Abstract
Cytotoxic lymphocytes are a subset of immune cells with key roles in host defense against viral infections, against tumors as well as in self-regulation of immunity. Cytotoxic lymphocytes kill target cells by triggering cell death pathways with direct cell-cell contacts. To ensure the specificity and efficiency of killing, cytotoxic lymphocytes form a specialized structure named cytotoxic immunological synapse (IS) with their target cell. The IS is composed of adhesion molecules, signaling molecules, and cytoskeletal components that coordinate the delivery of cytotoxic granules. Deficiencies in the proper functioning of the IS can lead to immune dysregulation that can clinically manifest in children as a severe and often lethal cytokine storm known as familiar hemophagocytic lymphohistiocytosis (fHLH). With respect to the pathophysiology of fHLH, many genetic mutations that disrupt the cytotoxic machinery have been identified. Consequently, a fundamental question arises regarding how such mutations affect cellular state and how this is integrated with signaling pathways to regulate cell decisions throughout IS maturation. The approach outlined in this thesis is an imaging-based characterization of IS formed in an experimental model system where NK cells, a sub-group of cytotoxic lymphocytes, are triggered to kill target cells in vitro. Imaging is performed in a high-throughput fashion in a multi-well plate format and multiplexed immunofluorescence is used to stain several markers known to be involved in the formation and functionality of the IS of NK cells. These include multiple signaling proteins, markers for cytotoxic granules and cellular state markers like cytoskeletal proteins and metabolic components. The result section of this thesis is divided into two main parts. The first part is related to the technical development of the imaging-based multiplexed assay, with particular emphasis on tackling weak cell adhesion on the bottom of imaging plates as well as triggering IS formation with suitable stimuli. In the second part, the established essay is used to functionally assess NK cells in 4 healthy donors and 2 patients deficient for SAP and Munc13-4. Using computational approaches, NK cells involved in IS are characterized through the polarization of their MTOC and other intracellular features specific for cytotoxic behavior. This multivariate analysis suggests that NK cells with defective vesicle fusion caused by MUNC13- deficiency show a distinct cellular state characterized by smaller cellular area and diminished cytoskeleton remodeling, along with a strongly suppressed MEK-ERK pathway. The interplay between this cellular state, signaling status and impaired vesicle fusion warrants further investigation. In conclusion, these frameworks and findings show the potential of multiplexing image-based assays to study cytotoxicity of NK cells and could pave the way to an exploration of the IS under various drug perturbations aimed at targeted manipulation of the spatial orchestration of intracellular events during the cytotoxic process.
Koovely, Danil