ROLE OF IGE IN IMMUNOSURVEILLANCE: MECHANISM AND POTENTIAL THERAPEUTIC APPLICATIONS

IgE is the class of immunoglobulins responsible for the protection against intestinal parasites and exerts a key role in the pathophysiology of allergic reactions. In addition, it has been demonstrated its involvement in the immune response against tumors in various animal models. According to our model, the function of IgE in the anti-tumor response is mediated by the binding of IgE with its high affinity receptor FcεRI expressed, in mice, on the surface of mast cells and basophils. Activation of FcεRI receptor leads to cell degranulation with release of preformed and newly synthesized mediators able to recruit effector cells that induce the establishment of a powerful inflammation at the tumor site. This inflammation, leading to cell death, could determine the processing of tumor antigens and the resulting immune response against tumor. Based on promising data obtained previously in our laboratories, regarding the adjuvant effect of exogenous IgE in the anti-tumor vaccination and on the many controversial epidemiological studies about a possible link between allergies and cancer protection, we decided to investigate the possible role of endogenous IgE in the immunosurveillance of tumor. The use of two transgenic mouse models, one knock-out for the production of IgE (IgE-KO mice) and the other high IgE producer (KN1 mice), allowed us to investigate the possible involvement of host endogenous IgE in the immunity against cancer. Either in the absence or with a normal amount of IgE, tumor growth, preceded by vaccination with irradiated TS/A tumor cells (mammary adenocarcinoma), is not hindered and is comparable to not immunized mice. Differently, in high IgE producer mice, a single immunization was sufficient to obtain a complete anti-tumor protection. Moreover, challenging mice with a different tumor cell line, N2C tumor cells (less aggressive than TS/A tumor cells), the anti-tumor protection is observed even without immunization in 100% of KN1 mice compared to IgE-KO and wild type control mice. To demonstrate that the protection observed in KN1 mice is due to the interaction of IgE with its high affinity FcRI receptor, we decided to delete the FcRI alpha gene in KN1 mice to remove their receptor. For this purpose we crossed KN1 mice with FcRI-KO mice, in order to obtain a double-mutant mouse model (DM), characterized by elevated levels of IgE but lacking in the expression of the high affinity receptor for IgE. The inoculation of N2C tumor cells in DM mice showed that the previously anti-tumor protection, observed in KN1 mice, has been widely lost and this is the fundamental point of this study because demonstrates that IgE-FcRI axis is the basis of the role of IgE in anti-tumor immune response. We also demonstrated, through an in vitro test of mediators release, the existence of tumor-specific IgE in the serum of KN1 and DM mice that were challenged in vivo with N2C tumor cells; moreover, the depletion of IgE from sera of KN1 and DM mice validate the specific contribution played by IgE in the mediators release. Another point of this study was to understand how IgE acts in the observed anti-tumor effect. KN1 mice depleted of CD8+ T cells prior to tumor cells inoculation lost the anti-tumor protection. This result allowed us to point out that the anti-tumor immunosurveillance, driven by IgE, is largely mediated by an immune response dependent on CD8+ T cells. Next, we decided to isolate tumor-specific IgE through hybridoma technology, starting from spleen or bone marrow of KN1 mice that previously rejected the tumor in vivo. We tried to isolate tumor-specific IgE to employ them either in preventive vaccination or in therapeutic treatment protocols, in mice with already developed tumors, but unfortunately we were able to isolate a very low number of IgE secreting hybridoma that didn't show any specificity against the tumor cells. Without any tumor-specific IgE, we planned a different approach for treating solid tumors, through intratumoral injection of rMVA-tmIgE (Modified Vaccinia Virus Ankara expressing a truncated form of the human membrane IgE), previously developed in our laboratories. This rMVA-tmIgE allowed to obtain tumor-infected cells expressing the Cε3Cε4 fragment of human IgE on their cell surface. Since MVA does not replicate among the infected cells without any effect on the surrounding host cells, the expression of Cε3Cε4 is restricted only in the tumor. Several studies have also demonstrated the efficacy of the intratumoral treatment (i.t.) with vectors based on MVA, showing a consequent reduction of the tumor mass volume and an increased survival of treated mice. For this reason, in the last part of this project, we have evaluated the ability of rMVA-tmIgE to express, in vivo, the human membrane IgE on the tumor cells surface after intratumoral treatment of wild type mice with the vaccine. N2C tumor mass, treated i.t. with 107 plaque-forming unit (pfu) of rMVA-tmIgE, showed 24 and 48 hours after treatment a good infection efficiency. Moreover, the ability of rMVA-tmIgE to express human IgE in vivo was evident just one day after the intratumoral treatment, even if the signal of the Cε3Cε4 expression was much lower compared to the signal of MVA infection. This preliminary data is the first evidence that rMVA-tmIgE can be used to treat solid tumors in the mouse model "humanized" for the high affinity receptor of IgE (FcεRIα-/-/hFcεRIα+). Indeed, our previous studies, based on preventive vaccination protocols, have shown the efficacy of rMVA-tmIgE in countering tumor growth in the “humanized” mice model. In the near future we will determine the effect of i.t. rMVA-tmIgE in humanized mice carrying a solid tumor We expect to achieve an inflammatory reaction against the tumor induced by the binding of IgE, expressed on tumor cells, with FcεRI receptor expressed on the surface of immune cells. We think that this initial IgE-dependent response would lead to long-term protection through the activation of an immune cascade where CD8+ T cells are surely involved.