Dottorato in SCIENZE DELLA VITA

Titolo della tesi: Eosinophils acquire immune checkpoint molecules through trogocytosis: implications in cancer immunity.

Trogocytosis is a cellular process whereby a cell acquires a membrane fragment from a donor cell in a contact-dependent manner allowing for transfer of surface proteins with functional integrity. Trogocytosis has been described in most immune cells, with the exception of eosinophils, and represents an important mechanism for the regulation of anti-tumor immune responses. Eosinophils play important roles in cancer immunity through release of soluble mediators and contact-dependent mechanisms. We previously reported that activation of eosinophils with the alarmin IL-33 promotes adhesion to cancer cells via the CD11b/CD18 integrin complex resulting in tumor cell killing. Here, we evaluated trogocytosis in eosinophils following activation with IL-33 and upon contact with tumor cells. Flow cytometry and confocal microscopy demonstrated that after 1-hour co-culture with tumor cells (EG.7-OVA, MC38, TC-1 and B16.F10) labelled with a membrane dye, IL-33 activated, but not resting, eosinophils acquire membrane fragments from target cells. Time-lapse video microscopy revealed that trogocytosis by activated eosinophils occurs rapidly, within 2 minutes after contact with the target tumor cell. Transmission electron microscopy (TEM) showed trogocytic invaginations at the interface between activated eosinophils and tumor cells. Blockade of CD11b/CD18 on eosinophils membrane significantly reduced trogocytosis, highlighting the role of this integrin complex for binding to target tumor cells and formation of an immunological synapse. Since in immune cell trogocytosis is typically mediated by receptor–ligand interactions, we studied the ability of eosinophils to acquire specific proteins expressed by tumor cells, but not by eosinophils, focusing on immune checkpoint molecules. Eosinophils do not express certain immune checkpoints, such as PD-1 and TIGIT, but acquired these molecules after 1-hour co-culture with PD-1/TIGIT-expressing EG.7-OVA lymphoma cells. In contrast, eosinophils expressed PD-L1 that was further up-regulated by IL-33. Blocking the PD-1/PD-L1 interaction with αPD-L1 antibody markedly reduced PD-1 trogocytosis, demonstrating that the acquisition of PD-1 operated by eosinophils is receptor-dependent. Furthermore, blocking the eosinophil-tumor interaction of CD11b/CD18 also reduced PD-1 trogocytosis, denoting the requirement of cell-cell contact for this process. Interestingly, stimuli capable of up-regulating either CD11b/CD18 (i.e., CCL11) or PD-L1 (i.e., LPS), were not sufficient in triggering PD-1 trogocytosis in eosinophils, suggesting the need for induction of both signals for this process to occur. Finally, we provide a model of immunological synapse that may occur during eosinophils trogocytosis. Overall, our findings demonstrate that eosinophils can acquire tumor-expressed molecules through trogocytosis and that this process is strongly enhanced by activation stimuli (i.e., IL-33) that promote CD11b/CD18-dependent cell adhesion and increase the expression of specific ligands. The acquisition of immune checkpoints by eosinophils in the tumor microenvironment may potentially affect immunotherapy response in cancer patients.