Receptor localization and dynamics of murine natural killer cells at single cell level : using advanced fluorescence microscopy

Natural Killer (NK) cells are immune cells and important for the defense against virally infected and malignant cells. NK cells are regulated by germline encoded activating and inhibitory receptors. Activating receptors specifically recognize ligands which are either encoded by infectious agents, or induced upon infection or cellular stress. Inhibitory receptors interact with self-ligands expressed on healthy cells, among them MHC class I. NK cells inspect the host cells by screening for alterations in activating and inhibitory ligand expression. The balance between input from activating and inhibitory receptors determines the NK cell response. NK cells undergo a process of functional maturation and acquisition of self-tolerance via sensing of the steady-state input through their receptors. This process is known as education. The cytotoxic activity of NK cells can be further increased by cytokines produced by other immune cells.

The aim of this thesis was to characterize the differences in receptor dynamics and localization between NK cells based on either cytokine activation or educational status. Fluorescence based advanced microscopy techniques were used to quantitate receptor dynamics and spatial organization.

In paper I, we investigated the influence of cytokine stimulation on the lateral diffusion of the inhibitory receptor Ly49A and its ligand MHC class I on NK cells within the cell membrane. The response to cytokine stimulation was heterogeneous among the NK cells. We characterized a subpopulation of NK cells with faster diffusion of both MHC class I and Ly49A. The receptor diffusion was established on primary NK cells using Fluorescence Correlation Spectroscopy. In paper II, a practical protocol for utilizing FCS on primary lymphocytes was presented. In Paper III, we showed that NKp46 and Ly49A were confined within microdomains on NK cells. The actin cytoskeleton and cholesterol composition of NK cells played important roles in initiating activating cell signaling. In Paper IV, we investigated the organization and clustering of activating and inhibitory receptors on educated and uneducated NK cells. We found that clusters of NKp46 and Ly49A were larger on uneducated NK cells. The nearest neighbour distances from activating to inhibitory receptors were not significantly different between educated and uneducated NK cells, thus the organization of inhibitory receptors in relation to the activating receptors do not seem to be of importance for the educational process.

In summary, the findings in this thesis enlightens the importance of altered receptor dynamics and organization on NK cells depending on the state of activation and education. Furthermore, receptor dynamics could be an important aspect for understanding NK cell function.

List of scientific papers

I. Cytokines Induce Faster Membrane Diffusion of MHC Class I and the Ly49A Receptor in a Subpopulation of Natural Killer Cells. Sunitha Bagawath-Singh, Elina Staaf, Arie Jan Stoppelenburg, Thiemo Spielmann, Taku Kambayashi, Jerker Widengren and Sofia Johansson. Front Immunol. Feb 4;7:16. 2016.
https://doi.org/10.3389/fimmu.2016.00016

II. Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy. Elina Staaf, Sunitha Bagawath-Singh, Sofia Johansson. J Vis Exp. 1;(120) 2017.
https://doi.org/10.3791/54756

III. Educated Natural Killer Cells Exhibit a More Dynamic Movement of NKp46 and a Higher Confinement of the Ly49A Receptor. E. Staaf, P. N. Hedde, Sunitha-Bagawath Singh, J. Piguet, E. Gratton, S. Johansson. [Submitted]

IV. Nano-structural Organization of Activating and Inhibitory Receptors in Murine Natural Killer cell Education. Sunitha Bagawath Singh, Jan Bergstrand, Daniel Rönnlund, Hans Blom, Jerker Widengren and Sofia Johansson. [Manuscript]