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Functional dichotomy in natural killer cell signaling: Vav1-dependent and -independent mechanisms.

Colucci F, Rosmaraki E, Bregenholt S, Samson SI, Di Bartolo V, Turner M, Vanes L, Tybulewicz V, Di Santo JP - J. Exp. Med. (2001)

Bottom Line: In vitro stimulation of surface receptors in Vav1-/- NK cells resulted in normal IFN-gamma production but reduced tumor cell lysis.Vav1 was found to control activation of extracellular signal-regulated kinases and exocytosis of cytotoxic granules.These results highlight fundamental differences between proximal signaling events in T and NK cells and suggest a functional dichotomy for Vav1 in NK cells: a role in cytotoxicity but not for IFN-gamma production.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Cytokines and Lymphoid Development, Pasteur Institute, 75015 Paris, France. cecco@pasteur.fr

ABSTRACT
The product of the protooncogene Vav1 participates in multiple signaling pathways and is a critical regulator of antigen-receptor signaling in B and T lymphocytes, but its role during in vivo natural killer (NK) cell differentiation is not known. Here we have studied NK cell development in Vav1-/- mice and found that, in contrast to T and NK-T cells, the absolute numbers of phenotypically mature NK cells were not reduced. Vav1-/- mice produced normal amounts of interferon (IFN)-gamma in response to Listeria monocytogenes and controlled early infection but showed reduced tumor clearance in vivo. In vitro stimulation of surface receptors in Vav1-/- NK cells resulted in normal IFN-gamma production but reduced tumor cell lysis. Vav1 was found to control activation of extracellular signal-regulated kinases and exocytosis of cytotoxic granules. In contrast, conjugate formation appeared to be only mildly affected, and calcium mobilization was normal in Vav1-/- NK cells. These results highlight fundamental differences between proximal signaling events in T and NK cells and suggest a functional dichotomy for Vav1 in NK cells: a role in cytotoxicity but not for IFN-gamma production.

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Vav1 controls ERK activation and granule exocytosis. (A) Purified NK and T cells were incubated with the indicated mAb and stimulated with cross-linking Abs. Cell lysates were immunoblotted with an mAb specific for the phosphorylated forms of ERK1/2. Total amounts of inactive ERK1/2 were assessed by polyclonal anti-ERK1/2. Stimulation with PMA generated comparable levels of phosphorylated ERK1/2 in wt and Vav1−/− cells. (B) Equal numbers of NK cells were stimulated with YAC-1 targets for 2 h and 30 min at 37°C. Supernatants were collected, and the specific release of esterase (granzyme A) was measured by using a BLT colorimetric assay, where the maximum release of esterase was obtained by freezing and thawing NK cells. Mean values ± SDs are shown from five independent experiments.
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Figure 7: Vav1 controls ERK activation and granule exocytosis. (A) Purified NK and T cells were incubated with the indicated mAb and stimulated with cross-linking Abs. Cell lysates were immunoblotted with an mAb specific for the phosphorylated forms of ERK1/2. Total amounts of inactive ERK1/2 were assessed by polyclonal anti-ERK1/2. Stimulation with PMA generated comparable levels of phosphorylated ERK1/2 in wt and Vav1−/− cells. (B) Equal numbers of NK cells were stimulated with YAC-1 targets for 2 h and 30 min at 37°C. Supernatants were collected, and the specific release of esterase (granzyme A) was measured by using a BLT colorimetric assay, where the maximum release of esterase was obtained by freezing and thawing NK cells. Mean values ± SDs are shown from five independent experiments.

Mentions: Vav1 appears essential in transducing TCR signals to the ERK pathway in T cells 26, although studies using T cells from two other Vav1 mutant mice did not support an essential role for Vav1 in ERK activation 1516. ERKs have been implicated in the control of both cytotoxicity and IFN-γ production by NK cells 343536. We therefore analyzed ERK1/2 phosphorylation in NK cells after stimulation of NK1.1 and compared it to CD3-initiated ERK1/2 phosphorylation in T cells. We found a reduced activation of ERK1 (eightfold) and ERK2 (2.5-fold) in Vav1−/− T cells (Fig. 7 A), confirming the essential role of Vav1 in TCR-mediated ERK activation 26. NK1.1-mediated ERK1 phosphorylation was 4.4-fold reduced in Vav1−/− NK cells, while no significant reduction in activation of ERK2 was detected (Fig. 7 A). However, stimulation of Vav1−/− NK cells via Ly49D resulted in reduced activation of both ERK1 (threefold) and ERK2 (twofold) kinases (data not shown). Collectively, these results suggest that Vav1 controls the ERK pathway in NK cells.


