Fig. 4.

Innate immune activation in macrophages and CD4⁺ T cells. a Macrophages and CD4⁺ T cells were transduced with HIV-1-GFP and assayed 3 days later for the indicated activation markers. b DCs, macrophages, and CD4⁺ T cells were challenged with HIV-1-GFP or the indicated mutants. When an essential viral component was disrupted within HIV-1-GFP, the factor in question was provided in trans during assembly in transfected HEK293 cells, as appropriate (see Methods). The upper panel shows flow cytometry of the DCs for GFP and CD86. The histograms show CD86 for DCs and macrophages or MX1 for CD4⁺ T cells. c 12-day spreading infections on CD4⁺ T cells with either macrophage-tropic or T cell-tropic, replication-competent HIV-1. d CD4⁺ T cells were stimulated for 3 days with PHA and IL2, and transduced with Tet-HIV-1 and rtTA3. Cells were then cultured without stimulation for 9 days. Doxycycline was then added at the indicated concentrations. Cells were assayed for GFP and MX1 3 days later. Shown are blood donor data representative of n = 6 (a), n = 4 (b–d). To determine significance, the MFI of individual flow cytometry samples was calculated as fold-change versus control. The exception being c where the MFI of only GFP+ cells was compared. When data from each donor replicate within a experiment were combined, the difference in MFI for all experimental vs control conditions was significant in all cases, p < 0.01; one-way ANOVA, Dunnett’s post-test against lentivector control for a–c or dox control for d