Extended Data Figure 3: Necrostatin-1 does not prevent lymphoid CD4 T-cell death and cell lysis in HIV-infected cultures.
From: Cell death by pyroptosis drives CD4 T-cell depletion in HIV-1 infection
a, b, Necrostatin was tested at 1 or 5 μM, a concentration that yields maximal inhibition without inducing toxicity (not shown). Pyroptosis shares cell death features with necroptosis which similarly leads to the release of intracellular contents into the extracellular space9. To test whether cell death involves necrotic signalling, we treated HIV-infected CD4 T cells with necrostatin, a specific inhibitor of RIP1, whose kinase activity is essential for programmed necroptosis to occur60. Concentrations of necrostatin that block necroptotic signalling (not shown) did not inhibit CD4 T-cell depletion in HIV-infected cultures (a), and did not prevent the release of intracellular contents into the culture medium, as indicated by LDH activity in the supernatants (b). Thus, although pyroptosis shares features with necroptosis, these data demonstrate that the signalling pathways linking caspase 1 activation to CD4 T-cell death are specific. Together, these findings indicate that the CD4 T-cell depletion and release of cytoplasmic contents in HIV-infected lymphoid cultures reflects pyroptosis rather than apoptosis or necroptosis. Error bars represent s.e.m. of at least three independent experiments using tonsil cells from at least three different donors. c, Caspase 1 inhibitors prevent CD4 T-cell death in HIV-infected splenic tissues. Splenic HLACs were cultured with no virus or were infected with HIV-1. The HIV-infected cultures were treated as indicated, either with no drugs, efavirenz (100 nM), the caspase 1 inhibitor Ac-YVAD-CMK (50 μM), or the caspases-3 inhibitor Z-DEVD-FMK (50 μM). After 4 days, viable CD4 T cells were counted by flow cytometry. Viable CD4 T cells are presented as the percentage remaining live CD4 T cells using CD8 T cells to normalize each HIV-infected or uninfected culture. Error bars represent s.e.m. from four independent experiments using tonsil cells isolated from four different donors.
