Cystatin F as a mediator of immune suppression in tumor microenvironment : doctoral thesis

Senjor, Emanuela (author), Kos, Janko (mentor)

Immunosuppressive 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.

• Language: English
• Keywords: cystatin F, glioblastoma, NK cells, immunosuppression, N-glycosylation
• Work type: Dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: FFA - Faculty of Pharmacy
• Place of publishing: Ljubljana
• Publisher: [E. Senjor]
• Year: 2022
• Number of pages: XX, 158 f.
• PID: 20.500.12556/RUL-140302
• UDC: 615.37:616-006.48(043.3)
• COBISS.SI-ID: 121229059
• Publication date in RUL: 14.09.2022

Secondary language

• Language: Slovenian
• Title: Cistatin F kot mediator imunske supresije v tumorskem mikrookolju : the interdisciplinary doctoral programme in Biomedicine Clinical Biochemistry and Laboratory Biomedicine

Abstract

Imunosupresivno mikrookolje povzroča zmanjšano delovanje citotoksičnih imunskih celic in prispeva k imunskemu pobegu rakavih celic. Naravne celice ubijalke (NK) so uporabne kandidatke za zdravljenje raka, ki se še ne uporabljajo v klinični praksi. Celice NK ciljajo tumorske celice s citotoksičnimi granulami, potem ko se ravnovesje signalov, ki jih sproži vezava ustreznih ligandov na aktivacijske in inhibitorne receptorje na njihovih površinah, prevesi v aktivacijo celic NK. Neaktivno shranjene efektorske molekule, grancime in perforin aktivirajo katepsini C, H in L. Cistatin F deluje kot modulator citotoksičnosti celic NK, saj lahko zavira omenjene katepsine in s tem oslabi citotoksično delovanje celic NK. Cistatin F je glikoziliran. Da lahko deluje, se mora pretvoriti iz dimerne v monomerno obliko, kar katalizira katepsin V. Nahaja se v endosomih/lizosomih, lahko pa se tudi izloča in internalizira v sosednje celice. Cistatin F se običajno izraža v imunskih celicah, vendar vedno več študij kaže tudi na njegovo izražanje v patoloških stanjih. Pokazali smo, da je izražanje cistatina F povečano v glioblastomu in da ga izražajo tako glioblastomske kot infiltrirajoče ter stalno prisotne imunske celice, kot npr. mikroglija. Poleg tega, so bile glioblastomske celice sposobne privzeti cistatin F z endocitozo, kar je prispevalo k njihovi zmanjšani občutljivosti na citotoksičnost celic NK. Dokazali smo tudi, da se N-glikozilacija cistatina F razlikuje med različnimi tipi celic in med celicami NK z različnim citotoksičnim potencialom. Visoko-manozna glikozilacija cistatina F je bila povezana z njegovo povečano lokalizacijo v lizosomih, interakcijo s katepsinom C in zmanjšanjem citotoksičnosti celic NK. Internalizacija cistatina F v celice NK je vplivala na njihovo delno anergijo. Analizirali smo tudi supervzbujene celice NK, ki imajo dober potencial za zdravljenje raka, saj so zanje značilni povečana citotoksičnost, izločanje citokinov in zmožnost proliferacije. Supervzbujene celice NK izkazujejo povečano izražanje in aktivnost katepsina C in grancima B, ter povečano izražanje neaktivnega dimernega cistatina F. Nazadnje smo identificirali še nizko-molekularni zaviralec katepsina V, ki je preprečil aktivacijo cistatina F in povečal citotoksičnost celic NK. Tako smo cistatin F opredelili kot pomemben dejavnik imunosupresije v tumorskem mikrookolju in zasnovali nove terapevtske pristope za izboljšanje imunske terapije raka.

• Keywords: cistatin F, glioblastom, celice NK, imunosupresija, N-glikozilacija, imunosupresivna biološka zdravila, rak (medicina), glioblastom