Figure 4. cGAS is essential for DC sensing of irradiated-tumor cells.

(A-C) BMDCs were cultured with 40Gy-pretreated MC38-SIY or non-irradiated-MC38-SIY cells. Subsequently purified CD11c⁺ cells were co-cultured with isolated CD8⁺ T cells from naive 2C mice for three days. (A) BMDCs from WT and Mb21d1^−/− mice were used for co-culture with irradiated and nonirradiated MC38-SIY cells. DC cross-priming activity was analyzed by ELISPOT assays. (B) BMDCs were transfected with a siRNA-non-targeting control or siRNA-Mb21d1. Two days later after the transfection, the BMDCs were harvested for the co-culture with irradiated and non-irradiated MC38-SIY cells. DC cross-priming activity was analyzed by ELISPOT assays. (C) WT and Mb21d1^−/− BMDCs were cultured with 40Gy-pretreated MC38-SIY cells. 10ng/ml IFN-β was added into the co-culture of Mb21d1^−/− BMDC and irradiated-MC38-SIY cells. 100μg/ml DMXAA was added to isolated Mb21d1^−/− CD11c⁺ cells for additional three hours incubation. DC cross-priming activity was analyzed by ELISPOT assays. (D) CD11c⁺ cells from WT or Mb21d1^−/− BMDCs after co-culture with irradiated or non-irradiated MC38-SIY cells were incubated for 2 days, and then subjected to ELISA assays for IFNβ level. (E) CD11c⁺ cells were sorted from tumors in WT mice at 72 hour after radiation. Real-time PCR assay was performed to quantify the Mb21d1 mRNA. cGAS-deficient mice are represented by Mb21d1^−/−. Representative data are shown from three (A-E) experiments. Data are represented as mean ± SEM. **P < 0.01 and ***P < 0.001 (Student's t test). See also Figure S4.