Exposure to IL-3 up-regulates functionally relevant surface molecules on pDCs and modulates their resistance to NK-mediated killing. (A) Peripheral-blood–derived pDCs were evaluated by immunofluorescence and cytofluorimetric analysis for the surface expression of CD86, HLA class I molecules, nectin-2, and CD48 both immediately after isolation from PBMCs and after 48 hours of culture in the presence of rhIL-3. The same surface markers were comparatively analyzed on iMDDCs and on matured MDDCs, induced by LPS. White profiles indicate isotypic negative control. Mean fluorescence intensity (MFI) is indicated in each histogram. One representative experiment is shown. (B) The same peripheral-blood–isolated pDCs were analyzed for their susceptibility to lysis by allogeneic polyclonal NK-cell lines at different E/T ratios. Specific ⁵¹Cr-release by pDCs was assessed both immediately after their isolation (□) and after 48 hours of culture in the presence of rhIL-3 (○). The cytolytic activity of polyclonally activated NK cells against pDCs was also evaluated in the presence of anti–HLA class I mAbs (▪ for freshly isolated pDCs, • for IL-3–activated pDCs, respectively). Each value represents the mean of triplicate experiments. The SD did not exceed 5%. Results are representative of at least 6 different experiments. (C) MDDCs were analyzed for their susceptibility to the same allogeneic polyclonally NK-cell lines that were used as effector cells against pDCs, at different E/T ratios. Specific ⁵¹Cr release was assessed against both iMDDCs (□) and LPS-matured MDDC (○). The cytolytic activity of polyclonally activated NK cells was also evaluated in the presence of anti–HLA class I mAbs (▪ iMDDCs, • mature MDDC, respectively). Each value represents the mean of triplicate experiments. The SD did not exceed 5%. Results are representative of at least 6 different experiments.