pDCs matured in the presence of IL-3 and IL-10 deliver enzymatically active GrB to CD4⁺ T cells. pDCs from healthy donors were purified and cultured for 60 hours in the presence of IL-3 and IL-10. Then, pDC (> 95% purity) were harvested and added to chamber slides containing purified immobilized CD4⁺ T cells (> 98% purity) stained with Cell Mask deep red membrane dye. Finally, GranToxiLux fluorogenic GrB substrate was added to the chamber slides and cocultures were analyzed for 2.5 hours using spinning disk confocal microscopy. Images were deconvolved using ImageJ software. (A) GrB⁺ pDCs are displayed in green-yellow, cell membranes in red, and active GrB internalized by immobilized CD4⁺ T cells in green. No green or yellow fluorescence was found when pDCs were cultured in the absence of IL-3 and IL-10 (not shown). (B) Shown is a sequence of detailed images of 1 individual CD4⁺ T cell before and during contact to a GrB⁺ pDCs (blue arrows indicate visible contact). Note the rapid diffusion of active GrB within the T cell after first contact to GrB⁺ pDCs.