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NKp46 Clusters at the Immune Synapse and Regulates NK Cell Polarization.

Hadad U, Thauland TJ, Martinez OM, Butte MJ, Porgador A, Krams SM - Front Immunol (2015)

Bottom Line: Concordantly, knock-down of NKp46 in primary human NK cells decreased recruitment of F-actin to the synapse.Live cell imaging experiments showed a linear correlation between NKp46 expression and lytic granules polarization to the immune synapse.Taken together, our data suggest that NKp46 signaling directly regulates the NK lytic immune synapse from early formation to late function.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Division of Abdominal Transplantation, Stanford University , Stanford, CA , USA ; The Shraga Segal Department of Microbiology and Immunology and Genetics, Ben-Gurion University of the Negev , Beersheba , Israel.

ABSTRACT
Natural killer (NK) cells play an important role in first-line defense against tumor and virus-infected cells. The activity of NK cells is tightly regulated by a repertoire of cell surface expressed inhibitory and activating receptors. NKp46 is a major NK cell-activating receptor that is involved in the elimination of target cells. NK cells form different types of synapses that result in distinct functional outcomes: cytotoxic, inhibitory, and regulatory. Recent studies revealed that complex integration of NK receptor signaling controls cytoskeletal rearrangement and other immune synapse-related events. However, the distinct nature by which NKp46 participates in NK immunological synapse formation and function remains unknown. In this study, we determined that NKp46 forms microclusters structures at the immune synapse between NK cells and target cells. Over-expression of human NKp46 is correlated with increased accumulation of F-actin mesh at the immune synapse. Concordantly, knock-down of NKp46 in primary human NK cells decreased recruitment of F-actin to the synapse. Live cell imaging experiments showed a linear correlation between NKp46 expression and lytic granules polarization to the immune synapse. Taken together, our data suggest that NKp46 signaling directly regulates the NK lytic immune synapse from early formation to late function.

No MeSH data available.


Related in: MedlinePlus

NKp46 spatial distribution at the immune synapse. Representative images of fixed human primary NK cells stained with DAPI for nuclear labeling (blue), anti-NKp46-biotin followed by streptavidin Alexa Fluor 647 (red), and DIC images. (A) Non-interacting primary NK cells. (B,C) NK cells co-cultured with target HeLa cells; with (B) or without (C) accumulation at the immune synapse. (D) NKp46 distribution analysis using automated particles detection. Upper panels show the enhanced magnified image from (B) of NKp46 staining (left) and DIC (right). The lower left panel shows detected objects in a masked image. Overlay is shown in lower right panel. Object 1 is the synapse area. (E) Summary of relative accumulation of NKp46 at the immune synapse from 24 NK-target interactions. Scale bars = 10 μm.
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Figure 1: NKp46 spatial distribution at the immune synapse. Representative images of fixed human primary NK cells stained with DAPI for nuclear labeling (blue), anti-NKp46-biotin followed by streptavidin Alexa Fluor 647 (red), and DIC images. (A) Non-interacting primary NK cells. (B,C) NK cells co-cultured with target HeLa cells; with (B) or without (C) accumulation at the immune synapse. (D) NKp46 distribution analysis using automated particles detection. Upper panels show the enhanced magnified image from (B) of NKp46 staining (left) and DIC (right). The lower left panel shows detected objects in a masked image. Overlay is shown in lower right panel. Object 1 is the synapse area. (E) Summary of relative accumulation of NKp46 at the immune synapse from 24 NK-target interactions. Scale bars = 10 μm.

Mentions: Organization of NK receptors in activating clusters is important for signaling (46). To examine the spatial distribution of endogenous NKp46 in activated primary human NK cells, we used a monoclonal antibody to NKp46 to examine NK cell interactions with HeLa target cells, The NKp46 receptor distributes homogenously on the cell membrane surface of NK cells that do not form immune synapses with target cells (Figure 1A). In contrast, confocal images of NK cells that bind and interact with target cells clearly demonstrate that the NKp46 receptor segregates in large clusters at the immune synapse (Figure 1B). In a few observed conjugations (3 out of 27), the NKp46 receptor segregated at the cell membrane but did not cluster at the immune synapse (Figure 1C). In order to quantify the portion of the receptor molecules that are accumulating at the immunological synapse, we used automated particle analysis. In one representative image (enhanced magnification of Figure 1B, lower panel), 14 objects were detected (white numbers), with objects one and four in the area of the immune synapse according to NK-target cell contact area in brightfield image (Figure 1D). Measuring the distribution of labeled NKp46 in 24 NK-target cell conjugations indicated that 58 ± 8% (mean ± SD) of the membrane-associated NKp46 receptor molecules are accumulating at the immune synapse (Figure 1E). These results clearly indicate that following NK cell conjugation, NKp46 segregates into distinct domains at the cell membrane. The enrichment of clusters at the immune synapse suggests that the receptor confinement is important for activation.


