Quantifying HIV Latency in Memory CD4+ T Cells

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)}.