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. 2021 Sep 30;9(9):e002569. doi: 10.1136/jitc-2021-002569

Figure 1.

Figure 1

Live oncolytic vaccinia virus fails to induce IFN-β, and CCL5 from infected bone marrow-derived dendritic cells (BMDCs), or B16-F10, or MC38 cells. (A) Schematic diagram of homologous recombination between a pCB-mGM-CSF plasmid and E3L∆83N vaccinia viral DNA at the thymidine kinase (TK) locus to generate the recombinant virus E3L∆83N-TK- (OV) and E3L∆83N-TK--mGM-CSF (OV-GM). mGM-CSF was expressed under the control of the vaccinia synthetic early and late promoter (PsE/L). (B) Fold changes of viral titers of recombinant viruses in murine B16-F10 melanoma cells at 72 h post-infection compared with those at 1 h post-infection. B16-F10 melanoma cells were infected with OV-TK+, OV, or OV-GM at an MOI of 0.01. Cells were collected at 1, 24, 48, and 72 h post-infection, and viral yields (log pfu) were determined by titration in BSC40 cells. (C) mGM-CSF expression of live OV-GM or heat-iOV-GM in B16-F10 or SK-MEL31 cells as verified by ELISA. Supernatants were collected at 24 hours post-infection. (D) BMDCs were infected with either live OV or live OV-GM at an MOI of 10, or with an equivalent amount of heat-iOV-GM, and supernatants were collected 22 h post-infection. The levels of secreted IFN-β, CCL5 and CXCL10 in the supernatants were determined by ELISA. (E) Western blot analyses of BMDCs from WT or STINGGt/Gt mice infected with either live OV-GM at an MOI of 10 or with an equivalent amount of heat-iOV-GM. The levels of p-TBK1, TBK1, p-IRF3, IRF3, and STING are shown. GAPDH was used as a loading control. hpi: hours post-infection. (F) Expression levels of DC surface markers, MHCI, CD40, CD86, and CD80, on BMDCs infected with either live OV, live OV-GM, or heat-iOV-GM as determined by FACS. NT: no treatment control. (G) The concentrations of secreted IFN-β and CCL5 in the supernatants of murine B16-F10 melanoma cells infected with either live OV or live OV-GM at an MOI of 10, or with an equivalent amount of heat-iOV-GM for 24 hours. (H) The concentrations of secreted IFN-β and CCL5 in the supernatants of murine MC38 colon adenocarcinoma cells infected with either live OV, live OV-GM, or heat-iOV-GM.