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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 expression correlates with reduced lytic granule polarization time. (A) Representative snapshots from live cell time-lapse imaging (Video S1 in Supplementary Material). NK92 cells expressing NKp46-IRES-GFP (green) were pre-loaded with LysoTracker DND-99 (gray) and co-incubated with DiD-labeled (red) 721.221 or HepG2 cells. Left panel shows time of initial effector–target interaction and right panel shows time of lytic granules polarization. (B) Summarized results for multiple individual effector-target show lytic granules polarization time per NK cell GFP MFI. Dashed lines show trends adapted by linear correlation test. Scale bar = 10 μm.
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Figure 4: NKp46 expression correlates with reduced lytic granule polarization time. (A) Representative snapshots from live cell time-lapse imaging (Video S1 in Supplementary Material). NK92 cells expressing NKp46-IRES-GFP (green) were pre-loaded with LysoTracker DND-99 (gray) and co-incubated with DiD-labeled (red) 721.221 or HepG2 cells. Left panel shows time of initial effector–target interaction and right panel shows time of lytic granules polarization. (B) Summarized results for multiple individual effector-target show lytic granules polarization time per NK cell GFP MFI. Dashed lines show trends adapted by linear correlation test. Scale bar = 10 μm.

Mentions: Distinct from cytotoxic T cells, NK cell induced cytolytic killing is largely dependent on microtubule-organizing center (MTOC) followed by lytic granule polarization to the immune synapse (47). To explore the effect of NKp46 surface expression on lytic granules polarization at the single cell level, we used NK92 cells expressing an NKp46-IRES-GFP construct in which GFP is a reporter for NKp46 expression level. We validated that GFP intensity correlates with surface expression of NKp46 (Figure S4 in Supplementary Material). Using NK cells pre-loaded with LysoTracker, we measured lytic granule polarization to the immune synapse with respect to time in NK – target cell conjugates using time-lapse live cell imaging (Figure 4A and Video S1 in Supplementary Material). Data collected from multiple interactions (n > 15) between NK92.p46-IRES-GFP cells and 721.221 target cells, which express low levels of NKp46 ligand (Figure S5C in Supplementary Material), showed no correlation between NKp46 receptor expression and time to polarization of the MTOC (R2 = 0.05, slope = −0.002). In contrast, when we analyzed interactions with HepG2 cells, which express high levels of NKp46 ligand (Figure S5A in Supplementary Material), we found that increased NKp46 receptor expression correlated with a linear reduction in the granule polarization time (R2 = 0.93, slope = −0.08) (Figure 4B). These results show that NKp46 plays a crucial role in the rapid recruitment of lytic granules to the NK-target cell interface.


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 expression correlates with reduced lytic granule polarization time. (A) Representative snapshots from live cell time-lapse imaging (Video S1 in Supplementary Material). NK92 cells expressing NKp46-IRES-GFP (green) were pre-loaded with LysoTracker DND-99 (gray) and co-incubated with DiD-labeled (red) 721.221 or HepG2 cells. Left panel shows time of initial effector–target interaction and right panel shows time of lytic granules polarization. (B) Summarized results for multiple individual effector-target show lytic granules polarization time per NK cell GFP MFI. Dashed lines show trends adapted by linear correlation test. Scale bar = 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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Figure 4: NKp46 expression correlates with reduced lytic granule polarization time. (A) Representative snapshots from live cell time-lapse imaging (Video S1 in Supplementary Material). NK92 cells expressing NKp46-IRES-GFP (green) were pre-loaded with LysoTracker DND-99 (gray) and co-incubated with DiD-labeled (red) 721.221 or HepG2 cells. Left panel shows time of initial effector–target interaction and right panel shows time of lytic granules polarization. (B) Summarized results for multiple individual effector-target show lytic granules polarization time per NK cell GFP MFI. Dashed lines show trends adapted by linear correlation test. Scale bar = 10 μm.
Mentions: Distinct from cytotoxic T cells, NK cell induced cytolytic killing is largely dependent on microtubule-organizing center (MTOC) followed by lytic granule polarization to the immune synapse (47). To explore the effect of NKp46 surface expression on lytic granules polarization at the single cell level, we used NK92 cells expressing an NKp46-IRES-GFP construct in which GFP is a reporter for NKp46 expression level. We validated that GFP intensity correlates with surface expression of NKp46 (Figure S4 in Supplementary Material). Using NK cells pre-loaded with LysoTracker, we measured lytic granule polarization to the immune synapse with respect to time in NK – target cell conjugates using time-lapse live cell imaging (Figure 4A and Video S1 in Supplementary Material). Data collected from multiple interactions (n > 15) between NK92.p46-IRES-GFP cells and 721.221 target cells, which express low levels of NKp46 ligand (Figure S5C in Supplementary Material), showed no correlation between NKp46 receptor expression and time to polarization of the MTOC (R2 = 0.05, slope = −0.002). In contrast, when we analyzed interactions with HepG2 cells, which express high levels of NKp46 ligand (Figure S5A in Supplementary Material), we found that increased NKp46 receptor expression correlated with a linear reduction in the granule polarization time (R2 = 0.93, slope = −0.08) (Figure 4B). These results show that NKp46 plays a crucial role in the rapid recruitment of lytic granules to the NK-target cell interface.

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