Maturation, Ag presentation capacity, and IL-12 production of DCs Maturation, Ag presentation capacity, and IL-12 production of DCs. (A) Cell-surface expression of CD80, CD86, and CD40 on BMDCs before (shaded area) or 24 h after (outlined area) stimulation, with LPS were examined by flow cytometry. Maturation, Ag presentation capacity, and IL-12 production of DCs. (A) Cell-surface expression of CD80, CD86, and CD40 on BMDCs before (shaded area) or 24 h after (outlined area) stimulation, with LPS were examined by flow cytometry. Representative flow cytometry profiles from three independent experiments, each using six WT and six PYNOD-KO mice, are shown. (B and C) BMDCs from WT and PYNOD-KO mice were pulsed with OVA in the presence of LPS for 6 h. OVA-specific naive CD4+ T cells from OT-II Tg mice were labeled with CFSE and cocultured with OVA-pulsed BMDCs. (B) Three days later, the proliferation of CD4+ T cells (CFSE dilution) was assessed by flow cytometry. Representative flow cytometry profiles from three independent experiments, each using six WT and six PYNOD-KO mice are shown. The vertical dashed lines are arbitrary lines that are provided as aid for comparison of CFSE dilution levels between the upper and lower profiles. (C) IFN-γ concentration in the culture supernatants was measured by ELISA. Averages of data obtained using two WT and two PYNOD-KO mice are shown. Consistent results were obtained in another similar experiment. (D) WT and PYNOD KO BMDCs were stimulated with 1 μM CpG ODN. Twelve hours later, secretion of IL-12p70 was determined by ELISA. Consistent results were obtained from three similar experiment. (E and F) WT and PYNOD KO BMDCs were stimulated with 1 μM CpG ODN for 2 and 4 h. mRNA expression of IL-12p35 and p40 were determined by quantitative RT-PCR. Consistent results were obtained from three similar experiments. (C–F) No significant differences were observed between WT and PYNOD-KO cells. Shinsuke Nakajima et al. ImmunoHorizons 2018;2:129-141 Copyright © 2018 The Authors