Pd 1 apc mih4

PBMCs were used for staining with fluorochrome-conjugated monoclonal antibodies against cell surface markers for T cell activation, exhaustion, and memory differentiation as described previously.²¹ Briefly, freshly isolated PBMCs were washed in phosphate buffered saline, adjusted to two million cells per stain, and incubated with titrated concentrations of the following antibodies in a staining volume of 50 μL in PBS: 1:100 CD3/BV605 (SK7; BD Biosciences), 1:100 CD4/V450 (RPA-T4; BD Biosciences), 1:100 CD8/V500 (RPA-T8; BD Biosciences), 1:50 PD-1/APC (MIH4; eBioscience), 1:25 CCR7/PE (150503; R&D Systems), 1:50 CD45RA/Alexa Fluor 700 (HI100; BioLegend), 1:25 HLA-DR/FITC (L243; BD Biosciences), and 1:25 CD38/PE-Cy7 (HIT2; BD Biosciences). After 20 min incubation at room temperature cells were washed twice in PBS and resuspended in PBS with 2% paraformaldehyde. Near-infrared LIVE/DEAD marker (Invitrogen) was used to exclude nonviable cells. Fluorescence minus one staining was used to determine the cutoff for PD1 high cells. Data were acquired on a LSR-II driven by FACSDiva software (BD) and analyzed using FlowJo software v8.8.6 (TreeStar, Ashland, OR). All FACS analyses were performed using freshly isolated PBMCs at the HIV Pathogenesis Programme, UKZN, Durban, South Africa.