Fig. 3
HIV-1 latently infecting U1 cells is activated upon challenge with exosomes from HIV-1 infected cells in a Nef-, TNFα-, and ADAM17-dependent manner. a Different amounts of exosomes (i.e., from 30 to 120 μU of AchE activity) purified from supernatants of the indicated cell lines were used to challenge 5 × 104 U1 cells in U-bottom 96 well plates. After 24 h, the cells were extensively washed, and the amount of released HIV-1 was measured in terms of CAp24 concentration in the supernatants, after additional 24 h. As a control, cells were left untreated (Ctrl) or treated with 100 ng/mL of recombinant TNFα. The results are the mean values + SD from five independent experiments carried out with duplicates. *p < 0.05. b ADAM17 activity detected in 1 mU of exosomes purified from the supernatants of the indicated U937-derived cell lines. Shown are mean amounts of active ADAM17 + SD detected in quadruplicate samples from a representative exosome preparation from each cell line. c Effects of TAPI-2 and anti-TNFα on the activation of latent HIV-1 induced by exosomes from HIV-1 expressing cells. U1 cells (5 × 104/condition) were challenged with 100 μU of exosomes from HIV-1 infected cells expressing wt Nef. Then, cells were left untreated or incubated in the presence of either 1 μM TAPI-2, 160 ng/mL of anti-TNFα neutralizing antibodies, or equivalent amounts of unrelated, isotype-specific IgGs. As a control, cells were treated with 100 ng/mL of recombinant TNFα in the presence or not of the anti-TNFα neutralizing antibodies. After 24 h, cells from all conditions were extensively washed, re-seeded in the appropriate conditions, and the amount of HIV-1 released in supernatants was measured as CAp24 concentration after additional 24 h. The results are the mean values + SD from three independent experiments carried out with duplicates. *p < 0.05