αifnγ apc 4s b3

For all experiments, frozen PBMCs were thawed and rested for 16-18h in complete RPMI 1640 media at 37°C in a 5% CO₂ incubator before starting the experiments. Phenotypic analyses of PBMCs were performed using surface antibody staining for 30 minutes at 4°C, using combinations of the following anti-human monoclonal antibodies: αCD3-PE/Dazzle594 (OKT3), αCD3-FITC (OKT3), αCD38-PE/Cy7 (HB-7), αCD19-BV650 (HIB19), αCD56-BV750 (5.1H11), αCD4-PerCP/Cy5.5 (OKT4), αCD8-AF700 (SK1), αCCR7-APC/Fire (G043H7), αCD45RO-BV711 (UCHL1), all purchased from BioLegend. After staining the cells with antibodies for the phenotypic markers cells were fixed in 1% paraformaldehyde and acquired in a flow cytometer. The functional capacity of T cells was determined by analyzing the intracellular cytokine production upon stimulation with overlapping HIV-Gag polyprotein peptides using αIFNγ-APC (4S.B3) and αTNFα-PE (MAb11) antibodies from BioLegend. After staining the cells for surface markers, cells were permeabilized with Cytofix/Cytoperm (BD Biosciences) followed by intra cellular cytokine staining and finally fixed in 1% paraformaldehyde. Live/dead cell discrimination was performed using Zombie Aqua fixable viability Kit (BioLegend). Data were acquired and compensated using Cytek Aurora (Cytek Biosciences) flow cytometer and further analyzed with FlowJo (version 10.7.1).