High-level expression of mammalian G-protein-coupled receptors (GPCRs) is a necessary step toward biophysical characterization and high-resolution structure determination. Even though many heterologous expression systems have been used to express mammalian GPCRs at high levels, many receptors are improperly trafficked or are inactive in these systems. En route to engineering a robust microbial host for GPCR expression, we have investigated the expression of 12 GPCRs in the yeast Saccharomyces cerevisiae, where all receptors are expressed at the mg/L scale. However, only the human adenosine A 2 a (hA 2 aR) receptor is active for ligandbinding and located primarily at the plasma membrane, whereas other tested GPCRs are mainly retained within the cell. Selective receptors associate with BiP, an ER-resident chaperone, and activated the unfolded protein response (UPR) pathway, which suggests that a pool of receptors may be folded incorrectly. Leader sequence cleavage of the expressed receptors was complete for the hA 2 aR, as expected, and partially cleaved for hA 2 bR, hCCR5R, and hD 2L R. Ligand-binding assays conducted on the adenosine family (hA 1 R, hA 2 aR, hA 2 bR, and hA 3 R) of receptors show that hA 2 aR and hA 2 bR, the only adenosine receptors that demonstrate leader sequence processing, display activity. Taken together, these studies point to translocation as a critical limiting step in the production of active mammalian GPCRs in S. cerevisiae.

Eosinophils are major effectors of Th2-related pathologies, frequently found infiltrating several human cancers. We recently showed that eosinophils play an essential role in anti-tumor responses mediated by immunotherapy with the ‘alarmin’ intereukin-33 (IL-33) in melanoma mouse models. Here, we analyzed the mechanisms by which IL-33 mediates tumor infiltration and antitumor activities of eosinophils. We show that IL-33 recruits eosinophils indirectly, via stimulation of tumor cell-derived chemokines, while it activates eosinophils directly, up-regulating CD69, the adhesion molecules ICAM-1 and CD11b/CD18, and the degranulation marker CD63. In co-culture experiments with four different tumor cell lines, IL-33-activated eosinophils established large numbers of stable cell conjugates with target tumor cells, with the polarization of eosinophil effector proteins (ECP, EPX, and granzyme-B) and CD11b/CD18 to immune synapses, resulting in efficient contact-dependent degranulation and tumor cell killing. In tumor-bearing mice, IL-33 induced substantial accumulation of degranulating eosinophils within tumor necrotic areas, indicating cytotoxic activity in vivo. Blocking of CD11b/CD18 signaling significantly reduced IL-33-activated eosinophils’ binding and subsequent killing of tumor cells, indicating a crucial role for this integrin in triggering degranulation. Our findings provide novel mechanistic insights for eosinophil-mediated anti-tumoral function driven by IL-33. Treatments enabling tumor infiltration and proper activation of eosinophils may improve therapeutic response in cancer patients.

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Interleukin-33 (IL-33) is an epithelial-derived cytokine that can be released upon tissue damage, stress, or infection, acting as an alarmin for the immune system. IL-33 has long been studied in the context of Th2-related immunopathologies, such as allergic diseases and parasitic infections. However, its capacity to stimulate also Th1-type of immune responses is now well established. IL-33 binds to its specific receptor ST2 expressed by most immune cell populations, modulating a variety of responses. In cancer immunity, IL-33 can display both pro-tumoral and anti-tumoral functions, depending on the specific microenvironment. Recent findings indicate that IL-33 can effectively stimulate immune effector cells (NK and CD8+ T cells), eosinophils, basophils and type 2 innate lymphoid cells (ILC2) promoting direct and indirect anti-tumoral activities. In this review, we summarize the most recent advances on anti-tumor immune mechanisms operated by IL-33, including the modulation of immune checkpoint molecules, with the aim to understand its potential as a therapeutic target in cancer.

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Salivary gland tumors are a heterogeneous group of neoplasms representing less than 10% of all head and neck tumors. Among salivary gland tumors, salivary duct carcinoma (SDC) is a rare, but highly aggressive malignant tumor resembling ductal breast carcinoma. Sublingual treatments are promising for SDC due to the induction of both local and systemic biological effects and to reduced systemic toxicity compared to other administration routes. In the present study, we first established that the sublingual administration of type I IFN (IFN-I) is safe and feasible, and exerts antitumor effects both as monotherapy and in combination with chemotherapy in transplantable tumor models, i.e., B16-OVA melanoma and EG.7-OVA lymphoma. Subsequently, we proved that sublingual IFN-I in combination with cyclophosphamide (CTX) induces a long-lasting reduction of tumor mass in NeuT transgenic mice that spontaneously develop SDC. Most importantly, tumor shrinkage in NeuT transgenic micewas accompanied by the emergence of tumor-specific cellular immune responses both in the blood and in the tumor tissue. Altogether, these results provide evidence that sublingual IFN holds promise in combination with chemotherapy for the treatment of cancer.

The alarmin IL-33 plays pleiotropic roles in allergy, autoimmunity and inflammation through binding to its specific receptor ST2 expressed by most hematopoietic cells. Emerging evidences suggest an involvement of this cytokine also in cancer immunity, although its function remains ill-defined. Eosinophils (EOS) are a rare blood population playing critical roles in allergic inflammation and parasitic responses. We recently showed that EOS play an essential role in anti-tumor responses against melanoma growth and pulmonary metastasis mediated by IL-33 in vivo.In the present study we analyzed the mechanisms by which IL-33 mediates tumor infiltration and antitumoral activities of EOS. We show that IL-33 indirectly stimulates the recruitment of EOS inducing tumor-derived chemokines CCL24 and CCL5. Furthermore, IL-33 directly activates EOS inducing the expression of adhesion molecules, such as the integrin CD11b, resulting in efficient contact-dependent tumor cell killing. In co-culture experiments, IL-33 activated EOS tightly bond to tumor cells, forming increased numbers of conjugates, with respect to resting eosinophils. Confocal laser-scanning microscopy (CLSM) of eosinophil-tumor cell conjugates revealed polarization of the pore-forming eosinophilic cationic protein (ECP) and of CD11b on the cell synapses exclusively in IL-33-activated, but not resting, EOS. Furthermore, we show that IL-33 activated EOS release larger amounts of extracellular vesicles (EV) with respect to resting EOS. Transmission electron microscopy (TEM) revealed increased degranulation and EV release of IL-33-activated EOS following cell contact with target tumor cells. Our results advocate for an eosinophil-mediated tumoricidal function promoted by IL-33, thus opening perspectives for novel cancer immunotherapy strategies. Citation Format: Fabrizio Mattei, Carla Buccione, Sara Andreone, Francesca Spadaro, Adele De Ninno, Jacopo Mancini, Cristiana Zanetti, Isabella Parolini, Francesca Iosi, Antonella Tinari, Valeria Lucarini, Annamaria Gerardino, Giovanna Ziccheddu, Luca Businaro, Claudia Afferni, Giovanna Schiavoni. IL-33 activates antitumoral toxicity in eosinophils through stimulation of contact-dependent degranulation [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A091.