Cytotoxic effects of HIV-1-specific CD8⁺ T cells can be preferentially mediated by a subset of cells producing both interferon-γ and TNF-α in responses to viral antigen stimulation. (A) PBMCs from study subject AC-16 were stimulated for 3 hours with the viral peptide B44-AW11 (AEQASQDVKNW). After labeling with fluorogenic interferon-γ- and TNF-α-catching reagents, HIV-1-specific CD8⁺ T cells were sorted according to their cytokine secretion pattern. The CD8+ T-cell populations producing both interferon-γ and TNF-α or interferon-γ but no TNF-α were then selectively tested in caspase-3-based cytotoxicity assays with peptide-pulsed BCLs at an E/T ratio of 1:1. Quadrants were set according to the caspase-3 activation in unloaded target cells. (B) Target cell apoptosis following incubation with sorted subsets of HIV-1-specific CD8⁺ T cells secreting either interferon-γ and TNF-α or interferon-γ only. Data reflect the means and standard deviations from 5 different epitope-specific CD8+ T-cell populations in 4 study subjects. (C) Target cell apoptosis induced by sorted TNF-α- and IFN-γ-secreting HIV-1-specific CD8⁺ T cells that were treated with concanamycin A (con A) or not. Data indicate means and standard deviations from 3 different experiments. (D) Intracellular expression of perforin in HIV-1-specific CD8⁺ T cells secreting either interferon-γ and TNF-α or interferon-γ only. Means and standard deviations from 12 different CD8⁺ T-cell populations are shown. (E) Proportion of HIV-1-specific CD8⁺ T cells with “effector” phenotype (CD45RA+/CCR7^-) within the subsets of cells secreting TNF-α and interferon-γ or interferon-γ alone. Data reflect means and standard deviations of 12 different CD8⁺ T-cell populations.