Close contact between TRAIL-expressing pDCs and apoptotic T cells in lymph nodes of patients infected with HIV-1. (A) A quantitative analysis of pDCs in lymph nodes of viremic patients infected with HIV-1 (n = 8) and controls (n = 5) was performed by immunofluorescence staining with an anti–BDCA-2 mAb combined with anti–HLA-DR, anti-CD45RA, or anti-CD123 mAbs. The quantity of TRAIL-expressing pDCs was analyzed by anti-TRAIL/anti–BDCA-2 staining. Data are given as absolute numbers of double-positive cells per square millimeter ± SEM. (B) Representative views of anti–BDCA-2/anti–HLA-DR, anti–BDCA-2/anti-CD45RA, and anti–BDCA-2/anti-CD123 stainings of a lymph node of a viremic patient infected with HIV-1. Arrows denote double-positive pDCs. (C-E) Immunofluorescence triple labeling of lymph nodes of patients infected with HIV-1 and HIV-1–seronegative controls with (C) TUNEL (FITC), anti-CD4 (TRITC), and anti–BDCA-2 (Cy5); (D) TUNEL (FITC), anti-CD3 (TRITC), and anti–BDCA-2 (Cy5); or (E) TUNEL (FITC), anti-TRAIL (TRITC), and anti–BDCA-2 (Cy5) showed BDCA-2⁺, TRAIL-expressing pDCs surrounded by apoptotic T cells in HIV-1 lymph nodes. Arrows denote BDCA-2⁺ pDCs. (F) To quantify the proximity of pDCs or monocytes to apoptotic T cells, the contacts of BDCA-2⁺ or CD14⁺ cells to TUNEL⁺CD3⁺ cells were enumerated in lymph nodes of HIV-infected patients and healthy controls. The data are given as absolute numbers of indicated cells in contact to apoptotic T cells ± SEM. (G) Immunofluorescence triple labeling of lymph nodes of viremic patients infected with HIV-1 with TUNEL (FITC), anti-TRAIL R1, or anti-TRAIL R2 (TRITC) and anti-CD3 (APC) denotes apoptotic T cells as TRAIL R1–expressing and TRAIL R2–deficient cells. Arrows denote triple-positive cells. (B-E,G) Original magnification ×400. Pictures were recorded with a Zeiss Axiovert 200M microscope (40×/1.30 oil objective), a HAL100 camera, and LSM510 software (release 3.0, update 3.2) in a 1024 × 1024 solution.