Role of IgE receptors in IgE antibody-dependent cytotoxicity and phagocytosis of ovarian tumor cells by human monocytic cells

Abstract

Antibodies directed against tumor-associated antigens are emerging as effective treatments for a number of cancers, although the mechanism(s) of action for some are unclear and still under investigation. We have previously examined a chimeric IgE antibody (MOv18 IgE), against the ovarian tumor-specific antigen, folate binding protein (FBP), and showed that it can direct human PBMC to kill ovarian cancer cells. We have developed a three-color flow cytometric assay to investigate the mechanism by which IgE receptors on U937 monocytes target and kill ovarian tumor cells. U937 monocytes express three IgE receptors, the high-affinity receptor, FcepsilonRI, the low-affinity receptor, CD23, and galectin-3, and mediate tumor cell killing in vitro by two mechanisms, cytotoxicity, and phagocytosis. Our results suggest that CD23 mediates phagocytosis, which is enhanced by upregulation of CD23 on U937 cells with IL-4, whereas FcepsilonRI mediates cytotoxicity. We show that effector : tumor cell bridging is associated with both activities. Galectin-3 does not appear to be involved in tumor cell killing. U937 cells and IgE exerted ovarian tumor cell killing in vivo in our xenograft model in nude mice. Harnessing IgE receptors to target tumor cells suggests the potential of tumor-specific IgE antibodies to activate effector cells in immunotherapy of ovarian cancer.

Fig. 1

Flow cytometric assessment of receptor expression and MOv18 IgE binding to wild type U937 cells. a IgE receptor expression in untreated (top) and IL-4 stimulated (bottom) U937 cells. Cells express CD23 (left) and FcεRI (right) [mAbs, anti-mouse IgG (Fab)₂–FITC, solid lines; IgG₁ isotype control Ab, anti-mouse IgG (Fab)₂–FITC, dashed lines]. b MOv18 IgE binding to untreated (top) and IL-4 stimulated (bottom) U937 cells (left panels), was reduced by IDEC-152 anti-CD23 mAb Fab (middle panels) and also by 15.1 anti-FcεRI (right panels) (MOv18 IgE, anti-IgE-FITC, solid lines; anti-IgE-FITC, dashed lines)

Fig. 2

Example of the in vitro three-color flow cytometric assay setup to analyze ADCC and ADCP. a Cell populations were labeled separately to setup instrument settings and determine where gates should be set. Live IGROV1 tumor cells were labeled with CFSE alone or with CFSE and PI. Dead IGROV1 cells were labeled with CFSE and PI. U937 cells were used as effector cells, either unstained, CD89-PE labeled or PI-stained. b Dot plots of mixed populations from which calculations were made: Region 1 (R1, green), total CFSE⁺ tumor cell targets. Region 2, (R2, orange), CFSE⁺ tumor cells present within PE-positive effector cells (phagocytosed). Region 3 (R3, red): intact tumor cells killed by ADCC are CFSE⁺/PI⁺

Fig. 3

CD23-mediated ADCP by wild type U937 cells and IgE. a Two-color flow cytometric dot plots detected ADCP of IGROV1 cells by U937 cells and MOv18 IgE after 1 and 2.5 h in culture. Left panels: CFSE-labeled IGROV1 (x axis, lower right) and U937 cells labeled with CD89-PE mAb (y axis, upper left). IGROV1 cells phagocytosed by U937 cells (CFSE⁺/PE⁺, upper right). Right panels: CFSE-labeled dead IGROV1 cells, labeled with Propidium Iodide (PI) (y axis, upper left) (CFSE⁺/PI⁺, upper right). b Quantitation of MOv18 IgE-mediated IGROV1 tumor cell killing by ADCC and ADCP after 1 and 2.5 h using unstimulated (top panel) and IL-4-treated (bottom panel) U937 cells. Cytotoxicity: black bars; phagocytosis: gray bars. Results are mean ± SD of six independent experiments. c ADCC and ADCP of IGROV1 tumor cells by unstimulated (top left) and IL-4 sitmulated (bottom left) U937 cells and after incubation of U937 cells with IDEC-152 Fab (top and bottom right panels) blocked tumor cell ADCP. Results are mean ± SD of six independent experiments. Significance of values compared to samples given MOv18 IgE by the Student’s t-test: ^n/s P > 0.05, *P < 0.05, **P < 0.005, ***P < 0.0005

