II flow cytometer. Results are presented as percentages of cells after gating out of dead cells and doublets. CD4⁺ and CD8⁺ T cells were identified as CD3⁺ cells, whereas CD14^+/− and CD16^+/− cells were identified as CD3⁻ and CD19⁻ populations. CD25⁺ CD27⁻ populations were gated on the CD4⁺ cells.

Before the experiment, CD19-depleted PBMCs were maintained in IL2-containing (20 ng/ml, SCI-Store, Russia) full RPMI-1640 medium. Then autologous CTV-labeled (cat No. C34557, Thermo Fisher Scientific) CD19-depleted PBMCs (10⁵ cells) were co-cultured with previously differentially activated and non-activated B cells (10⁵ cells). After 5 days of co-incubation, supernatants were collected separately to determine pro- and anti-inflammatory cytokine levels by ELISA (cat No. A-8752 for IFNγ, A-8756 for TNF, A-8766 for IL1β, A-8768 for IL6, and A-8774 for IL10, Vector-Best, Russia). Cells were stained with antibodies to the main phenotypic markers of NK- and T cells: CD3-FITC (Sorbent, Russia), CD4-APC (clone RPA-T4, lot No. B307926, BioLegend), CD8-PE-Cyanine7 (clone SK1, lot No. B276851, BioLegend), CD16-PE (Sorbent), cell viability was determined using Viability Dye-eFluor780 (Thermo Fisher Scientific). Frequencies of proliferating CD3^-CD16⁺ NK-cells, CD3⁺CD4⁺ T helpers, CD3⁺CD8⁺ T killers were determined by flow cytometry following the loss of CTV signal to determine the suppressive capacity of B cells. Cells were then analyzed by flow cytometry using a FACS Canto II (BD Biosciences, USA), FlowJo software version 10 (TreeStar) was used for analysis.