There is a growing interest in adoptive transfer of ex vivo-activated lymphocytes aiming to improve and exploit their anti-tumor responses. Natural Killer (NK) cells are an innate lymphoid cell type which represents about 10-15% of circulating lymphocytes in the blood. Due to their ability to detect and kill infected and transformed cells, NK cells are crucial for host immunity. Many clinical trials are using ex vivo-cultivated NK cells (1), however, because of the immunosuppressive effect of the tumor, NK cell functionality is impaired (2, 3). This results in reduced responsiveness against tumor cells. To use NK cells effectively for tumor immunotherapy, a reversal of functional defects is of paramount importance. Preclinical NK-cell studies demonstrate that it is possible to produce NK cells which have recovered the phenotype of efficient tumor cytotoxicity. Our group has developed a system where we can expand these NK cells in a clinically compatible manner. These cells have a high, specific cytotoxicity against autologous and allogeneic tumors in vitro and in vivo. Even though promising results of safety, viability and anti-tumor responses have been reported, the best source of NK cells and optimal expanding conditions need to be determined. It is crucial that we optimize NK cell processing methods to achieve sufficient numbers of NK cells with the most efficient tumor cytotoxicity and clinical responses. For this purpose, we turn our attention to exosomes, 30-200 nm vesicles shed from cells as a means of cell communication. Research into the processing and characterization of exosomes has been increasing exponentially as their importance in cell signaling is revealed. It is now well established that exosomes play many roles in cancer and cell differentiation. We will utilize the known properties of exosomes to enhance the differentiation of NK cells. The exosome cargo of NK cells will be evaluated for miRNA content. These exosomes will then be introduced to hematopoietic stem cells at progressive stages of differentiation to NK cells to evaluate their effect on differentiation. We hypothesize that the cargo content of NK cell-derived exosomes can accelerate and promote NK cell lineage commitment during in vitro CD34+ cell derived NK cell differentiation. The data will be analyzed for differentiation time, phenotype of differentiated cells and functionality of the final product. The project should produce numerous replications for robust statistical analysis. This endeavor has great potential to provide insights on NK cell expansion that can efficiently eliminate tumor cells, improving the manufacturing of cell therapy products. Moreover, the evaluation of NK exosome cargo will provide a detailed overview and possibly mechanistic explanations for the tumor-induced dysfunctions of NK cells.