Rhil 15

CEM-NKr CCR5⁺ cells (49 (link)– (link)51 (link)) (NIH AIDS reagents program) were infected with vesicular stomatitis virus glycoprotein (VSV-g)-pseudotyped HIV-1 (strain JRCSF, 0.5 IU/cell). Infection was facilitated with Polybrene (4 μg/ml) and spinoculation (800 × g for 45 min at room temperature [RT]). Cells were then incubated for 2 days at 37°C and media (RPMI supplemented with 10% fetal bovine serum [FBS], l-glutamine, and penicillin/streptomycin [Pen/Strep]) exchanged once 6 h after infection. NK cells were isolated from buffy coats from healthy donors using the RosetteSep NK cell enrichment kit (STEMCELL Technologies) and stimulated with rhIL-15 (1 ng/ml; STEMCELL Technologies) at 37°C overnight. Infected CEM-NKr CCR5⁺ cells were labeled with CellTrace far red cell proliferation kit (Thermo Fisher) before 1:50 diluted plasma sample were added together with the NK cells. After 4 h of incubation at 37°C, cells were stained with LIVE/DEAD Fixable Blue Dead Cell Stain kit (Thermo Fisher). Cells were then fixed and permeabilized with Fixation/Permeabilization Solution kit (BD) and stained for intracellular HIV-p24 (Beckman Coulter, CA, USA; clone KC57). Cell killing was calculated as frequency of viable, p24⁺ CEM-NKr CCR5⁺ cells compared to a negative control without antibodies (phosphate-buffered saline [PBS]).