Fig. 6

Exosomes from HIV-1 productively infected primary cells activates latent HIV-1 infecting primary CD4⁺ T lymphocytes. Untouched CD4⁺ T lymphocytes from PBMCs of healthy donors were challenged by spinoculation with (VSV-G) ΔenvHIV-1 in the presence or not of AZT. As a control, conditions with unchallenged CD4⁺ T lymphocytes (Ctrl) were included. After 48 h, cells were extensively washed, and then left in culture for additional 24 h. Afterwards, exosome challenge was performed as described below. a FACS analysis for the presence of both GM1 and CD63 in nanovesicles recovered from pools of AchE positive fractions of iodixanol gradients loaded with nanovesicle pellets from supernatants of either CD4⁺ T lymphocytes or MDMs infected with (VSV-G) wtHIV-1. Vesicles were bound to aldehyde/sulfate latex beads and then labeled with FITC-conjugated CTX-B and PE-conjugated anti-CD63 mAb. b Intracytoplasmic CAp24 FACS analysis of CD4⁺ T lymphocytes 72 h post-infection. The results are expressed as mean HIV-1 Gag⁺ percentage calculated after challenge of CD4⁺ T lymphocytes from three healthy donors analyzed in duplicate conditions. c Intracytoplasmic CAp24 FACS analysis of HIV-1 latently infected CD4⁺ T lymphocytes 24 h after spinoculation with 100 μU of exosomes purified from HIV-1 infected CD4⁺ T lymphocytes or MDMs. As a control, the cells were challenged with equivalent amounts of exosomes from uninfected CD4⁺ T lymphocytes or MDMs. CD4⁺ T lymphocytes were also treated with PMA + ionomycin (PMA) or left untreated (Ctrl). In addition, CD4⁺ T lymphocytes originally challenged with (VSV-G) ΔenvHIV-1 in the presence of AZT were treated with either PMA + ionomycin or exosomes from HIV-1 infected cells, i.e. CD4⁺ T lymphocytes and MDMs. The results are reported as mean percentage values + SD of duplicate cultures of CD4⁺ T lymphocytes from three healthy donors. *p < 0.05. The same experiment whose results are shown in the panel C has been conducted with CD4⁺ T lymphocytes from two healthy donors which were latently infected with ΔenvHIV-1 in the presence or not of AZT. Panel d reports the mean percentage values of HIV-1 expressing cells 72 h after challenge. Then, cells were challenged with three doses of each exosome preparation, i.e., from 10 to 100 μU, or with PMA as a control. In addition, cell challenged with 100 μU of exosomes were treated with either TAPI-2, anti-TNFα Abs, or isotype matched IgGs soon after exosome challenge. The results calculated as mean percentage values of duplicate cultures of CD4⁺ T lymphocytes from each donor are shown in panel e, f. ADAM17 activity detected in 1 mU of exosomes purified from the supernatants of both uninfected or HIV-1 infected CD4⁺ T lymphocytes and MDMs. Shown are mean values of ng of active ADAM17 detected in quadruplicate samples from a representative exosome preparation from each cell culture