Fig 1: 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)
Fig 2: A CD206 TriTE with bivalent CD3 binding retains specificity for M2 macrophages and overcomes ascites suppression. a Monocyte-derived macrophages were polarised as indicated, CFSE-stained, and co-cultured for 96 h with T cells at increasing E:T ratios and BiTE/TriTE concentrations. % Live cells were calculated with propidium iodide staining and Celigo image cytometry, with values displayed as a heat map. b T cells were co-cultured with monocyte-derived macrophages and BiTEs/TriTEs in the presence or absence of 50% ascites fluid from seven different patients (Patients 1, 2, 3, 4, 6, 7 and 8). 96 h later, CD25 expression was determined by flow cytometry. C, CFSE-stained monocyte-derived macrophages were treated with T cells (10:1 E:T ratio) and BiTEs/TriTEs in medium alone or 50% ascites supernatant from seven different patients (Patients 1, 2, 3, 4, 6, 7 and 8). 96 h later, cells were stained with proprodium iodide and analysed with a Celigo image cytometer to calculate % live cells. Data show mean ± SD of biological triplicates (b, c). Statistical significance was assessed by two-way ANOVA followed by Bonferroni post-hoc analysis, with each treatment being compared to the relevant “Mock” condition (b, c) (*, P < 0.05; **, P < 0.01; ***, P < 0.001)
Fig 3: CD206- and FRβ-targeting BiTEs activate primary human T cells to kill autologous M2-polarised macrophages. a Schematic representations of CD206- and FRβ-targeting BiTEs. b, Western blot analysis of supernatants from HEK293A cells 48 h after transfection with BiTE expression plasmids. Blots were probed with a mouse anti-His primary antibody, followed by an HRP-conjugated anti-mouse secondary antibody. c Human MDMs were polarised as indicated, stained with CFSE, and treated with T cells (10:1 E:T ratio) and increasing concentrations of BiTEs. Macrophage killing was assessed 96 h later by propidium iodide staining and Celigo image cytometry. d MDMs were stained with CFSE and treated with the indicated concentrations of BiTE in the presence or absence of T cells (10:1 E:T ratio). 96 h later, cytotoxicity was assessed by propidium iodide staining and analysis with a Celigo image cytometer. e T cell activation in the presence or absence of target cells was assessed by flow cytometric measurement of CD25 expression 96 h after BiTE addition. Data show mean ± SD of biological triplicates (c, d and e). Statistical analysis was performed by two-way ANOVA with Bonferroni post-hoc tests comparing with the relevant “Mock” condition (d and e) (*, P < 0.05; **, P < 0.01; ***, P < 0.001)
Fig 4: Human malignant ascites supernatant suppresses CD206 BiTE activity but not FRβ BiTE activity. a CFSE-stained MDMs were co-cultured with T cells (10:1 E:T ratio) and the indicated BiTEs, in the presence or absence of 50% ascitic supernatant from three patients (Patients 1, 2 and 5). 96 h later, percentage live MDMs was determined with propidium iodide staining and a Celigo image cytometer. b T cell activation was assessed by flow cytometric analysis of CD25 expression after 96 h of co-culture with MDMs and the indicated BiTEs, in the presence or absence of 50% ascitic supernatant from three patients (Patients 1, 2 and 5). c Quantities of IL-6, IL-10 and total (active and latent) TGF-β in malignant ascites fluid from six different patients, as determined by enzyme-linked immunosorbent assay. Normal serum (NS) pooled from three healthy donors was included as a control. d Quantities of soluble CD206 in malignant ascites fluid from nine different patients was determined by enzyme-linked immunosorbent assay. Pooled NS was used as a control. Each condition was measured in biological triplicate and represented as mean ± SD (a-d). Statistical significance was assessed by one-way ANOVA with Dunnett’s post-hoc analysis compared with “Pooled NS” (c, d), or two-way ANOVA followed by Bonferroni post-hoc analysis, with each treatment being compared to the “Mock” condition within the relevant group (a and b). (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, non-significant)
Fig 5: Addition of a second T cell-binding domain to the parental CD206 BiTE. a Schematic representations of CD206-targeting TriTEs. b Western blotting analysis of supernatants from HEK293A cells transfected with TriTE expression plasmids 48 h previously. Blots were probed with a mouse anti-His primary antibody and an HRP-conjugated anti-mouse secondary antibody. c BiTE and TriTE binding to recombinant CD206 protein, as determined by ELISA using a mouse anti-His primary antibody and an HRP-conjugated anti-mouse secondary antibody. d-f T cells were cultured for 96 h with the indicated BiTEs/TriTEs at a dose of 50 nM in the presence or absence of target cells. T cell activation was assessed by measuring CD25 expression by flow cytometry, with representative histograms displayed in (d) and geometric MFI values displayed in (e). IFN-γ levels in the supernatants were quantified by ELISA (f). Data show mean ± SD of biological triplicates (c, e and f). Statistical significance was assessed by two-way ANOVA followed by Bonferroni post-hoc analysis, with each treatment being compared to the “Mock” condition within the relevant group (e and f) (*, P < 0.05; **, P < 0.01; ***, P < 0.001)
Supplier Page from RayBiotech for Human MMR ELISA