Cd3 brilliant violet 711 clone okt3

PBMCs were thawed, resuspended in R-10 medium and added to 96-well plates at 500,000 cells/well in 200 μl/well of R-10 medium. Peptides were added at the same concentrations as in ELISPOT assays for 6 hour stimulations in the presence of 1 ug/ml each brefeldin A (GolgiPlug, BD Biosciences) and monensin (GolgiStop, BD Biosciences), and 2 μl/well of anti-CD107a-PerCP-Cy5.5 (clone H4A3, Biolegend). Cells were then washed in 1% FBS PBS and surface stained with fluorochrome-conjugated antibodies to CD3 Brilliant Violet 711 clone OKT3, CD4 Brilliant Violet 650 clone OKT4, CD8-Alexa Fluor 700 clone HIT8a, CD62L-PE-Cy5 clone DREG-56, CD45RA-Alexa Fluor 488 clone HI100, PD-1-Brilliant Violet 605 clone EH12.2H7 (all from Biolegend), Tim-3-PE (clone 344823, R&D Systems), as well as fixable aqua viability dye (Life Technologies). Cells were fixed and permeabilized using the BD Cytofix/Cytoperm system, following the manufacturer’s instructions. Permeabilized cells were then stained with anti-IFN-γ-APC (BD Pharmingen, clone B27) and anti-MIP-1-β-APC-H7 (BD Pharmingen, clone D21-1351). Cells were analyzed on a BD LSR Fortessa X20 flow cytometer with FACSDIVA software. Data were analyzed using FlowJo software, TreeStar.

The CD4 + T-cell subsets were sorted from frozen aliquots of peripheral blood mononuclear cells (PBMCs) stored for 4–16 years which contained 50–100 × 10⁶ cells that were collected from 5 of the participants at approximately years 3, 5, 10 on ART (Visit ID 1–3). At approximately 15 years of therapy, CD4+ T-cells were isolated from a leukapheresis for all 6 participants (Visit ID 4). Participant 2518 had a second leukapheresis sample collected for this study 2 years later at approximately 17 years on therapy (Visit ID 5). The cells were sorted using the following antibodies: CD3-Brilliant Violet 711 (clone OKT3, BioLegend), CD4-APC-eFluor 780 (clone OKT4, Thermo Fisher), CD14-V500 (clone M5E2, BD# 561391), LIVE/DEAD Aqua marker (Invitrogen# L34957), CD45RA-PECF594 (clone HI100, BD Biosciences), and HLA-DR− Brilliant Violet 421 (clone L243, BioLegend). CD3+CD4+ T-cells were gated on memory cells defined as CD45RA negative. HLA-DR± cells were then sorted on a BD FACS ARIA-II (Supplementary Figure 1). The HLD-DR± populations were sorted to >99% purity. Following sorting, the cell subsets were processed at 4°C and stored as a dry cell pellet at −80°C until analysis.