Figure 4

HIV-1 induced T cell Caspase-1 activity and death in non-productively infected LP CD4+ T cells. (A) Detection of active Caspase-1 in T cells. At 2 and 6 dpi, LPMCs were harvested and incubated for 30 min with the CaspaLux-1 Substrate (Oncoimmunin) then surface stained to allow for identification of CD3+CD8- T cells by flow cytometry. Cells were gated from a tight lymphocyte gate that excluded cellular debris. Cleaved CaspaLux-1-E₁D₂ was detected in CD3+CD8- T cells using the FITC channel. (B) Representative histograms of Caspalux-1-E₁D₂ staining. (C) HIV-1 induced Caspase-1 activity in LP CD4+ T cells. The fold change in median Caspase-1 activity is shown at 6 dpi. Significance was determined using the Wilcoxon matched-pairs signed rank test. (D). YVAD (Caspase-1; 25 μM), DEVD (Caspase-3; 25 μM) or a DMSO vehicle control were added at 0 and 2 dpi and CD4+ T cell survival was evaluated at 4 dpi. LP CD4+ T cell survival was normalized to parallel mock infections. The number of cells per well in a representative donor with mock (white bar) or HIV-1 (grey bar) infection with DMSO (left), DEVD (center) and YVAD (right). The percent depletion from mock are indicated for each condition. (E) The percent depletion from mock infected controls are shown in the presence of DMSO (white box), DEVD (light grey), and YVAD (dark grey) (n = 6). Box and whiskers indicate the median and range respectively. Significant differences in the percent depletion from the DMSO vehicle were determined using non-parametric repeated measures ANOVA. Dunn’s multiple comparison tests were used to compare selected pairs of columns as shown. Overall ANOVA, p = 0.0003. *p = 0.02–0.03.