Figure 5. Hif1α is required for DC immunogenicity.

Conditional Hif-1α^flox/−. CD11c-Cre⁺ (HIF1α^−/−) or HIF1α^+/− control mice were injected with 50 µg poly IC IP. 14 h later, spleen were harvested, CD11c⁺MHCII⁺B220⁻DX5⁻ DCs cell-sorted and seeded in a Seahorse XF-24 analyzer. (A) OCR in real time after sequential addition of oligomycin, FCCP, and antimycin-A/rotenone. (B) ECAR values. Data represent means ± SD of cuadriplicates. Data are representative of three experiments. (C) As in (A) but cells were incubated with MitoTracker Red CMXRos or MitoTracker Green FM and analyzed by flow cytometry. Histogram is representative of three independent experiments. (D) Hif1α^−/− and control mice were injected with 50 µg of poly IC and 14 h later splenic DCs were incubated with CellRox Deep Red reagent for 30 min at 37°C or (E) live purified CD11c⁺ MHCII⁺ DCs were lysed and ATP levels were measured by Elisa (n = 3). (F) Splenic DCs were stained with 7-AAD after 14 h stimulation with poly IC in vivo. Histogram is representative of three independent experiments. (G) HIF1α^−/− or HIF1α^+/− control mice were stimulated with 50 µg of poly IC. 18 h later, CD11c⁺ MHCII⁺ DCs were purified by cell-sort, fixed, and added in graded numbers to 2×10⁵ allogeneic Balb/C T cells. T cell proliferation was detected by CFSE dilution of CD3⁺ cells. Data are representative of three experiments. (H) Mice were primed and boosted 4 wk apart with 5 µg α-DEC-p24 and 50 µg poly IC. 1 wk later, IFN-γ secretion in gated CD3⁺ CD4⁺ T cells from spleen, was measured in response to gag p24 peptide mix or control gag p17 peptide mix. Bars represent the mean ± SD from two experiments with six mice total.