Single cell studies of natural killer cell immune surveillance

Abstract

Our immune system protects us from microbes such as viruses, bacteria and parasitic worms. The immune system also plays a key role in fighting cancer and in carrying out a function termed ‘immune surveillance’, natural killer (NK) cells patrol tissues and participate in the elimination of transformed cells and established tumors. By forming a tight contact to a abnormal cell such as a tumor cell, i.e. target cell, followed by secretion of toxic compounds the NK cell is able to kill the target cell. This process requires the formation of a lytic immune synapse which is a specialized interface formed between the NK cell and the target cell.

NK cell populations are heterogeneous as they are composed of individual cells that differ in their phenotype and functional responses. The work presented in this thesis aimed to study the functional heterogeneity within NK cell populations. In addition, we also set out to reveal what aspects of NK cell cytotoxicity that are influenced by the functional maturity, i.e. the level of education, of individual NK cells. Moreover, we were interested in identifying NK cells that are especially effective in killing tumor cells, so called ‘serial killers’. The interest in serial killers is mainly due to their potential use in cancer therapies. For these purposes we have employed a microwell array system in combination with time-lapse imaging to perform long-term studies of the functional responses of individual NK cells. Furthermore, another aim was to determine the influence of the spatial distribution of ligands on NK cell responses. Here arrays of spatially separated ligands were patterned into artificial immune synapses (AIS) and NK cells interacting with AIS were followed using time-lapse imaging. Results from the single cell analysis performed in the presented investigations has provided important insights to the dynamics of NK cell migration behavior and interactions with target cells.

In paper I we characterized NK cells and found that these generally displayed a binary commitment as they were dedicated to a ‘kill’ or ‘no kill’ type of behavior. Furthermore, the results showed that NK cells can kill target cells via a fast or slow process which may have different effects on surrounding cells and tissues.

In paper II we compared NK cells activated with the cytokine IL-2 to non-activated, resting NK cells in terms of their migration behavior, ability to form conjugates, and killing of tumor cells. The results showed that IL-2 activation of NK cells induces a migratory phenotype and enhances their ability to form conjugates and kill tumor cells.

In paper III we compared the migration behavior, ability to form conjugates, and killing by NK cells at different levels of education. Here the results revealed heterogeneity in the migration behavior and cytotoxic response within the defined subsets of uneducated and educated NK cells. Still, the frequencies of NK cells that formed conjugates and killed target cells were significantly higher among educated NK cells compared to uneducated NK cells. In paper IV NK cells were imaged while interacting with ligands patterned into spatially separated AIS. We found that NK cells interacting with AIS composed of ligands for the activating receptors LFA-1 and CD16 displayed different morphologies and migration responses.