The immune system provides defense against infectious agents such as viruses, bacteria and parasites. Besides eliminating extracellular agents, the immune system also constantly monitors our own cells for signs of transformation, including tumor development and virus infection. This process, called immune surveillance, is mediated in part by natural killer (NK) cells. NK cells sense transformation through the interaction of surface receptors with proteins on the surface of the diseased cell. The efficient binding of these receptors results in the formation of a tight contact between the two cells, called an immune synapse. If danger signals dominate in the synapse, the NK cell has the potential to deliver toxic compounds and to bind to specific death receptors at the target cell surface, resulting in the induction of target cell death. Apart from the ability to eliminate transformed cells, NK cells also have an immuno-regulatory function by directly killing other immune cells and by secreting pro- and anti-inflammatory cytokines.

Because of these roles, NK cells are of special interest in the growing field of cancer immunotherapy, where the function of immune cells is enhanced to defeat tumor cells. Clinical trials using NK cell-centered therapy have shown promising results against blood-borne cancer, yet progress has been limited against solid tumors. One possible explanation is related to the locally immuno-suppressive environment created by the solid tumor, for which improved research models are necessary. Besides, there is growing evidence of pronounced heterogeneity in the function of individual cells amidst the NK cell pool. Improving our understanding of NK cell biology thus requires advances in dedicated single-cell assays. For this purpose, our research group has previously developed miniaturized multi-well chips where individual cells can be confined and followed by microscopy over periods of several days. Using these microchips, a peculiar group of highly potent NK cells has been identified, which are able to kill several target cells in a row and contribute disproportionately to the overall cytotoxicity, and are therefore referred to as serial-killing NK cells.

The work presented in this thesis is focused on developing and applying microscopy-based single-cell assays to the study of NK cell functional heterogeneity, with a particular focus on the mechanistic aspects of cytotoxicity. In Paper I, we investigated the formation and outcome of immune synapses in single cells, using micro-patterning to create distinct spatial distributions of ligands. We observed that synapse formation was guided by the overall shape of the ligands while local signaling regulated the final steps of exocytosis. Paper II is dedicated to the study of the cytotoxic mechanisms used by individual NK cells and their regulation, in particular comparing serial-killing NK cells and moderate killers. Using dedicated fluorescent reporters, we identified a switch between two commonly used killing pathways, degranulation and death ligand engagement, and proposed a model for the underlying process. This topic was further detailed in Paper III, where the contribution of these cytotoxic mechanisms under additional antibody stimulation was studied. The investigation was conducted in a newly developed single-use plastic microchip, designed to enable the generation of multiple simultaneous two- and three-dimensional cell cultures while retaining high imaging performance. In Paper IV, we implemented single-cell retrieval from the silicon-glass microwells. We characterized the performance of our setup and demonstrated its potential at identifying and retrieving rare populations defined by functional readouts. 

Together, these studies further demonstrate the importance of single-cell analysis in the field of immunology. Besides advancing our understanding of NK cell biology, these developments may prove valuable in developing improved immunotherapies.