Fas ligation enhances endocytosis by neutrophils and activation and proliferation of antigen-specific T cells. (A) DCs that were treated with medium, Jo-2, isotype Ab, or LPS for 12 hours were incubated with neutrophils for 20 hours. For separate Jo-2 DC/PMN (polymorphonuclear neutrophil) coculture, we seeded Jo-2–treated DCs on the upper chamber, whereas neutrophils were seeded onto the bottom of a 1.0-μm pore sized transwell. Then, 2 μL Fluospheres per well was added into the coculture system, and 3 hours later the cells were collected and stained with CD11c-PE to gate CD11c^- neutrophils. Cells were detected by FACS, and the FL1 fluorescence of the CD11c^- cells neutrophils was analyzed. (B) Purified CD4 T cells were cocultured with DCs treated as indicated in the presence of OVA_323-339 peptide at a ratio of 1:10 (DC/T) in round-bottom 96-well plates for 5 days. Then cells were collected and double stained with anti-CD4–FITC and 7-AAD and counted by FACS. Supernatants were collected at 24 hours for assay of IL-2 and IFN-γ by ELISA. (C) Purified CD4 T cells were cocultured with DCs treated as indicated in the presence of OVA_323-339 peptide at a ratio of 1:10 (DC/T) in the IFN-γ ELISpot plate (3 × 10⁴ T cells/200 μL/well) for 24hours. Then, following the manufacturer's instruction, the microplate was developed and dried in the air. Photos were taken using an Olympus 52X12 dissection microscope with a Cohu high-performance CCD camera (Cohu, San Diego, CA) and Leica QWin imaging software (Hamburg, Germany).