MHC-dependent killing of CD8⁺ T cells by imDCs is predominantly mediated by perforin and not by the NO system. (A) Representative perforin (green) immunostaining in CB6/F1 and PKO imDCs together with Hoechst 33342 (blue) nuclear staining. (b) Perforin (70 kD) expression was determined by Western blotting. (C-D) CD4⁺ TEA or 2C CD8⁺ alloreactive T cells stained with calcein AM green were incubated for 5 hours in the absence (blue) or presence of CB6/F1 (yellow), FVB (red), or PKO (green) imDCs treated or not with the NO inhibitor L-NAME (100 ng/mL) for 20 minutes at 37°C at a 4:1 cell ratio. At the end of the culture, the total number of live stained CD4⁺ TEA or 2C CD8⁺ T cells was evaluated by FACS. Average ± SD (n ≥ 3) shown. (E-G) The presence of perforin⁺CD11c DC under steady state. (E) In situ immunostaining for perforin (red) in the spleens and lymph nodes of CD11c-eYFP mice. Arrows indicate cells that coexpress both markers. Splenocytes from mice expressing eYFP-CD11c were isolated by FACS based on high expression of eYFP, MHC-II, and negative staining for CD3. (F) Phenotype of the enriched cells. (G) Sorted cells were spun onto a slide using Cytospin, fixed, and immunostained for perforin (red). (H-I) Typical immunostaining and quantification of perforin⁺ DCs in the spleens of mice 14 days after infusion of GM-CSF–secreting B-16 melanoma cells.