Latent HIV-1 Reactivation by Anti-PD-1 Antibody

Different cell concentrations (fivefold limiting dilutions, i.e., 5× 10⁵, 1 × 10⁵, 2 × 10⁴ and 4 × 10³ cells of sorted viable blood resting memory (CD45RA^-HLA-DR^-CD25^-CD69^-) CD4 T cells from aviremic ART treated HIV-infected individuals were co-cultured with autologous irradiated CD8-depleted blood mononuclear cells (10⁶ cells/ml), as previously described [44 (link)], in presence or in absence of blocking anti-PD-1 Mab, Pembrolizumab, at 5 μg/mL or isotype control (Eureka Therapeutics). As a positive control, sorted cells were stimulated for 3 days with anti-CD3 and anti-CD28 mAb-coated plates (10 μg/ml) in presence of IL-2 (50 units/ml). Supernatants were collected at days 0, 5 and 14. Medium was replaced at day 5. Quantification of replication competent HIV-1 was performed at 14 by assessing HIV-1 RNA levels by COBAS AmpliPrep/TaqMan HIV-1 Test (Roche; Switzerland) as previously described [44 (link)]. Wells with detectable HIV-1 RNA (≥200 HIV-1 RNA copies/ml) were referred to as HIV-1 RNA-positive wells. The frequency of latently HIV-1 infected CD4 T cells reactivated by IC MAbs was estimated by conventional limiting dilution methods using Extreme Limiting Dilution analysis (http://bioinf.wehi.edu.au/software/elda/) [63 (link)] and expressed in RNA-unit per million (RUPM) as previously described [44 (link)].