Extended Data Figure 7: Inhibitors of caspase 1, but not of NLRP3, prevent CD4 T-cell death by HIV-1.

a, Quantitative evaluation of bioactive IL-1β secreted in HIV-infected CD4 T-cell cultures using ELISA. Isolated tonsillar CD4 T cells were left uninfected or infected with HIV in the presence of the indicated drugs. Four days after infection, supernatants were filtered through 0.22-μm filter plates and subjected to IL-1β ELISA analysis. A total of 200 μl of supernatant from 2 million isolated CD4 T cells was used for each condition. The assay was performed as described by the manufacturer’s instructions (R&D Systems). Bioactive IL-1β was detected in supernatants of HIV-infected cultures, at levels comparable to those in uninfected cells treated with nigericin. Treatments of HIV-infected cultures with viral or caspase 1 inhibitors, but not caspase 3 inhibitor, reduced accumulation of IL-1β in the supernatants to levels comparable to those detected in uninfected cultures. These finding demonstrate that caspase 1 activation is specifically required for the release of bioactive IL-1β in lymphoid CD4 T cells infected with HIV-1. Error bars represent s.e.m. of three independent experiments using tonsil cells from at least three different donors. b, Inhibitors of caspase 1 and the NLRP3 inflammasome prevent release of mature IL-1β induced by nigericin, but not CD4 T-cell death by HIV-1. Because nigericin engages the NLRP3 inflammasome to activate caspase 1 in lymphoid CD4 T cells, we sought to determine if NLRP3 also similarly controls caspase 1 activity in response to HIV-1 infection. Cell cultures were treated with four separate NLRP3 inhibitors including CRID3⁶⁴, parthenolide⁶⁵, and the sulfonylureas glyburide⁶⁶ and glimepiride. Treatments with CRID3, parthenolide or sulfonylureas (not shown) completely inhibited NLRP3-dependent release of mature IL-1β by nigericin, but had no effect on IL-1β release triggered by HIV infection of lymphoid CD4 T-cell cultures (Fig. 3f). c, Treatments with CRID3, parthenolide or sulfonylureas did not prevent HIV-1-mediated CD4 T-cell death. These results suggest that the NLRP3 inflammasome does not control the caspase-1-mediated death responses in lymphoid CD4 T cells abortively infected with HIV-1. Cell death results are represented as ratios of viable CD4 versus CD8 T cells in each HIV-infected or uninfected culture. Error bars represent s.e.m. of four independent experiments using tonsil cells from four different donors.