Figure 5

TNF-matured LCs transmit both X4 and R5 HIV-1. (A) Epidermal sheets were pretreated with different stimuli including Pam3CSK4 and TNF for 4 hours, pulsed with low and high titers of X4 and R5 HIV-1 for 5 hours (A) At day 3 emigrant LCs were collected, cocultured with CCR5 Jurkat T cells and HIV-1 transmission was determined after 3 days by measuring intracellular HIV-1 p24 or GFP expression by flow cytometry. Using T cell-marker CD3 and LC-marker CD1a, infection of the target cells were exclusively analyzed. Error bars represent the mean ± SEM of at least 3 independent experiments. (B) Epidermal sheets were pretreated with Pam3CSK4 and TNF for 4 hours or left untreated as control, following exposure to low or high titers of X4 and R5 HIV-1 in presence or absence of AZT for 5 hours. After 3 days, emigrated LCs were cocultured with CCR5 Jurkat T cells and transmission rate was determined at day 6 by flow cytometry. Error bars represent the mean ± SEM of at least 3 independent experiments. (C) Treatment of LCs with stimuli highly increased infection. Epidermal sheets were pretreated with Pam3CSK4 and TNF for 4 hours then pulsed with X4 HIV-1 for 5 hours. Infection of emigrant LCs was determined at 6–7 days by intracellular p24 staining in combination with LC-marker CD1a by flow cytometric analysis. Error bars represent the mean ± SEM of 3 independent experiments.