Figure 4

HIV-1 replicates in unstimulated CD4⁺T lymphocytes treated with exosomes from F12/Hut-78 cells. A. FACS analysis of challenged lymphocytes. 10⁵ CD4⁺ T lymphocytes were challenged with 60 μU of exosomes from F12/HIV-1 cells, and then infected with HIV-1. Alternatively, exosomes and HIV-1 were added to cells at the same time, or cells were first infected and then challenged with the exosomes. As control, cells were infected with or without PHA. Three days later, the cells were scored for the expression of HIV-1 Gag by FACS analysis. In the upper panels, shown are the dot-plots from a representative experiment. Percentages of HIV-1 Gag positive cells are indicated. In the bottom panel, shown are the fold increases of the percentages of HIV-1 CAp24-positive cells compared to cultures treated with HIV-1 alone. Shown are the mean of fold increases + SD as calculated from five independent experiments with duplicates. *p < 0.05. B. Dose–response effect of exosomes. Shown are the mean of fold increases + SD as calculated from three independent experiments with triplicates. *p < 0.05. C. Effects of AZT. Cultures were run in the absence (Nil) or in the presence of 10 μM AZT. Shown are the mean of fold increases + SD as calculated from three independent experiments with duplicates. *p < 0.05. D. Detection of infectious HIV-1. 10⁵ CD4⁺ T lymphocytes were challenged with 60 μU of exosomes from either Hut-78 or F12/Hut-78 cells, and then infected with HIV-1 with or without AZT. Three days later, co-cultures with Rev-CEM cells were undertaken. After additional 3 days, GFP positive Rev-CEM cells were scored by FACS. Shown are the mean of fold increases of GFP⁺ Rev-CEM cells from exosome-treated cells compared to co-cultures with lymphocytes treated with HIV-1 alone, as calculated from two independent experiments with duplicates.