Macaque DCs, in contrast to PBLs and macrophages, are permissive for HIV-1 infection. (A) Human and macaque PBLs were activated using 20, 10, or 1 μg/mL phytohemagglutinin (human) or 10, 5, and 1 μg/mL concanavalin A (macaque), referred to as high (Hi), intermediate (Mi), and low (Lo) activation stimuli, respectively. Cells (0.2 × 10⁶) were then transduced with 10-fold dilutions (ng of p24/100 μL) of HIV-1 lentiviral vector TRIP ΔU3 CMV GFP. The percentage of GFP⁺ cells was evaluated by flow cytometry 4 days after transduction. Results are the mean of 3 experiments carried out on 3 separate human donors and 5 separate rhesus macaques plus or minus SD. (B) Representative flow cytometric data of transduced human and macaque PBLs (120 ng of p24 TRIP ΔU3 CMV GFP). (C) The activation phenotype of PBLs was monitored before transduction by HLA-DR surface labeling and flow cytometry. (D) Macaque and human PBLs were nucleofected with 3 μg of anti-TRIM5α or anti-Luc siRNA according to the manufacturer's instructions (Amaxa Biosystems, Gaithersburg, MD), and infected after 6 hours with 60 ng p24/300 μL of (VSV-G) NL43 GFP Δenv virus. The percentage of GFP⁺ cells was evaluated by flow cytometry 4 days after infection. (E) Images show phase contrast and GFP fluorescence of transduced macaque DCs acquired with a standard microscope (Carl Zeiss, Jena, Germany) with a 40× objective (LD Plan-Neofluar 40×/10.6 Corr) without immersion. Images were acquired using a Carl Zeiss AxioCam camera and AxioVision software. (F) Representative flow cytometric data of human and macaque DCs transduced or not with 120 ng of p24 TRIP ΔU3 CMV GFP. (G) Human and macaque DCs (0.2 × 10⁶) were transduced with 10-fold dilutions (ng p24/100 μL) of TRIP ΔU3 CMV GFP. The percentage of GFP⁺ cells was assessed by flow cytometry 4 days after transduction. Results are the mean plus or minus SD of 3 experiments carried out on 3 separate human donors and 5 separate rhesus macaques. (H) PBLs and DCs were isolated and activated/differentiated from human or Indian rhesus macaque blood. Cells (5 × 10⁵) were infected in 96-well plates with 2, 0.2, or 0 ng of p24 antigen/100 μL of (VSV-G) LAI-Luc pseudotyped virus. Luciferase activity in infected cells was measured 3 dpi by plate luminometer. (I) Human and macaque macrophages (0.6 × 10⁶) were transduced with increasing doses of TRIP ΔU3 CMV GFP vector. At 7 days after transduction, cells were observed by epifluorescence microscopy (Carl Zeiss) for phase contrast and GFP fluorescence using a 10× objective (120 ng/100 μL dose shown). Cells were then gently detached using ethylenediaminetetraacetic acid, and the percentage of CD14⁺GFP⁺ cells was assessed by flow cytometry.