Fix perm kit

Single-cell suspensions were incubated on ice for 30 min with a combination of antibodies specific to cell surface markers for identification of lymphocyte subsets. These antibodies are anti-NK1.1 (clone PK136), anti-CD3 (clone 145–2C11), anti-CD8α (clone 53–6.7), anti-NKG2D (clone CX5), anti-CD44 (clone IM7), anti-CD25 (clone PC61), anti-Gr1 (clone RB6-8C5), and anti-CD11b (clone ICRF44). All antibodies used for flow cytometry analyses were purchased from Biolegend (San Diego, CA, USA). Tetramer staining was performed with 2 μg/ml of PE-labeled H-2D^b/ gp100_25-33 tetramer at 37 °C for 20 min and followed by surface marker staining. For intracellular staining, cells were stained with surface markers followed by fixation and permeabilization with BD Perm/Fix kits and antibodies specific to intracellular molecules. Cells were analyzed using the BD Fortessa. Data were analyzed using the FlowJo software (Tree Star).

Cell staining was done on ice in the dark, and centrifugation steps were performed at 340 g, 4°C for 5 min. Cells were pelleted by centrifugation, resuspended in PBS with 1% BSA, and incubated with Fc Block (BD Biosciences; 1:50) for 10 min. Cells were fixed and permeabilised using the Fix-Perm Kit (BD Biosciences) according to the manufacturer’s protocol. The cells were then incubated with the granzyme C antibody (clone SCF1D8 labelled with PE, Biolegend) for 30 min in the dark at 4°C washed twice, and acquired on a FACSCanto II. Data were analysed using FlowJo software.

To determine endogenous intracellular IL-10 production, PBMC were cultured for 48 hours; during the final 5 hours of incubation the cultures were supplemented with Brefeldin A (1∶100, BD), PMA (50 ng/ml, Invivogen) and Ionomycin (1 ug/ml, Invivogen). After incubation the cells were washed, stained for viable cells (LIVE/DEAD Aqua Fixable Dead Cell Stain Kit, Invitrogen), surface stained, fixed/permeabilized (Fix/Perm Kit BD Biosciences) and stained for intracellular IL-10 (IL-10-AF-647, eBioscience). To determine spontaneous expression of IL-10 by Bregs from HIV-infected individuals and healthy controls, PBMCS were incubated overnight, stimulated for the final 5 hours and stained as described for healthy controls. The following antibodies were used for immunophenotyping of PBMC: CD19-ECD (Beckman Coulter), PD-L1-PE-Cy7 (eBioscience), CD24-PE, CD38-FITC, HLA-DR-PE-Cy7, CD4-Pacific Blue, CD8-APC-H7, Lineage-1-FITC, CD11c- AF-700, HLA-ABC-PE-C7 and CD107a-PE-C5 (BD, Bioscience). HIV-specific CD8⁺ T cells were identified by binding to MHC-1-APC Dextramer® (Immudex) and HIV-infected CD4+ T cells were identified by binding to KC-57-PE antibody (Beckman Coulter). All samples were acquired on an LRSII (BD, Bioscience) flow cytometer and the data was analyzed using FlowJo software (Tree Star Inc).

Cell surface and intracellular molecular expressions were evaluated by flow cytometry using CytoFLEX (BeckmanCoulter, U.S.A.). Fluorescein-conjugated mouse anti-human antibodies were used, including CD3-Alexa Fluor 700, CD3-BV650, CD8-BV786, CD8-PerCP/Cy5.5, CD45RA-APC/CY7, CCR7-PE/CY7,CD27-PE, CD28-BV421, CD69-APC/CY7, CD103-BV605, CXCR3-BV510, HLA-DR-APC, CD39-BV421, PD-1-PE, CD127-PE/CY7, CD62L-PE/CY7, Perforin-APC, Granzyme B-PE, IFN-γ-PE, and IL-4-APC (Biolegend, UK). For cell-surface staining, single-cell suspensions were stained on ice for 30 min in PBS with 1% fetal bovine serum (FBS). For intracellular staining, cells were fixed and permeabilized using the Fix/Perm kit (BD Biosciences, U.S.A.). To detect intracellular cytokines, CD8⁺T cells were stimulated for 6 h with phorbol 12-myristate 13-acetate (PMA; 1 μg/mL; Sigma) and ionomycin (1 μg/mL; Sigma), and 4 h with GolgiStop (1 μL/mL; BD Biosciences) in a round-bottom 96-well plate. Thereafter, cells were harvested, stained for surface expression, and then fixed and permeabilized for intracellular staining. Flow cytometry data was analyzed using FlowJo software (BD, UK) and CytoExpert software (Beckman Coulter, U.S.A.).