Cytotoxic activity of freshly isolated pDCs from viremic patients infected with HIV-1. (A) Isolated pDCs from (1) viremic patients infected with HIV-1 and (2) nonviremic patients infected with HIV-1, and (3) healthy controls were cultured with CD4⁺ T cells from the respective patient groups for an europium-TDA release assay at the effector/target cell ratios indicated. Data represent means of duplicate wells ± SEM from 1 of 3 independent experiments. (B) Freshly isolated monocytes, pDCs, NK cells, and CD8⁺ T cells were incubated with autologous CD4⁺ T cells from viremic patients infected with HIV-1 for an europium-TDA release assay at the effector/target cell ratios indicated. Data represent means of duplicate wells ± SEM from 2 independent experiments. (C) pDCs from a viremic patient infected with HIV-1 as effector cells were preincubated with indicated neutralizing antibodies and, thereafter, with autologous CD4⁺ T cells. Cytotoxicity was determined by an europium-TDA release assay at the effector/target cell ratios indicated. Data represent means of duplicate wells ± SEM from 4 independent experiments. (D) To elucidate the relevance of different cell populations and cytotoxic molecules for the killing of CD4⁺ T cells in peripheral blood, we either removed pDCs, monocytes, or cytotoxic T cells or added the inhibitory substances indicated (neutralizing antibodies, concanamycin A) before a 12-hour culture period of PBMCs from (1) viremic patients infected with HIV-1 and (2) nonviremic patients infected with HIV-1 and (3) healthy controls. Early apoptosis of CD4⁺ T cells was determined by Annexin V/7-AAD/CD4/CD3 quadruple FACS stainings before and after the culture. Data represent means of duplicate wells ± SEM from 1 of 2 independent experiments.