NKp46 Clusters at the Immune Synapse and Regulates NK Cell Polarization.

Hadad U, Thauland TJ, Martinez OM, Butte MJ, Porgador A, Krams SM - Front Immunol (2015)

NKp46 spatial distribution at the immune synapse. Representative images of fixed human primary NK cells stained with DAPI for nuclear labeling (blue), anti-NKp46-biotin followed by streptavidin Alexa Fluor 647 (red), and DIC images. (A) Non-interacting primary NK cells. (B,C) NK cells co-cultured with target HeLa cells; with (B) or without (C) accumulation at the immune synapse. (D) NKp46 distribution analysis using automated particles detection. Upper panels show the enhanced magnified image from (B) of NKp46 staining (left) and DIC (right). The lower left panel shows detected objects in a masked image. Overlay is shown in lower right panel. Object 1 is the synapse area. (E) Summary of relative accumulation of NKp46 at the immune synapse from 24 NK-target interactions. Scale bars = 10 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4585260&req=5

Figure 1: NKp46 spatial distribution at the immune synapse. Representative images of fixed human primary NK cells stained with DAPI for nuclear labeling (blue), anti-NKp46-biotin followed by streptavidin Alexa Fluor 647 (red), and DIC images. (A) Non-interacting primary NK cells. (B,C) NK cells co-cultured with target HeLa cells; with (B) or without (C) accumulation at the immune synapse. (D) NKp46 distribution analysis using automated particles detection. Upper panels show the enhanced magnified image from (B) of NKp46 staining (left) and DIC (right). The lower left panel shows detected objects in a masked image. Overlay is shown in lower right panel. Object 1 is the synapse area. (E) Summary of relative accumulation of NKp46 at the immune synapse from 24 NK-target interactions. Scale bars = 10 μm.
Mentions: Organization of NK receptors in activating clusters is important for signaling (46). To examine the spatial distribution of endogenous NKp46 in activated primary human NK cells, we used a monoclonal antibody to NKp46 to examine NK cell interactions with HeLa target cells, The NKp46 receptor distributes homogenously on the cell membrane surface of NK cells that do not form immune synapses with target cells (Figure 1A). In contrast, confocal images of NK cells that bind and interact with target cells clearly demonstrate that the NKp46 receptor segregates in large clusters at the immune synapse (Figure 1B). In a few observed conjugations (3 out of 27), the NKp46 receptor segregated at the cell membrane but did not cluster at the immune synapse (Figure 1C). In order to quantify the portion of the receptor molecules that are accumulating at the immunological synapse, we used automated particle analysis. In one representative image (enhanced magnification of Figure 1B, lower panel), 14 objects were detected (white numbers), with objects one and four in the area of the immune synapse according to NK-target cell contact area in brightfield image (Figure 1D). Measuring the distribution of labeled NKp46 in 24 NK-target cell conjugations indicated that 58 ± 8% (mean ± SD) of the membrane-associated NKp46 receptor molecules are accumulating at the immune synapse (Figure 1E). These results clearly indicate that following NK cell conjugation, NKp46 segregates into distinct domains at the cell membrane. The enrichment of clusters at the immune synapse suggests that the receptor confinement is important for activation.

Bottom Line: Concordantly, knock-down of NKp46 in primary human NK cells decreased recruitment of F-actin to the synapse.Live cell imaging experiments showed a linear correlation between NKp46 expression and lytic granules polarization to the immune synapse.Taken together, our data suggest that NKp46 signaling directly regulates the NK lytic immune synapse from early formation to late function.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Division of Abdominal Transplantation, Stanford University , Stanford, CA , USA ; The Shraga Segal Department of Microbiology and Immunology and Genetics, Ben-Gurion University of the Negev , Beersheba , Israel.

ABSTRACT
Natural killer (NK) cells play an important role in first-line defense against tumor and virus-infected cells. The activity of NK cells is tightly regulated by a repertoire of cell surface expressed inhibitory and activating receptors. NKp46 is a major NK cell-activating receptor that is involved in the elimination of target cells. NK cells form different types of synapses that result in distinct functional outcomes: cytotoxic, inhibitory, and regulatory. Recent studies revealed that complex integration of NK receptor signaling controls cytoskeletal rearrangement and other immune synapse-related events. However, the distinct nature by which NKp46 participates in NK immunological synapse formation and function remains unknown. In this study, we determined that NKp46 forms microclusters structures at the immune synapse between NK cells and target cells. Over-expression of human NKp46 is correlated with increased accumulation of F-actin mesh at the immune synapse. Concordantly, knock-down of NKp46 in primary human NK cells decreased recruitment of F-actin to the synapse. Live cell imaging experiments showed a linear correlation between NKp46 expression and lytic granules polarization to the immune synapse. Taken together, our data suggest that NKp46 signaling directly regulates the NK lytic immune synapse from early formation to late function.

No MeSH data available.


Related in: MedlinePlus