Fig. 4

Potential role for FcεRI in IgE-mediated tumor cell killing in wild-type U937 cells. a Tumor cell killing by IL-4-stimulated U937 and MOv18 IgE used at suboptimal concentration (top left) and ADCC blocked by 15.1 anti-FcεRI mAb Fab (top right), ADCP blocked by IDEC-152 anti-CD23 mAb Fab (bottom left) and both ADCC and ADCP reduced by simultaneous incubation of U937 cells with both mAb Fabs (bottom right). b Flow cytometric evaluation on the effect of sFcεRIα on IgE binding to IL-4-stimulated U937 cells. MOv18 IgE bound to IL-4-stimulated U937 cells (left) and sFcεRIα prevented binding of IgE to U937 cells (right) (MOv18 IgE, anti-IgE-FITC, solid lines; anti-IgE-FITC alone, dashed lines). c Tumor cell killing by unstimulated U937 (top left) and ADCC blocked by sFcεRIα (top right). MOv18 IgE-mediated ADCC and ADCP in IL-4-stimulated U937 (bottom left) were reduced by sFcεRIα (bottom right). Cytotoxicity: black bars; phagocytosis: gray bars. Results are mean ± SD of six independent experiments. Significance of values compared to samples given MOv18 IgE by the Student’s t-test: ^n/s P > 0.05, *P < 0.05, **P < 0.005, ***P < 0.0005

Fig. 5

FcεRI αγ₂-transfected U937 cells: receptor expression, MOv18 IgE binding and ADCC/ADCP assays. a IgE receptor expression in FcεRI αγ₂-transfected U937 cells: cells do not express CD23 (left) but express the sFcεRI αγ₂ complex (right) [mAbs, anti-mouse IgG (Fab)₂–FITC, solid lines; IgG₁ isotype control Ab, anti-mouse IgG mAb (Fab)₂–FITC, dashed lines]. b MOv18 IgE binding to FcεRI αγ₂-transfected U937 cells (left) was blocked by sFcεRIα (middle) but not affected by IDEC-152 anti-CD23 mAb Fab (right) (MOv18 IgE, anti-IgE-FITC, solid lines; anti-IgE-FITC alone, dashed lines). c ADCC and ADCP by FcεRI αγ₂-transfected U937 cells: MOv18 IgE-mediated tumor cell killing (left) was blocked by sFcεRIα (middle) but was unaffected by IDEC-152 CD23mAb Fab (right). Cytotoxicity: black bars; phagocytosis: gray bars. Results are mean ± SD of four independent experiments. Significance of values compared to samples given MOv18 IgE by the Student’s t-test: ^n/s P > 0.05, *P < 0.05, **P < 0.005, ***P < 0.0005

Fig. 6

Potential role for galectin-3 in IgE-mediated tumor cell killing in wild-type U937 cells. a Flow cytometric histograms show lack of surface galectin-3 expression (left) and intracellular galectin-3 (right) in permeabilised U937 cells [anti-galectin-3 mAb B2C10, anti-mouse IgG (Fab)₂–FITC, solid lines; IgG₁ isotype control Ab, anti-mouse IgG (Fab)₂–FITC, dashed lines]. b Effect of lactose treatment on MOv18 IgE ADCC and ADCP by U937 cells and MOv18 IgE. Tumor cell killing by untreated U937 cells (left) and following incubation of cells with 25 mM lactose (right). Cytotoxicity: black bars; phagocytosis: gray bars. Results are mean ± SD of four independent experiments

Fig. 7

Superimposed bright-field and fluorescent images of IGROV1-U937 interactions. CFSE-stained IGROV1 tumor cells (green) and CD89-PE-labeled U937 cells (red) after 2.5 h in culture. a Untreated wild type U937 cells (red) given MOv18 IgE (right) were in contact with IGROV1 cells (green, white arrow) but not when incubated with NIP IgE (right). Treatment with IDEC-152 Fab reduced E : T contact. E : T was further reduced when MOv18 IgE was given sFcεRIα. b IL-4-stimulated wild type U937 cells (red) showed enhanced contact with IGROV1 (green) when given MOv18 IgE and extensive phagocytosis of IGROV1 (green CFSE inside U937, yellow-orange, white arrows). Reduced E : T contact was observed with NIP IgE. IDEC-152 Fab reduced phagocytosis (lack of green fluorescence inside U937, red). E : T contact and phagocytosis of IGROV1 were reduced with sFcεRIα. c FcεRI αγ₂-transfected U937 cells incubated with MOv18 IgE (red) were in contact with IGROV1 cells (green, white arrow), but not with NIP IgE. IDEC-152 mAb Fab prior to assays did not affect E : T cell contact, which was reduced with sFcεRIα. Original magnification ×40. Scale bars 20 μm

Fig. 8

Effect of MOv18 IgE and monocytic cells on survival of ovarian carcinoma xenograft-bearing nude mice. Survival following transplantation of HUA xenografts into nude mice and treatment with monocytic U937 cells either a untreated or b pre-treated with IL-4 prior to i.p. injections with or without MOv18 IgE. Treatments were given on the day of tumor challenge and again 2-week following tumor challenge. Horizontal bars indicate mean survival (days). Significance of values by the Student’s t-test: ^n/s P > 0.05, *P < 0.05, **P < 0.005, ***P < 0.0005