Mechanisms for the inhibitory effects of LRDCs on the antigen-specific CD4 T-cell proliferation. (A-C) CD4 T cells of DO11.10 × C57BL/6 F1 hybrid mice were labeled with 5 μM CFSE and cocultured with cDCs or/and LRDCs in the presence of OVA323-339 peptide for 5 days; the divisions of CFSE (A) and intracellular staining for IFN-γ secretion (C) of CD4 T cells were then analyzed by FACS. PGE2 and IFN-γ released in the supernatant were assayed by ELISA (B). In the experiments using the transwell system, LRDCs were seeded into the upper chamber of the transwell with CD4 T cells and cDCs in the bottom of a 24-well plate. (D) A variety of neutralizing antibodies or blocking reagents were added into the coculture system, and the inhibitory functions of LRDCs were assayed. (E) Detection of T-cell apoptosis in the coculture system. (F,G) LRDCs or/and cDCs derived from IFN-γ knockout mice were used in the coculture system, CD4 T-cell proliferation was detected by FACS (F), and the level of IFN-γ in supernatant was assayed by ELISA (G). Data represent the mean plus or minus SD of triplicate wells and are representative at least from 3 independent experiments. *P < .001; **P < .01; ***P > .05.
