IgE/FcεRI-Mediated Antigen Cross-Presentation by Dendritic Cells Enhances Anti-Tumor Immune Responses

Abstract

Epidemiologic studies discovered an inverse association between immunoglobulin E (IgE)-mediated allergies and cancer, implying tumor-protective properties of IgE. However, the underlying immunologic mechanisms remain poorly understood. Antigen cross-presentation by dendritic cells (DCs) is of key importance for anti-tumor immunity because it induces the generation of cytotoxic CD8⁺ T lymphocytes (CTLs) with specificity for tumor antigens. We demonstrate that DCs use IgE and FcεRI, the high-affinity IgE receptor, for cross-presentation and priming of CTLs in response to free soluble antigen at low doses. Importantly, IgE/FcεRI-mediated cross-presentation is a distinct receptor-mediated pathway because it does not require MyD88 signals or IL-12 induction in DCs. Using passive immunization with tumor antigen-specific IgE and DC-based vaccination experiments, we demonstrate that IgE-mediated cross-presentation significantly improves anti-tumor immunity and induces memory responses in vivo. Our findings suggest a cellular mechanism for the tumor-protective features of IgE and expand the known physiological functions of this immunoglobulin.

Figure 1. IgE/FcεRI-mediated antigen uptake allows for cross-priming of CTLs in response to free soluble antigen at low dose

(A) Binding and uptake of fluorescently-labeled OVA (OVA^AF647) by DCs that were pre-loaded with OVA-specific IgE (histogram: red line). Representative histogram overlay. See also Figure S1. (B–G) CD8⁺ T cell priming via IgE. (B) Schematic overview of IgE/FcεRI-independent (no IgE) and IgE/FcεRI-dependent (plus IgE) antigen sampling. (C) In vivo T cell proliferation assay. Splenic DCs from IgE_R-TG and WT mice (no or plus IgE) were pulsed in vitro with NP-OVA (0.05 µg/ml) and injected into WT recipients. Prior to DC injection, recipients received CFSE-labeled CD8⁺ OT-I T cells. Representative FACS plots and quantification ((3 independent experiments, ≥ 2 mice were per experiment). (D) In vivo killing assay, dots represent individual mice (n=2). (E) In vitro T cell proliferation assay. Triplicates +/− SEM of a representative experiment (n≥5). Granzyme B production was determined by ELISA. Bd = below detection limit. Triplicates +/− SEM, representative experiment (n=3). (F) Signaling induced by NP-BSA through IgE/FcεRI cross-linking does not augment cross-presentation resulting from IgE-independent antigen uptake. DCs were loaded with NP-specific IgE (+) or not (−) prior to simultaneous stimulation with NP-BSA (50 µg/ml) and the indicated amounts of non-haptenized OVA. (G) Comparison of IgE- and IgG-mediated antigen cross-presentation after incubation of DCs with free soluble antigen, or immune complexes (IC). Schematic overview: (I) DCs were pre-incubated with monomeric IgE or IgG1, washed, and treated with free soluble antigen, (II) IgE or IgG1 was pre-incubated with antigen to form ICs and then added to DCs. NT= not treated with antigen. Triplicates of representative experiment (n=2).

Figure 2. Cellular mechanisms of IgE/FcεRI -mediated cross-presentation

(A) The IgE/FcεRI-mediated cross-presentation depends on endosomal trafficking. DCs pre-loaded with IgE or not, were incubated with antigen in the presence of primaquine. OVA peptide (SIINFEKL) was used as control. Representative experiment (n=2). (B–E) The IgE/FcεRI-mediated antigen cross-presentation pathway operates independently of MyD88 and IL-12. (B–C) DCs from MyD88^−/− mice expressing FcεRI (MyD88^−/− × IgE_R-TG) induce efficient in vitro T cell proliferation and granzyme B production after IgE-dependent antigen uptake. See also Figure S2. (D) Antigen-specific IgE/FcεRI-crosslinking does not induce IL-12 in DCs. Splenic DCs were stimulated as indicated. (E) Effects of exogenously added IL-12 on the cross-priming capacity of DCs. (B–E) DC were incubated with 0.05 µg/ml OVA. Biological triplicates of representative experiments (n=3).

Figure 3. IgE/FcεRI-mediated cross-presentation is executed by CD8α− DCs and is blunted by IL-4

(A) Evaluation of FcεRI expression by human DC subsets. mRNA expression values were extracted from published publically available microarray data (NCBI GEO data repository GSE35459). (B) FcεRI expression on mouse CD8α⁺ and CD8α⁻ DCs from IgE_R-humanized mice. Histogram overlays of FcεRI (black lines) and isotype control (grey filled). (C–D) FACS-sorted CD8α⁻ and CD8α⁺ splenic DCs were compared for their ability to induce granzyme B producing OT-I T cells following IgE/FcεRI-independent and IgE/FcεRI-dependent antigen uptake. Data are shown as mean ± SEM of triplicates, representative experiment (n=3). (E) In vitro CTL generation in the presence of IL-4, representative experiment (n=2). See also Figure S3.

Figure 4. IgE/FcεRI-mediated cross-presentation promotes anti-tumor immunity

(A) Outline of the tumor protection experiment. (B) Vaccination with DCs loaded with tumor-specific antigen via IgE/FcεRI increases tumor-free survival. Control (CTRL) mice did not receive DCs. Unprimed DCs were loaded with IgE but not incubated with antigen. (Pooled data of 2 experiments; 20 mice per group, CTRL and unprimed n=10 mice). Tumor cells were injected subcutaneously (s.c.). (C) Tumor-free mice from the experiments shown in (B) were re-challenged with OVA-expressing B16 tumor cells and monitored for tumor growth. (D–E) Tumor-specific IgE mediates tumor protection in vivo. (D) Overview of tumor experiment after passive immunization with IgE. Mice, in which expression of FcεRI was restricted to DCs, were treated with OVA-specific IgE or DNP-specific IgE and OVA-expressing B16 cells were injected i.v. (E) Tumor count in lungs. Quantification (5 mice per group) and representative images are shown.