Fig. 5

HIV-1 latently infecting primary CD4⁺ T lymphocytes is activated upon challenge with exosomes from HIV-1 infected cells. Untouched CD4⁺ T lymphocytes isolated from PBMCs of four healthy donors were challenged by spinoculation with (VSV-G) ΔenvHIV-1 in the presence or not of AZT. As 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. a Intracytoplasmic CAp24 FACS analysis of CD4⁺ T lymphocytes 72 h post-infection. The results are the mean values + SD calculated after challenge of CD4⁺ T lymphocytes from four healthy donors in duplicate conditions. b Intracytoplasmic CAp24 FACS analysis of HIV-1 latently infected CD4⁺ T lymphocytes 24 h after spinoculation with 100 μU of exosomes purified from either uninfected U937 cells or their counterpart chronically infected with ΔnefHIV-1 and expressing or not wt Nef. As control, cells were treated with 10 ng/mL of PMA+ 0.5 μg/μL of 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 U937 cells expressing wt Nef. Shown are the results calculated as mean percentage values of triplicate cultures of CD4⁺ T lymphocytes from each donor. The inter-donor mean values + SD are also presented. *p < 0.05. The same experiments whose data are shown on panels a and b have been reproduced using PBMCs from two healthy donors and three doses (from 10 to 100 μU) of exosomes from cells infected with ΔnefHIV-1 and expressing wt Nef. In addition, CD4⁺ T quiescent lymphocytes challenged with 100 μU of exosomes were treated with either TAPI-2, anti-TNFα Abs, or isotype matched IgGs soon after the exosome challenge. c Intracytoplasmic CAp24 FACS analysis of CD4⁺ T lymphocytes 72 h post-infection. The results are the mean values + SD calculated after challenge of CD4⁺ T lymphocytes from two healthy donors in duplicate conditions. d Intracytoplasmic CAp24 FACS analysis of HIV-1 latently infected CD4⁺ T lymphocytes 24 h after spinoculation with the exosomes. As control, cells were treated with 10 ng/mL of PMA+ 0.5 μg/μL of 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. Shown are the results calculated as mean percentage values of duplicate cultures of CD4⁺ T lymphocytes from each donor