Cd8 pb

Briefly, cells were incubated for 6 h at 37°C in 5% CO₂ with pools of overlapping 15-mer long peptides at a final concentration of 2 μM each, stimulated with SEB (Sigma) at 1 μg/ml or left unstimulated (negative control). The secretion inhibitor brefeldin A (Sigma) was added for the final 5 h of culture. After the 6 h incubation, surface staining for CD3-PercP, CD4-PECy7, CD8-PB (unless otherwise stated, staining reagents, instrumentation and software were from BD Biosciences) and live/dead fixable aqua dye (Life Technologies) was performed and then followed by intracellular staining for IFNγ-APC, TNFα-FITC, MIP-1β-PE, CD154-APC-eFluor780 (eBiosciences, San Diego, CA). Analysis was done on a two-laser CANTO II flow cytometer (BD Biosciences) and data analyzed with FlowJo software (TreeStar Inc., Ashland, OR). Spectral compensation was performed for each experiment with each individual mAb used in the surface and intracellular cytokines staining using compensation particles (BD Biosciences).

To determine intracellular cytokine production, PBMC of PWH were cultured in RPMI 1640 supplemented with 10% FCS, penicillin (100 U/ml), streptomycin (100 U/ml), anti-CD28 (2 μg/ml) and anti-CD29 (1 μg/ml) alone or stimulated with either HIV-1 clade B gag peptide pool (2 μg/ml; NIH AIDS reagent program, 12425) or with staphylococcal enterotoxin B (SEB; 2 μg/ml). To all cultures, FitC-labelled CD107a, BD GolgiStop and BD GolgiPlug (BD Biosciences) were added simultaneously. After 6 h incubation at 37 °C, cells were washed with PBS supplemented with 0.5% BSA and then stained extracellularly for 30 min at 4 °C in the dark with CD3 V500, CD4 BV650 and CD8 PB (BD Biosciences) followed by another wash step. For intracellular staining, cells were fixed and permeabilized using the BD cytofix/cytoperm kit (BD Biosciences) followed by staining for 30 min at 4 °C in the dark using the following fluorescently labelled antibodies: IL-2 PE, IFN-y APC AlexaFluor750, Mip1B PE-Cy7 and TNF-a AlexaFluor700. Before flow cytometry analysis, PBMC were washed using PBS supplemented with 0.5% BSA and fixed with CellFIX (BD Biosciences). Fluorescence was measured on the LSRFortessa (BD Biosciences) and marker expression levels were analysed using FlowJo Version 10.8.1 (TreeStar).

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).

Heparinized peripheral blood samples were collected from the subjects, and mononuclear cells (PBMCs) were obtained by Ficoll gradient centrifugation following the manufacturer's protocol (GE Healthcare Bio‐Sciences AB, Uppsala, Sweden). PBMCs were incubated for 15 min with human γ‐globulins at 4°C for FcR‐blocking. Then, the cells were washed and immediately labeled with specific antibodies or isotype controls for 30 min at 4°C. Specifically, antibodies against the following molecules were used: Vα24‐PE and Vβ11‐FITC (Beckman Coulter, Brea, CA), CD3‐PerCPCy5.5, CD4‐PE, (BioLegend, San Diego, CA), CD8‐PB (BD Pharmingen, Franklin Lakes, NJ), CRTAM‐APC (R&D, Minneapolis, MN), and CD69‐PETR (Beckman Coulter). The following isotype controls were used: IgG1‐PE, IgG2a‐FITC, IgG1‐PErCPCy5.5 (all from BioLegend), IgG1‐PB, (BD Pharmingen), IgG2b‐APC (R&D), and IgG1‐PETR (Beckman Coulter). After incubation, the cells were washed with PBS/2% FBS and were fixed with 4% paraformaldehyde in PBS.