CD103^hi iCD103-DCs cross-present cell-associated antigens. (A) GM-DCs, FL-DCs, and iCD103-DCs were seeded at equal numbers. OVA-Alexa647 was added for 10 minutes at 37°C. Controls were kept on ice or were untreated. The uptake of OVA was quantified by flow cytometry. Plots display CD11c expression vs OVA-Alex647 signal intensity among CD11c⁺ GM-DCs (top), CD11c⁺B220^– FL-cDCs (middle), and CD11c⁺B220^– iCD103-cDCs (bottom). Numbers indicate percentages of DCs that have actively internalized OVA-Alexa647. (B-D) CD11c⁺ GM-DCs, CD11c⁺B220^–CD103^hi iCD103-DCs, lung CD103⁺ DCs, and lung CD11b⁺ DCs were FACS-sorted. DCs were co-cultured at varying ratios with CD8⁺CellViolet⁺ .OTI T cells in the presence of (B) irradiated OVA-coated B2m^−/− splenocytes or (C-D) irradiated B16-OVA melanoma cells and CpG. Proliferation was quantified by flow cytometry after 5 days of culture. (B,D) The graphs show the numbers of proliferated CD8β⁺ OTI cells (mean values of duplicate wells) in response to (B) splenocytes or (D) B16-OVA cells. (C) Representative histograms show CellViolet dilution of CD8β⁺ OTI cells in response to B16-OVA. Wells without DCs (No DCs) or without DCs and B16-OVA cells (CpG only) served as controls. Results are representative of 2 independent experiments (A-D).