Fig. 6

CD206- and FRβ-targeting T cell engagers activate endogenous ascites T cells to kill ascites macrophages. a-e Total unpurified ascites cells from five different patients were cultured for five days with 50 nM BiTEs/TriTEs in medium only or 50% ascites supernatant from the same patient sample. a, b Cells were stained with anti-CD11b, anti-CD64, anti-CD80 and anti-CD86 antibodies, as well as a LIVE/DEAD fixable stain, then analysed by flow cytometry. a % Live residual CD11b⁺CD64⁺ cells were calculated relative to “Mock”-treated samples. b 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 Activation of endogenous CD4⁺ and CD8⁺ ascites T cells was assessed by flow cytometric measurement of CD25 expression. d IFN-γ levels in the culture supernatants were determined by ELISA. e Numbers of CD4⁺ and CD8⁺ cells were determined through addition of counting beads to samples immediately prior to antibody staining. Fold-changes in CD4⁺ and CD8⁺ cell count were calculated relative to “Mock”-treated samples. Data show the grand mean ± SD of five individual patient means (calculated from biological triplicate). Statistical significance was assessed by two-way ANOVA followed by Bonferroni post-hoc analysis, with each treatment being compared to the relevant “Mock” condition (a, c-e) (*, P < 0.05; **, P < 0.01; ***, P < 0.001)