Reactivation of HIV-1 Latency in CD4+ T Cells

Resting CD4⁺ T cells cultures latently infected with HIV-1 were established and then reactivated as previously described with some modifications^{40 (link)}. Briefly, purified CD4⁺ T cells cultivated with 29 nM CCL19 for 1–3 days were infected by spinoculation with 300 ng p24/10⁶ cells of NL4-3 HIV-1 (NIH AIDS Reagent Program) pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) at 1200 g for 2 h, washed, and placed back in culture in complete medium alone at 5 × 10⁶/ml cell concentration. At day 3 post-infection, latently infected CD4⁺ T cells were collected and seeded at 3 × 10⁶/ml in complete medium alone or containing an HDACi (335 nM VOR, 10 nM ROM, 20 nM PAN or 100 nM ENT), 1 µM PRO, or HDACi + PRO combinations and supplemented or not with 12.5 ng/ml IL-15; also, a culture with 10 ug/ml PHA was set for maximal HIV reactivation. After 48 h of culture, cells were washed and reseeded at 2 × 10⁶/ml in the same initial conditions; finally, the intracellular p24 accumulation in T cells was analyzed by FACS and concentration of p24 in the culture medium was measured by ELISA (INNOTEST HIV Antigen mAb; FUJIREBIO, Japan) according to manufacturer’s protocol, 18 h and 5 days after the second stimulation, respectively.