Granzyme B activity in coculture of natural killer cells and tumour cells

Naravne celice ubijalke so ključne celice imunskega sistema, ki spadajo v vejo prirojene imunosti in s citotoksičnostjo branijo sistem pred patogeni in tumorsko spremenjenimi celicami. Naravne celice ubijalke povezujejo vejo adaptivne imunosti z vejo prirojene imunosti, saj ob svoji aktivaciji v okolje sproščajo pro-vnetne citokine, ki aktivirajo še ostale celice imunskega sistema. S svojo citotoksičnostjo naravne celice ubijalke skrbijo za odstranjevanje nevarnosti v sistemu. V svoji notranjosti vsebujejo posebne sekretorne granule, v katerih se nahaja vrsta citolitičnih molekul, med njimi so grancimi (A, B, H, K, M), perforin and granulizin, ki v tarčni celici povzročijo celično smrt. Ena izmed pomembnejših molekul, ki se nahajajo v sekretornih granulah in eden glavnih mediatorjev celične smrti v tarčnih celicah je grancim B. Grancim B je serinska proteaza, ki se kot neaktiven encim nahaja v sekretornih granulah naravnih celic ubijalk. Ob aktivaciji celice se granule skupaj z grancimom B, perforinom in ostalimi molekulami z eksocitozo sprostijo v citosol tarčne celice. V citosolu poteče kaskada dogodkov, ki vodi v sestavljanje kompleksa, imenovanega apoptosom in iniciacijo celične smrti. Grancim B pa ne igra le vloge pri celični smrti, temveč so njegovo povečano koncentracijo v zunajceličnem matriksu odkrili pri različnih avtoimunih obolenjih. V prihodnosti bi lahko grancim B postal pomemben biomarker tako za napoved napredovanja različnih avtoimunih boleznih, kot tudi za napoved uspešnosti zdravljenja z imunoterapijo. V diplomskem delu smo preverjali možnost detekcije grancima B s fluorescenčnim substratom in vitro ter dokazali povišano aktivnost v kokulturi naravnih celic ubijalk in tumorskih celic. Izražanje grancima B v kokulturi smo detektirali z imunodetekcijo po prenosu Western.Natural killer cells are key cells of the immune system that belong to the innate immunity branch and defend the system against pathogens and tumour cells with their cytotoxicity. Natural killer cells link adaptive immunity to innate immunity by releasing pro-inflammatory cytokines into the environment upon activation, which then activate other cells of the immune system. Through their cytotoxicity, natural killer cells ensure that threats in the system are eliminated. They contain secretory granules consisting of a series of cytolytic molecules, such as granzymes (A, B, H, K, M), perforin and granulysin. They are released into the cytosol of the target cell by degranulation and induce apoptosis. One of the most important molecules found in secretory granules and a major mediator of cell death in target cells is granzyme B. Granzyme B is a serine protease that is found as an inactive enzyme in the secretory granules of natural killer cells. Upon activation of the cell, the granules with granzyme B, perforin and other molecules, are released into the cytosol of the target cell by exocytosis. A cascade of events takes place in the cytosol, leading to the assembly of a complex called the apoptosome and the initiation of cell death. Granzyme B not only plays a role in cell death, but its increased concentration in the extracellular matrix has been found in various autoimmune diseases. In future, granzyme B could become an important biomarker both for predicting the progression of various autoimmune diseases and for the prediction of immunotherapy treatments success. In this thesis, we tested the in vitro detection of granzyme B with a fluorescent substrate and demonstrated increased proteolytic activity in a coculture of natural killer cells and tumour cells. Granzyme B expression in coculture was detected by immunodetection after Western transfer