Cystatin F as a mediator of immune suppression in tumor microenvironmentSenjor, Emanuela (Avtor) Kos, Janko (Mentor) cystatin FglioblastomaNK cellsimmunosuppressionN-glycosylationImmunosuppressive microenvironment causes decreased function of cytotoxic immune cells and contributes to the immune escape of cancer cells. Natural killer (NK) cells represent a very promising candidate for cancer therapy. They target tumor cells with granule-mediated cytotoxic pathway when the balance between activatory and inhibitory signals caused by the interaction of suitable ligands with their receptors is disrupted towards the activation of NK cells. Effector molecules, granzymes and perforin are stored in their inactive form and are activated from precursor forms by cathepsins C, H, and L. Cystatin F acts as a modulator of NK cell cytotoxicity as it can inhibit those cathepsins, thereby attenuating NK cell cytotoxicity. Cystatin F is glycosylated and is activated from dimeric to monomeric form by cathepsin V. It is localized in endosomes/lysosomes, but can also be secreted and further internalized to bystander cells. Cystatin F is normally expressed by immune cells, but several studies implicate cystatin F expression to other cell types, in particular under various pathological conditions. We have shown that cystatin F expression is increased in glioblastoma by either glioblastoma tumor or immune cells, both infiltrating immune cells and resident immune cells such as microglia. Moreover, glioblastoma cells were able to internalise cystatin F, which contributed to the decreased susceptibility of glioblastoma cells to NK cell cytotoxicity. We demonstrated that N-glycosylation profile of cystatin F differed between cell types and between NK cells of different cytotoxic potential. High-mannose glycosylation was associated with increased localization in lysosomes, cathepsin C interaction and attenuation of NK cell cytotoxicity. Further, cystatin F internalisation to NK cells by means of endocytosis, enhanced split anergy of NK cells. We characterised super-charged NK cells, potent candidates for cancer therapy, with increased cytotoxicity, cytokine secretion and ability to proliferate. Super-charged NK cells had increased expression and activity of cathepsin C and granzyme B and increased expression of inactive dimeric cystatin F. Finally, we identified a small molecular inhibitor of cathepsin V, which prevented activation of cystatin F from inactive dimeric form and increased NK cell cytotoxicity. We have therefore established cystatin F as an important mediator of immunosuppression in the tumor microenvironment and proposed novel therapeutic approaches to improve cancer immunotherapy by targeting cystatin F.[E. Senjor]20222022-09-14 07:58:52Doktorska disertacija140302sl