a) Doxycycline-inducible shRNA-mediated knockdown of cGAS (TSA shcGAS) or STING (TSA shSTING) in TSA cells completely abrogated IFNβ release and Ifnb1 and Mx1 gene expression induced by viral infection and 8GyX3 radiation in vitro. ( b– f) Mice with TSA shcGAS, or non-silencing shRNA (TSA shNS) in the irradiated tumour and TSA shNS in the abscopal tumour were treated with doxycycline, 8GyX3 and anti-CTLA4. IFNγ production by TDLN cells ( b) and percentage of IFNγ + CD8 + T cells in spleen ( c) in response to CD8 epitope AH1A5 (full circles) or control peptide MCMV (open circles). Each symbol represents one animal. Horizontal lines indicate the mean of antigen-specific (solid lines) or control (dashed lines). ( d) Representative fields (× 200) showing CD8 + cells (green), DAPI+ nuclei (blue), and RFP+ TSA cells (red), and mean number±s.d. of CD8 + cells per field in abscopal tumours harvested at day 22 from 8GyX3+anti-CTLA4-treated mice with irradiated TSA shcGAS (red squares) or TSA shNS (blue squares) tumours. White bars, 25 um. ( e) Growth of irradiated and abscopal tumour in mice with TSA shNS cells treated with 0Gy (black), 8GyX3 (green), 8GyX3+anti-CTLA4 (blue), and mice with TSA shcGAS cells treated with 0Gy (dashed line), 8GyX3 (yellow), 8GyX3+anti-CTLA4 (red). ( f) Survival of mice from 8GyX3+anti-CTLA4 that rejected the irradiated and abscopal tumour ( n=4) and were rechallenged at day 100 with a tumorigenic inoculum of TSA cells, together with a group of naïve mice ( n=5). ( a- d) Duplicate; * P<0.05; ** P<0.005; *** P<0.0005: t-test; n=3. ( e) Duplicate; * P<0.05; ** P<0.005: comparison of irradiated tumour outgrowth; two-way ANOVA; n=7; ## P<0.005: comparison of abscopal tumour outgrowth; two-way ANOVA; n=7. All data are mean±s.e.m.