Receptor localization and dynamics of murine natural killer cells at single cell level : using advanced fluorescence microscopy
Author: Bagawath Singh, Sunitha
Date: 2018-01-18
Location: Hörsal Atrium, Nobels väg 12B, Karolinska Institutet, Solna
Time: 9.30
Department: Inst för mikrobiologi, tumör- och cellbiologi / Dept of Microbiology, Tumor and Cell Biology
View/ Open:
Thesis (1.635Mb)
Abstract
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.
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 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.
Fulltext (DOI)
Pubmed
View record in Web of Science®
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.
Fulltext (DOI)
Pubmed
View record in Web of Science®
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]
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.
Fulltext (DOI)
Pubmed
View record in Web of Science®
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.
Fulltext (DOI)
Pubmed
View record in Web of Science®
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]
Institution: Karolinska Institutet
Supervisor: Johansson, Sofia
Co-supervisor: Kärre, Klas; Widengren, Jerker
Issue date: 2017-12-21
Rights:
Publication year: 2018
ISBN: 978-91-7676-891-4
Statistics
Total Visits
Views | |
---|---|
Receptor ...(legacy) | 401 |
Receptor ... | 209 |
Total Visits Per Month
September 2023 | October 2023 | November 2023 | December 2023 | January 2024 | February 2024 | March 2024 | |
---|---|---|---|---|---|---|---|
Receptor ... | 2 | 3 | 1 | 0 | 0 | 1 | 0 |
File Visits
Views | |
---|---|
Thesis_Sunitha_Bagawath_Singh.pdf | 167 |
Thesis_Sunitha_Bagawath_Singh.pdf(legacy) | 162 |
Top country views
Views | |
---|---|
United States | 163 |
Sweden | 158 |
Germany | 71 |
China | 48 |
Russia | 14 |
France | 10 |
India | 10 |
South Korea | 9 |
Finland | 6 |
United Kingdom | 6 |
Top cities views
Views | |
---|---|
Ashburn | 47 |
Stockholm | 30 |
Wilmington | 20 |
Beijing | 19 |
Woodbridge | 17 |
Houston | 16 |
Uppsala | 15 |
Lane | 14 |
Solna | 13 |
Menlo Park | 9 |