Limits...
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 over-expression increases F-actin rearrangement at the immune synapse. (A) Representative micrograph of NK92 cell co-incubated with CFSE-labeled (green) target cell, fixed and permeabilized, and stained for F-actin using Rhodamine Phalloidin (red) and DAPI (blue) for nucleus labeling. Scale bar = 10 μm. (B) Flow cytometry analysis of NKp46 expression NK92 (dashed line), NK92.p46 (solid line) cells, and isotype antibody staining (gray-filled graph). (C) Calculated ratio of synapse specific F-actin (Figure S2 in Supplementary Material) is presented for multiple effector-target conjugations from three independent experiments (x, n = 32) as log values and is representative of three repeated experiments. Graph bars show mean ± SEM. *p < 0.05, Student’s t-test.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4585260&req=5

Figure 2: NKp46 over-expression increases F-actin rearrangement at the immune synapse. (A) Representative micrograph of NK92 cell co-incubated with CFSE-labeled (green) target cell, fixed and permeabilized, and stained for F-actin using Rhodamine Phalloidin (red) and DAPI (blue) for nucleus labeling. Scale bar = 10 μm. (B) Flow cytometry analysis of NKp46 expression NK92 (dashed line), NK92.p46 (solid line) cells, and isotype antibody staining (gray-filled graph). (C) Calculated ratio of synapse specific F-actin (Figure S2 in Supplementary Material) is presented for multiple effector-target conjugations from three independent experiments (x, n = 32) as log values and is representative of three repeated experiments. Graph bars show mean ± SEM. *p < 0.05, Student’s t-test.

Mentions: Cytoskeletal rearrangement that leads to formation of a dense F-actin mesh at the immune synapse is a central step in NK cell-mediated cytotoxicity (42). Over-expression of NKp46 increases NK cytotoxicity against HeLa cells (Figure S1 in Supplementary Material). To test the specific effect of NKp46 signaling on F-actin recruitment to the immune synapse, we developed a quantitative conjugation assay that allowed us to measure F-actin accumulation at the immune synapse (Figure 2A; Figure S2 in Supplementary Material). Staining of NK-target conjugations with Phalloidin show that NKp46 over-expression (NK92.p46; blue) in an NK cell line increased the amount of F-actin at the immune synapse by 30% as compared to staining of low NKp46 expressing cells (NK92; red), suggesting that cytoskeletal rearrangement is influenced by NKp46 signaling (Figures 2B,C). Importantly, there were no significant differences in the expression of other NK cell-activating receptors between NK92 and NK92.p46 cells (Figure S3 in Supplementary Material).


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 over-expression increases F-actin rearrangement at the immune synapse. (A) Representative micrograph of NK92 cell co-incubated with CFSE-labeled (green) target cell, fixed and permeabilized, and stained for F-actin using Rhodamine Phalloidin (red) and DAPI (blue) for nucleus labeling. Scale bar = 10 μm. (B) Flow cytometry analysis of NKp46 expression NK92 (dashed line), NK92.p46 (solid line) cells, and isotype antibody staining (gray-filled graph). (C) Calculated ratio of synapse specific F-actin (Figure S2 in Supplementary Material) is presented for multiple effector-target conjugations from three independent experiments (x, n = 32) as log values and is representative of three repeated experiments. Graph bars show mean ± SEM. *p < 0.05, Student’s t-test.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: NKp46 over-expression increases F-actin rearrangement at the immune synapse. (A) Representative micrograph of NK92 cell co-incubated with CFSE-labeled (green) target cell, fixed and permeabilized, and stained for F-actin using Rhodamine Phalloidin (red) and DAPI (blue) for nucleus labeling. Scale bar = 10 μm. (B) Flow cytometry analysis of NKp46 expression NK92 (dashed line), NK92.p46 (solid line) cells, and isotype antibody staining (gray-filled graph). (C) Calculated ratio of synapse specific F-actin (Figure S2 in Supplementary Material) is presented for multiple effector-target conjugations from three independent experiments (x, n = 32) as log values and is representative of three repeated experiments. Graph bars show mean ± SEM. *p < 0.05, Student’s t-test.
Mentions: Cytoskeletal rearrangement that leads to formation of a dense F-actin mesh at the immune synapse is a central step in NK cell-mediated cytotoxicity (42). Over-expression of NKp46 increases NK cytotoxicity against HeLa cells (Figure S1 in Supplementary Material). To test the specific effect of NKp46 signaling on F-actin recruitment to the immune synapse, we developed a quantitative conjugation assay that allowed us to measure F-actin accumulation at the immune synapse (Figure 2A; Figure S2 in Supplementary Material). Staining of NK-target conjugations with Phalloidin show that NKp46 over-expression (NK92.p46; blue) in an NK cell line increased the amount of F-actin at the immune synapse by 30% as compared to staining of low NKp46 expressing cells (NK92; red), suggesting that cytoskeletal rearrangement is influenced by NKp46 signaling (Figures 2B,C). Importantly, there were no significant differences in the expression of other NK cell-activating receptors between NK92 and NK92.p46 cells (Figure S3 in Supplementary Material).

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