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- Bagawath-Singh, Sunitha, et al.
(författare)
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Cytokines Induce Faster Membrane Diffusion of MHC Class I and the Ly49A Receptor in a Subpopulation of Natural Killer Cells
- 2016
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Ingår i: Frontiers in Immunology. - : Frontiers. - 1664-3224. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Cytokines have the potential to drastically augment immune cell activity. Apart from altering the expression of a multitude of proteins, cytokines also affect immune cell dynamics. However, how cytokines affect the molecular dynamics within the cell membrane of immune cells has not been addressed previously. Molecular movement is a vital component of all biological processes, and the rate of motion is, thus, an inherent determining factor for the pace of such processes. Natural killer (NK) cells are cytotoxic lymphocytes, which belong to the innate immune system. By fluorescence correlation spectroscopy, we investigated the influence of cytokine stimulation on the membrane density and molecular dynamics of the inhibitory receptor Ly49A and its ligand, the major histocompatibility complex class I allele H-2D(d), in freshly isolated murine NK cells. H-2D(d) was densely expressed and diffused slowly in resting NK cells. Ly49A was expressed at a lower density and diffused faster. The diffusion rate in resting cells was not altered by disrupting the actin cytoskeleton. A short-term stimulation with interleukin-2 or interferon- alpha + beta did not change the surface density of moving H-2D(d) or Ly49A, despite a slight upregulation at the cellular level of H-2D(d) by interferon-alpha + beta, and of Ly49A by IL-2. However, the molecular diffusion rates of both H-2D(d) and Ly49A increased significantly. A multivariate analysis revealed that the increased diffusion was especially marked in a subpopulation of NK cells, where the diffusion rate was increased around fourfold compared to resting NK cells. After IL-2 stimulation, this subpopulation of NK cells also displayed lower density of Ly49A and higher brightness per entity, indicating that Ly49A may homo-cluster to a larger extent in these cells. A faster diffusion of inhibitory receptors could enable a faster accumulation of these molecules at the immune synapse with a target cell, eventually leading to a more efficient NK cell response. It has previously been assumed that cytokines regulate immune cells primarily via alterations of protein expression levels or posttranslational modifications. These findings suggest that cytokines may also modulate immune cell efficiency by increasing the molecular dynamics early on in the response.
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| 2. |
- Bagawath Singh, Sunitha
(författare)
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Receptor localization and dynamics of murine natural killer cells at single cell level : using advanced fluorescence microscopy
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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.
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| 3. |
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| 4. |
- Staaf, Elina, et al.
(författare)
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Educated natural killer cells show dynamic movement of the activating receptor NKp46 and confinement of the inhibitory receptor Ly49A
- 2018
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Ingår i: Science Signaling. - : AMER ASSOC ADVANCEMENT SCIENCE. - 1945-0877 .- 1937-9145. ; 11:517
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Tidskriftsartikel (refereegranskat)abstract
- Educated natural killer (NK) cells have inhibitory receptors specific for self major histocompatibility complex (MHC) class I molecules and kill cancer cells more efficiently than do NK cells that do not have such receptors (hyporesponsive NK cells). The mechanism behind this functional empowerment through education has so far not been fully described. In addition, distinctive phenotypic markers of educated NK cells at the single-cell level are lacking. We developed a refined version of the image mean square displacement (iMSD) method (called iMSD carpet analysis) and used it in combination with single-particle tracking to characterize the dynamics of the activating receptor NKp46 and the inhibitory receptor Ly49A on resting educated versus hyporesponsive murine NK cells. Most of the NKp46 and Ly49A molecules were restricted to microdomains; however, individual NKp46 molecules resided in these domains for shorter periods and diffused faster on the surface of educated, compared to hyporesponsive, NK cells. In contrast, the movement of Ly49A was more constrained in educated NK cells compared to hyporesponsive NK cells. Either disrupting the actin cytoskeleton or adding cholesterol to the cells prohibited activating signaling, suggesting that the dynamics of receptor movements within the cell membrane are critical for the proper activation of NK cells. The faster and more dynamic movement of NKp46 in educated NK cells may facilitate a swifter response to interactions with target cells.
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