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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 knockdown in primary NK cells modifies F-actin accumulation.(A) NKp46 surface expression analysis 72 h post NKp46 siRNA (blue solid line), negative control siRNA (red solid line), untreated primary NK cells (black dashed line), and isotype control staining (gray-filled graph). Scale bar = 10 μm (B) Summarized MFI for surface NKp46 in NKp46 siRNA (blue), negative control siRNA (red), untreated primary cells (black) treated cells. (C) Representative micrographs of NKp46 siRNA-treated primary NK cells that were incubated with HeLa (upper panel) or HepG2 (lower panel) target cells, fixed and permeabilized, and stained for F-actin (red) and DAPI (blue). (D) Results summarized for different effector–target interactions synapse (n > 30) log values F-actin ratios of representative experiment of two repeated experiments Graph bars show mean ± SEM. *p < 0.05, Student’s t-test.
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Figure 3: NKp46 knockdown in primary NK cells modifies F-actin accumulation.(A) NKp46 surface expression analysis 72 h post NKp46 siRNA (blue solid line), negative control siRNA (red solid line), untreated primary NK cells (black dashed line), and isotype control staining (gray-filled graph). Scale bar = 10 μm (B) Summarized MFI for surface NKp46 in NKp46 siRNA (blue), negative control siRNA (red), untreated primary cells (black) treated cells. (C) Representative micrographs of NKp46 siRNA-treated primary NK cells that were incubated with HeLa (upper panel) or HepG2 (lower panel) target cells, fixed and permeabilized, and stained for F-actin (red) and DAPI (blue). (D) Results summarized for different effector–target interactions synapse (n > 30) log values F-actin ratios of representative experiment of two repeated experiments Graph bars show mean ± SEM. *p < 0.05, Student’s t-test.

Mentions: To validate the role of endogenous NKp46 in rearrangement of the cytoskeleton, we knocked down NKp46 in primary human peripheral blood NK cells using siRNA. Analysis of cell surface NKp46 following electroporation showed that NKp46 siRNA (blue line) almost completely abolished receptor levels, while control siRNA (red line) has no effect (Figures 3A,B). Importantly, there was no observed difference in cell growth or morphology between the control siRNA group and NKp46 siRNA group (data not shown). Analysis of interactions between NK cells and HeLa or HepG2 cells showed that knockdown of NKp46 resulted in the recruitment of 40 and 45%, respectively less F-actin to the immune synapse, whereas NK cells that received control siRNA recruited the same amount of F-actin to the immune synapse as untreated NK cells (Figures 3C,D). These results further support the observation that NKp46 contributes to cytoskeletal rearrangement at the immune synapse.


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 knockdown in primary NK cells modifies F-actin accumulation.(A) NKp46 surface expression analysis 72 h post NKp46 siRNA (blue solid line), negative control siRNA (red solid line), untreated primary NK cells (black dashed line), and isotype control staining (gray-filled graph). Scale bar = 10 μm (B) Summarized MFI for surface NKp46 in NKp46 siRNA (blue), negative control siRNA (red), untreated primary cells (black) treated cells. (C) Representative micrographs of NKp46 siRNA-treated primary NK cells that were incubated with HeLa (upper panel) or HepG2 (lower panel) target cells, fixed and permeabilized, and stained for F-actin (red) and DAPI (blue). (D) Results summarized for different effector–target interactions synapse (n > 30) log values F-actin ratios of representative experiment of two repeated experiments Graph bars show mean ± SEM. *p < 0.05, Student’s t-test.
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Figure 3: NKp46 knockdown in primary NK cells modifies F-actin accumulation.(A) NKp46 surface expression analysis 72 h post NKp46 siRNA (blue solid line), negative control siRNA (red solid line), untreated primary NK cells (black dashed line), and isotype control staining (gray-filled graph). Scale bar = 10 μm (B) Summarized MFI for surface NKp46 in NKp46 siRNA (blue), negative control siRNA (red), untreated primary cells (black) treated cells. (C) Representative micrographs of NKp46 siRNA-treated primary NK cells that were incubated with HeLa (upper panel) or HepG2 (lower panel) target cells, fixed and permeabilized, and stained for F-actin (red) and DAPI (blue). (D) Results summarized for different effector–target interactions synapse (n > 30) log values F-actin ratios of representative experiment of two repeated experiments Graph bars show mean ± SEM. *p < 0.05, Student’s t-test.
Mentions: To validate the role of endogenous NKp46 in rearrangement of the cytoskeleton, we knocked down NKp46 in primary human peripheral blood NK cells using siRNA. Analysis of cell surface NKp46 following electroporation showed that NKp46 siRNA (blue line) almost completely abolished receptor levels, while control siRNA (red line) has no effect (Figures 3A,B). Importantly, there was no observed difference in cell growth or morphology between the control siRNA group and NKp46 siRNA group (data not shown). Analysis of interactions between NK cells and HeLa or HepG2 cells showed that knockdown of NKp46 resulted in the recruitment of 40 and 45%, respectively less F-actin to the immune synapse, whereas NK cells that received control siRNA recruited the same amount of F-actin to the immune synapse as untreated NK cells (Figures 3C,D). These results further support the observation that NKp46 contributes to cytoskeletal rearrangement at the immune synapse.

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