IgE Activates Monocytes from Cancer Patients to Acquire a Pro-Inflammatory Phenotype

Simple Summary When activated by tumour antigen-specific IgEs, monocytes may contribute to the restriction of cancer growth in animal models of cancer. In this study, we investigated the effects of IgE stimulation on the activation state of human monocytes from healthy subjects and from patients with cancer. Cross-linking of cognate Fc receptors by IgE on human monocytes potentiated: (a) upregulation of activatory and down regulation of regulatory monocyte cell surface markers; (b) phosphorylation of intracellular protein kinases in monocytes previously described to be downstream of the mast cell and basophil FcεRI signalling pathway; (c) ovarian cancer patient monocyte-mediated cytotoxic killing of tumour cells and release of pro-inflammatory mediators, potentially associated with favourable patient survival. IgE can therefore activate human monocytes to acquire a pro-inflammatory phenotype capable of mediating effector functions against tumour cells. This may contribute to the mechanism of cancer immunotherapy using IgE antibodies. Abstract IgE contributes to host-protective functions in parasitic and bacterial infections, often by monocyte and macrophage recruitment. We previously reported that monocytes contribute to tumour antigen-specific IgE-mediated tumour growth restriction in rodent models. Here, we investigate the impact of IgE stimulation on monocyte response, cellular signalling, secretory and tumour killing functions. IgE cross-linking on human monocytes with polyclonal antibodies to mimic formation of immune complexes induced upregulation of co-stimulatory (CD40, CD80, CD86), and reduced expression of regulatory (CD163, CD206, MerTK) monocyte markers. Cross-linking and tumour antigen-specific IgE antibody-dependent cellular cytotoxicity (ADCC) of cancer cells by cancer patient-derived monocytes triggered release of pro-inflammatory mediators (TNFα, MCP-1, IL-10, CXCL-10, IL-1β, IL-6, IL-23). High intratumoural gene expression of these mediators was associated with favourable five-year overall survival in ovarian cancer. IgE cross-linking of trimeric FcεRI on monocytes stimulated the phosphorylation of intracellular protein kinases widely reported to be downstream of mast cell and basophil tetrameric FcεRI signalling. These included recently-identified FcεRI pathway kinases Fgr, STAT5, Yes and Lck, which we now associate with monocytes. Overall, anti-tumour IgE can potentiate pro-inflammatory signals, and prime tumour cell killing by human monocytes. These findings will inform the development of IgE monoclonal antibody therapies for cancer.


IgE Cross-Linking and Cytokine Stimulation by Monocytic and Tumor Cells
Prior to IgE cross-linking or cytokine stimulation, U937 monocytes were primed for 48 h with 50 ng/mL IL-4, to upregulate CD23 cell surface expression, then were passaged and re-stimulated with IL-4 for a further 48 h. U937, IGROV1, and A375 cells were plated at a density of 1 × 10 6 cells/mL in a 24-well plate (0.5 × 10 6 cells per well).
For IgE cross-linking, U937 cells were stimulated with 5µg/mL IgE, or media control, for 1 hour at 37 °C. Following washing, cross-linking was stimulated with 5µg/mL polyclonal goat anti-human IgE at 37 °C for 1 h. Cells were washed and resuspended in RLT buffer for RNA isolation by RNeasy Kit (Qiagen;74106).
For cytokine stimulation, TNFα, MCP-1, or IL-10 were added separately in each well at final concentrations of 10 ng/mL or combinations of TNFα and MCP-1 at final concentrations of 10 ng/mL each. Cells were incubated at 37 °C for 3 h for gene expression analysis by qPCR, or for 10 h for cytokine secretion analysis by ELISA. For qPCR analysis, cells were washed and resuspended in RLT buffer for RNA isolation by RNeasy Kit (Qiagen; 74106).

Tumor Cell Cytotoxicity and Phagocytosis Assay
Antibody-dependent cellular cytotoxicity and phagocytosis (ADCC/ADCP) of IGROV1 cells was quantified by adapting a previously-described three-colour flow cytometric method [24]. Primary monocytes isolated from healthy volunteers and cancer patients (effector cells) were incubated with IGROV1 (target cells), and 5µg/mL antibodies (Effector:Target cell ratio 3:1).

Cytokine ELISA
TNFα, MCP-1 and IL-10 in cell culture supernatants were measured using cytokine sandwich enzyme-linked immunosorbent assays (R&D Systems; DY210-05, DY279B-05, DY217B-05) following the manufacturer's instructions. Plates were read using a Flurostar ® Omega Spectrophotometer (BMG Labtech).  Figure S2. Immune mediators secreted upon IgE cross-linking on monocytes. Additional cytokines and chemokines (Luminex) measured in cell culture supernatants following cross-linking of IgE on the surface of primary monocytes isolated from healthy volunteer blood. Error bars represent standard error of mean (SEM) of n = 3 independent experiments. A student's t-test was performed to assess significance. Figure S3. Exposure to IgE does not alter FcεRI levels on human monocytes. FcεR expression of primary monocytes isolated from healthy volunteers following 24 h stimulation with no stimulation, IgE, IL-4 or a combination of IgE and IL-4 stimulation (n = 1). Figure S4. Immune mediators secreted upon IgE-mediated killing of tumour cells by human monocytes. Additional cytokine and chemokines (Luminex) measured in cell culture supernatants from IgE-mediated ADCC/ADCP assays with primary monocytes isolated from healthy volunteers (HV) (n = 4) and from ovarian cancer patients (OCP) (n = 3) (independent experiments). Error bars represent standard error of mean (SEM). A One way-ANOVA with Tukey's post-test was performed to assess significance.
0.16 † Newly associated kinases downstream of FcεRI signaling, based on data shown in Figure 3 and recent literature as referenced within the table.  Table S3. Clinical characteristics of healthy volunteers (n = 34) and ovarian cancer patients (n = 110) used for evaluation of total serum IgE levels ( Figure 4A (iv)).