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Abstract

Environmental conditions significantly influence the phenotype and functionality of immune cells. Specifically, hypoxic conditions (oxygen concentration below 1%) prevalent in tissues and organs impact both the adaptive and innate immune systems. The transcription factor hypoxia-inducible factor 1-alpha (HIF-1α) plays a pivotal role in regulating immune cell adaptation to hypoxia. Natural killer (NK) cells are essential in tumor surveillance, as they target tumor cells by producing cytokines, chemokines, and cytolytic granules. Nevertheless, NK cells infiltrating solid tumors with hypoxic environments often lose their functionality. In addition, the high sensitivity of human primary NK cells to in vitro culturing conditions poses a challenge for classical transduction and transfection methods, hindering the study and generation of improved NK cells for immunotherapies. In this study, I investigated the effects of hypoxia and HIF-1α on human NK cells. My findings indicate that NK cells exposed to hypoxia (1 % oxygen) exhibited reduced IFNγ production, diminished degranulation response to cytokine stimuli and tumor targets, and impaired proliferation compared to NK cells exposed to normoxia (21 % oxygen). I explored the potential rescue of human NK cell functionality under hypoxia by genetically targeting HIF1A using CRISPR/Cas9 ribonucleoprotein-based transfection. HIF-1α-targeted NK cells, under hypoxia, restored IFNγ production upon IL-12/18 stimulation compared to mock control cells. To assess the practical applicability of HIF-1α-targeted NK cells, I employed patient-derived organoid lines from colorectal adenocarcinoma patients. The results revealed an enhanced cytotoxicity in HIF-1α-targeted NK cells against specific patient-derived organoids, particularly when combining Chimeric Antigen Receptor (CAR) targeting HER-2 with HIF-1α targeting. Additionally, I investigated the impact of hypoxia on cytokine-induced memory-like (CML) NK cells. CML NK cells exposed to hypoxia for seven days displayed a reduced IFNγ production upon IL-12/18 stimulation compared to normoxic controls. However, degranulation and cytokine production during K562 co-culture remained unaffected. These results suggest an oxygen sensitivity in CML NK cells, potentially regulated by HIF-1α, which warrants further investigation. Furthermore, I developed an efficient transfection protocol for human primary NK cells using electroporation and CRISPR/Cas9 ribonucleoprotein with dye-labelled sgRNAs. This allowed successful targeting of surface molecules such as TRAIL, NKG2A, TIGIT, CD112R, DNAM-1, and CD96, enabling the sorting of double and triple knockout populations of NK cells. In summary, my study highlights the importance of oxygen concentrations for human primary NK cells and introduces a cytotoxic NK cell product: HER-2 CAR HIF-1α NK cells, which may hold promise for CML NK cells. Moreover, I established an effective transfection system for human primary NK cells, facilitating the generation of gene edited NK cells to enhance tumor target killing.