Fig. 7

EnAd expressing FRβ-targeting BiTEs activates endogenous ascites T cells to kill ascites macrophages. a DLD-1 cells were infected with parental or armed EnAd, and viability assessed four days later by MTT assay. % Live cells was calculated relative to “Mock”-treated cells. b DLD-1 cells were infected with armed EnAd at 100 vp/cell. Supernatants were harvested 72 h later and analysed for BiTE expression by western blotting using anti-His primary antibody and an HRP-conjugated anti-mouse secondary antibody. c-g Total unpurified ascites cells from five patients were infected with 100 vp/cell parental or BiTE-expressing EnAd for five days with or without autologous fluid. c Activation of endogenous CD4⁺ and CD8⁺ ascites T cells was assessed by flow cytometric measurement of CD25 expression. d CD4⁺ and CD8⁺ cell numbers were determined by adding counting beads to samples immediately prior to antibody staining. Fold-changes in CD4⁺ and CD8⁺ cell count were calculated relative to “Mock”-treated samples. e, g Cells were stained with anti-CD11b, anti-CD64, anti-CD80 and anti-CD86 antibodies, and a LIVE/DEAD fixable stain, then analysed by flow cytometry. e % Live residual CD11b⁺CD64⁺ cells were calculated relative to “Mock”-treated samples. f IFN-γ levels in the supernatants were determined by ELISA. g Fold-changes in geometric MFI values of CD64, CD80 and CD86 on live CD11b⁺CD64⁺ ascites cells were calculated relative to “Mock”-treated samples for each patient sample. c-f Data show the grand mean ± SD of five individual patient means (calculated from biological triplicate). c-f Statistical significance was assessed by two-way ANOVA followed by Bonferroni post-hoc analysis, with each treatment being compared to the relevant “Mock” condition (c-f) (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (c-g) (*, P < 0.05; **, P < 0.01; ***, P < 0.001)