Cd8 pb clone rpa t8

Flow cytometry was performed on a LSRFortessa Flow Cytometer (BD Biosciences) and the data were analyzed using FlowJo software. The following antibodies and regents were used: CD8 PB (clone RPA-T8) was from BD Biosciences; CD45RA PE-Cy7 (clone H2100), CCR7 APC (clone 3D12), CD38 PE-Cy7 (clone HIT2) were from eBioscience; CD3 PE/Dazzle594 (clone UCHT1), CD25 PE (clone BC96), HLA-DR ECD (clone L243), T-bet PE (clone 4B10) were from BioLegend; p24 FITC (clone KC57) was from Beckman; Live/Dead™ blue dye (Invitrogen) was used for distinguishing live and dead cells. Measurements of cytokines in cell culture medium were performed with BD™ Cytometric Bead Array Th1/2/17 kit according to the manufacturer’s protocol.

Cryopreserved TIL or PBMCs were thawed, washed in FACS (Fluorescence Activated Cell Sorting) Wash Buffer [1X Dulbecco’s phosphate buffered saline (PBS) with 1% Bovine Serum Albumin]and stained for cell surface markers; CD8 PB (clone RPA-T8, BD (BD Biosciences)), MART-1 dextramer APC (Immudex), CCR7 PerCP-Cy5.5 (clone G043H7 BioLegend), CD45RA FITC (clone HI100, BD), CD27 APC-H7 (clone M-T271, BD), CD28 PE-Cy7 (clone CD28.2, BD), CD3 PE (clone SK7, BD). A viability stain was included for dead cell exclusion (the fixable dye Live/dead stain-Aqua, Invitrogen, or 7-AAD (7-Aminoactinomycin D) for fresh stains). The cells were fixed and acquired using a BD FACS Canto II flow cytometer. Live cells were gated based on fluorescence minus one controls. The data were analyzed using Flow Jo software (BD).

CD4+ T cells were obtained by negative magnetic selection from 300 × 10⁶ PBMCs and stained with the following antibodies: Live/Dead Aqua Cell Stain (ThermoFisher Scientific cat.L34957), CD8 PB (clone RPA-T8; BD cat.558207), CD14 V450 (clone MΦP9; BD cat.560349), CD45RA APC-H7 (clone HI100; BD cat.560674), α4/CD49d PE-Cy7 (clone 9F10; Biolegend cat.304313) and β1/CD29 BB515 (clone MAR4; BD cat.564565). Viable memory CD4+ T cells expressing VLA-4 (CD8-/CD14^- CD45RA^- α4^high β1^high) or not (CD8-/CD14^- CD45RA^- α4^low/− β1^low/−) were sorted in 5 mL FACS tubes. Cells were rested for 2 h at a final concentration of 1.5 million per mL, prior to serial dilution in culture plates (Costar) coated with 2.5 µg/ml anti-CD3 (clone OKT3) and 1 µg/ml anti-CD28 (clone CD28.2) antibodies as described elsewhere^{50 (link)}. MOLT-4 CCR5 + target cells (NIH HIV Reagent Program cat. ARP-4984) were added 2 days post-sort at a final concentration of 0.5 × 10⁶ cells/mL and the culture was maintained for 21-days. Supernatants were collected at day 7, 11, 14, 18, and 21 for soluble HIV-p24 protein quantification by ELISA^{92 (link)}. Infectious units per million of cells (IUPM) were determined for each population based on the number of positive wells for soluble p24 protein (http://silicianolab.johnshopkins.edu/)^{93 (link)}.