Fig. 6.

Oxidized mtDNA ingested by DCs activates the TBK1-IRF3-IFN-β pathway in DCs, which leads to CD8+ T cell proliferation. a Mitochondrial DNA isolated from EG7 cells was directly irradiated with X-rays. BMDCs were subsequently stimulated with mtDNA for 3 h, and the amounts of pTBK1, total TBK1, pIRF3, total IRF3, and β-actin were measured by immunoblotting. DMXAA was used as a positive control. b BMDCs were stimulated with directly irradiated mtDNA for 18 h in the presence of lipofectamine, and the supernatants were collected to measure IFN-β levels by ELISA. c BMDCs, which were cultured with untreated EG7 cells or apoEG7 cells for 3 h, were labeled with anti-CD11c and anti-IFN-β antibodies and evaluated by flow cytometry. The percentages of CD11c⁺IFN-β⁺ cells in CD11c⁺ BMDCs are shown. d Skin cells collected as described in Fig. 5a were labeled with anti-CD11c, anti-CD45 and anti-IFN-β antibodies and assessed by flow cytometry. The percentages of CD45⁺CD11c⁺IFN-β⁺ DCs among CD45⁺ cells are shown (n = 3). e, f EG7 cells treated with irradiation or freeze-thaw cycles (F/T) were cultured with BMDCs from WT mice for 18 h (loaded DCs). Subsequently, CD11c⁺ cells purified from the loaded DCs were incubated with sorted CFSE-stained CD8⁺ T cells from OT-I mice (DCs:CD8⁺ T cells = 1:5). We estimated the proliferation of the CD8⁺ T cells by measuring the percentages of CFSE^lowCD8⁺ T cells in CD8⁺ T cells by flow cytometry. Data are representative of three experiments and presented as the mean ± SEM. *p < 0.05, **p < 0.01 (Student’s t-test)