Channel Catfish (Ictalurus punctatus, Rafinesque, 1818) leukocyte immune-type receptors (IpLITRs) are a family of proteins sharing structural and phylogenetic relationships with mammalian immune receptors. Based on their predicted signalling potential and ligand-binding properties, IpLITRs may be important in the control of immune cell effector responses in fish. The main objectives of this thesis were to determine how stimulatory IpLITRs activate cells and to develop assays for the screening of IpLITR ligands. Using cellular transfections, coimmunoprecipitation, and flow cytometry, I determined that stimulatory IpLITRs associate with specific adaptor molecules, which is required for their surface expression and signalling ability. These adaptors assemble with IpLITRs via their charged transmembrane regions and contain cytoplasmic tails encoding tyrosines that may initiate kinase pathways leading to immune cell activation. This study represents the first step towards elucidating how IpLITRs turn on immune cells. Combined with the development of assays to identify IpLITR ligands, my work sets the stage for further investigations into the functional characterization of these receptors. / Physiology, Cell and Developmental Biology

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Background Immune-stimulatory agents, like agonists of the innate immune receptor RIG-I, are currently tested in clinical trials as an intratumoral treatment option for patients with unresectable melanoma, aiming to enhance anti-tumor T cell responses. Switching of melanoma toward a dedifferentiated cell state has recently been linked to T cell and therapy resistance. It remains to be determined whether RIG-I agonists affect melanoma differentiation, potentially leading to T cell resistance. Methods Patient metastases-derived melanoma cell lines were treated with the synthetic RIG-I agonist 3pRNA, and effects on tumor cell survival, phenotype and differentiation were determined. Transcriptomic data sets from cell lines and metastases were analyzed for associations between RIG-I (DDX58) and melanoma differentiation markers and used to define signaling pathways involved in RIG-I- driven dedifferentiation. The impact of 3pRNA-induced melanoma dedifferentiation on CD8 T cell activation was studied in autologous tumor T cell models. Results RIG-I activation by 3pRNA induced apoptosis in a subpopulation of melanoma cells, while the majority of tumor cells switched into a non-proliferative cell state. Those persisters displayed a dedifferentiated cell phenotype, marked by downregulation of the melanocytic lineage transcription factor MITF and its target genes, including melanoma differentiation antigens (MDA). Transition into the MITFlow/MDAlow cell state was JAK-dependent, with some cells acquiring nerve growth factor receptor expression. MITFlow/MDAlow persisters switched back to the proliferative differentiated cell state when RIG-I signaling declined. Consistent with our in vitro findings, an association between melanoma dedifferentiation and high RIG-I (DDX58) levels was detected in transcriptomic data from patient metastases. Notably, despite their dedifferentiated cell phenotype, 3pRNA-induced MITFlow/ MDAlow persisters were still efficiently targeted by autologous CD8 tumor-infiltrating T lymphocytes (TILs). Conclusions Our results demonstrate that RIG-I signaling in melanoma cells drives a transient phenotypic switch toward a non-proliferative dedifferentiated persister cell state. Despite their dedifferentiation, those persisters are highly immunogenic and sensitive toward autologous TILs, challenging the concept of melanoma dedifferentiation as a general indicator of T cell resistance. In sum, our findings support the application of RIG-I agonists as a therapeutic tool for the generation of long-term clinical benefit in non-resectable melanoma.