Figure 4.

Dendritic cells (DCs) from NKG2D-deficient mice have impaired responsiveness to Toll-like receptor (TLR) ligands. Dendritic cells were isolated from lung and spleen and derived from bone marrow and treated with 20 ng/mL of interferon γ for 2 hours before being treated with 1 µg/mL of polyinosinic:polycytidylic acid (TLR 3), 1 µg/mL of lipopolysaccharide (LPS) (TLR 4), 100 ng/mL of SSRNA40 (TLR 7), and 50 ng/mL of CpG oligodeoxynucleotides 1826 (TLR 9) for 16 hours. The supernatant was collected, and interleukin 12 (IL-12) p40 was measured by enzyme-linked immunosorbent assay (A). Data are shown as the mean ± SEM of at least 5 independent experiments. *P < .05, ** P < .01. B, Schematic flow cytometry plot of intracellular staining of IL-12 p40 after LPS treatment. Macrophages were gated as CD11c⁺ autofluorescence^high, and DCs were gated as CD11c⁺, autofluorescence^low. C, Freshly isolated DCs were pulsed with 100 ng/mL of OVA peptide for 1 hour, then cultured with carboxyfluorescein succinimidyl ester-labeled OT-1 T cells at 2:10 ratio. The OT-1 T cell proliferation index was measured after 5 days. Data are shown as average proliferative index ± SEM from 4 independent experiments. Abbreviations: BMDC, bone marrow-derived dendritic cell; DC, dendritic cell; IL-12, interleukin 12; KO, knockout; MAC, macrophage; OVA, ovalbumin; TLR, Toll-like receptor; WT, wild-type.