Modulation of HIV-1 Production by Immune Checkpoint Ligands

Sorted LN PD-1^high and PD-1^- memory (CD45RA^-) CD4 T-cell populations (2 × 10⁵ cells) were cultured for 6 days at 37°C and 5% CO₂ in 96-well U-bottom plates coated with anti-CD3 (BD) and anti-CD28 (BD) (10 μg/ml) in presence or in absence of recombinant IC ligands at 100 μg/mL in complete RPMI. In some experiments, the combination of two IC-ligands was also tested. Of note, all experiments were performed in presence of 75 nM emtricitabine to prevent re-infection of stimulated CD4 T cells. Supernatants were collected at days 6 and quantification of HIV-1 production was performed by assessing HIV-1 RNA levels by COBAS AmpliPrep/TaqMan HIV-1 Test (Roche; Switzerland) as previously described [44 (link)].