Functional dichotomy in natural killer cell signaling: Vav1-dependent and -independent mechanisms.

Colucci F, Rosmaraki E, Bregenholt S, Samson SI, Di Bartolo V, Turner M, Vanes L, Tybulewicz V, Di Santo JP - J. Exp. Med. (2001)

Vav1 controls ERK activation and granule exocytosis. (A) Purified NK and T cells were incubated with the indicated mAb and stimulated with cross-linking Abs. Cell lysates were immunoblotted with an mAb specific for the phosphorylated forms of ERK1/2. Total amounts of inactive ERK1/2 were assessed by polyclonal anti-ERK1/2. Stimulation with PMA generated comparable levels of phosphorylated ERK1/2 in wt and Vav1−/− cells. (B) Equal numbers of NK cells were stimulated with YAC-1 targets for 2 h and 30 min at 37°C. Supernatants were collected, and the specific release of esterase (granzyme A) was measured by using a BLT colorimetric assay, where the maximum release of esterase was obtained by freezing and thawing NK cells. Mean values ± SDs are shown from five independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2193296&req=5

Figure 7: Vav1 controls ERK activation and granule exocytosis. (A) Purified NK and T cells were incubated with the indicated mAb and stimulated with cross-linking Abs. Cell lysates were immunoblotted with an mAb specific for the phosphorylated forms of ERK1/2. Total amounts of inactive ERK1/2 were assessed by polyclonal anti-ERK1/2. Stimulation with PMA generated comparable levels of phosphorylated ERK1/2 in wt and Vav1−/− cells. (B) Equal numbers of NK cells were stimulated with YAC-1 targets for 2 h and 30 min at 37°C. Supernatants were collected, and the specific release of esterase (granzyme A) was measured by using a BLT colorimetric assay, where the maximum release of esterase was obtained by freezing and thawing NK cells. Mean values ± SDs are shown from five independent experiments.
Mentions: Vav1 appears essential in transducing TCR signals to the ERK pathway in T cells 26, although studies using T cells from two other Vav1 mutant mice did not support an essential role for Vav1 in ERK activation 1516. ERKs have been implicated in the control of both cytotoxicity and IFN-γ production by NK cells 343536. We therefore analyzed ERK1/2 phosphorylation in NK cells after stimulation of NK1.1 and compared it to CD3-initiated ERK1/2 phosphorylation in T cells. We found a reduced activation of ERK1 (eightfold) and ERK2 (2.5-fold) in Vav1−/− T cells (Fig. 7 A), confirming the essential role of Vav1 in TCR-mediated ERK activation 26. NK1.1-mediated ERK1 phosphorylation was 4.4-fold reduced in Vav1−/− NK cells, while no significant reduction in activation of ERK2 was detected (Fig. 7 A). However, stimulation of Vav1−/− NK cells via Ly49D resulted in reduced activation of both ERK1 (threefold) and ERK2 (twofold) kinases (data not shown). Collectively, these results suggest that Vav1 controls the ERK pathway in NK cells.

Bottom Line: In vitro stimulation of surface receptors in Vav1-/- NK cells resulted in normal IFN-gamma production but reduced tumor cell lysis.Vav1 was found to control activation of extracellular signal-regulated kinases and exocytosis of cytotoxic granules.These results highlight fundamental differences between proximal signaling events in T and NK cells and suggest a functional dichotomy for Vav1 in NK cells: a role in cytotoxicity but not for IFN-gamma production.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Cytokines and Lymphoid Development, Pasteur Institute, 75015 Paris, France. cecco@pasteur.fr

ABSTRACT
The product of the protooncogene Vav1 participates in multiple signaling pathways and is a critical regulator of antigen-receptor signaling in B and T lymphocytes, but its role during in vivo natural killer (NK) cell differentiation is not known. Here we have studied NK cell development in Vav1-/- mice and found that, in contrast to T and NK-T cells, the absolute numbers of phenotypically mature NK cells were not reduced. Vav1-/- mice produced normal amounts of interferon (IFN)-gamma in response to Listeria monocytogenes and controlled early infection but showed reduced tumor clearance in vivo. In vitro stimulation of surface receptors in Vav1-/- NK cells resulted in normal IFN-gamma production but reduced tumor cell lysis. Vav1 was found to control activation of extracellular signal-regulated kinases and exocytosis of cytotoxic granules. In contrast, conjugate formation appeared to be only mildly affected, and calcium mobilization was normal in Vav1-/- NK cells. These results highlight fundamental differences between proximal signaling events in T and NK cells and suggest a functional dichotomy for Vav1 in NK cells: a role in cytotoxicity but not for IFN-gamma production.

Show MeSH
Related in: MedlinePlus