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The p110 delta of PI3K plays a critical role in NK cell terminal maturation and cytokine/chemokine generation.

Guo H, Samarakoon A, Vanhaesebroeck B, Malarkannan S - J. Exp. Med. (2008)

Bottom Line: Further, p110 delta(D910A/D910A) NK cell-mediated antiviral responses through natural cytotoxicity receptor 1 were reduced.Analysis of signaling events demonstrates that p110 delta(D910A/D910A) NK cells had a reduced c-Jun N-terminal kinase 1/2 phosphorylation in response to NKG2D-mediated activation.These results reveal a previously unrecognized role of PI3K-p110 delta in NK cell development and effector functions.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Immunology, Blood Research Institute, Medical College of Wisconsin, Milwaukee, WI 53226, USA.

ABSTRACT
Phosphatidylinositol 3-kinases (PI3Ks) play a critical role in regulating B cell receptor- and T cell receptor-mediated signaling. However, their role in natural killer (NK) cell development and functions is not well understood. Using mice expressing p110 delta(D910A), a catalytically inactive p110 delta, we show that these mice had reduced NK cellularity, defective Ly49C and Ly49I NK subset maturation, and decreased CD27(High) NK numbers. p110 delta inactivation marginally impaired NK-mediated cytotoxicity against tumor cells in vitro and in vivo. However, NKG2D, Ly49D, and NK1.1 receptor-mediated cytokine and chemokine generation by NK cells was severely affected in these mice. Further, p110 delta(D910A/D910A) NK cell-mediated antiviral responses through natural cytotoxicity receptor 1 were reduced. Analysis of signaling events demonstrates that p110 delta(D910A/D910A) NK cells had a reduced c-Jun N-terminal kinase 1/2 phosphorylation in response to NKG2D-mediated activation. These results reveal a previously unrecognized role of PI3K-p110 delta in NK cell development and effector functions.

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Defect in cytokine production occurs at the transcriptional level, and IL-12/IL-18–mediated generation of cytokine/chemokine is intact in p110δD910A/D910A NK cells. (a) IFN-γ–encoding mRNA levels were reduced in p110δD910A/D910A NK cells. NK cells were stimulated with anti-NKG2D mAb for 6 h, and mRNA was extracted and IFN-γ–encoding transcripts were quantified. (b) NK cell cytokine and chemokine production upon stimulation with a suboptimal concentration of anti-NKG2D mAbs in the presence or absence of IL-12, IL-18, or both. Data presented are means ± SD and are representative of three independent experiments.
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fig8: Defect in cytokine production occurs at the transcriptional level, and IL-12/IL-18–mediated generation of cytokine/chemokine is intact in p110δD910A/D910A NK cells. (a) IFN-γ–encoding mRNA levels were reduced in p110δD910A/D910A NK cells. NK cells were stimulated with anti-NKG2D mAb for 6 h, and mRNA was extracted and IFN-γ–encoding transcripts were quantified. (b) NK cell cytokine and chemokine production upon stimulation with a suboptimal concentration of anti-NKG2D mAbs in the presence or absence of IL-12, IL-18, or both. Data presented are means ± SD and are representative of three independent experiments.

Mentions: NK cells can generate inflammatory cytokines and chemokines such as IFN-γ, GM-CSF, macrophage inflammatory protein (MIP) 1α, MIP-1β, and regulated upon activation, normal T cell expressed and secreted (RANTES) (46). Therefore, we tested CD3−NK1.1+ NK cells for cytokine generation. Freshly purified CD3−NK1.1+ NK cells from WT/WT spleens generated an optimal amount of cytokines and chemokines when activated by plate-bound anti-NKG2D, anti-Ly49D, and anti-NK1.1 mAbs. However, NK cells from p110δD910A/D910A mice were significantly impaired in generating these cytokines and chemokines (Fig. 6 a). Thus, the ex vivo–purified NK cells that use NKG2D–DAP10 complexes for signaling depend on p110δ to generate cytokine response. Next, we tested the role of p110δ in cytokine and chemokine generation by IL-2–activated splenic NK cells. As seen in Fig. 6 b, WT/WT NK cells produced a large amount of IFN-γ or GM-CSF when stimulated with anti-NKG2D, anti-Ly49D, and anti-NK1.1 mAbs. However, p110δD910A/D910A NK cells were severely impaired to produce these cytokines. Further, generation of the chemokines MIP1-α, MIP1-β, and RANTES from p110δD910A/D910A NK cells was also significantly reduced (Fig. 6 b). Similar reductions in the cytokines and chemokines were observed in the BM-derived p110δD910A/D910A NK cells (Fig. S7, available at http://www.jem.org/cgi/content/full/jem.20072327/DC1). This substantial reduction could be caused by the inability of the p110δD910A/D910A NK cells to produce cytokines or simply by a defect in cytokine secretion. To distinguish between these two possibilities, IFN-γ production in response to anti-NKG2D mAb was measured by intracellular staining. The percentage of IFN-γ–positive cells among gated NK1.1+ populations in p110δD910A/D910A NK cells was significantly lower compared with WT/WT NK cells (P = 0.017; Fig. 7 a) Inactivation of p110δ reduced the number and functions of NK cells, particularly, the Ly49C/I subsets. Therefore, to determine the cytokine defects in individual Ly49 subsets, we analyzed the levels of intracellular IFN-γ in each one of them. All of the subsets from p110δD910A/D910A mice generated a significantly less amount of intracellular IFN-γ (P < 0.007; Fig. 7 b), and we did not see any preferential reduction in Ly49C/I subsets. This indicates that although p110δ regulates both of the effector functions, it is critically required for cytokine generation. To further investigate whether the defect is at the transcriptional level, we quantified the amounts of IFN-γ–encoding mRNA before and after plate-bound anti-NKG2D mAb activation. Fig. 8 a demonstrates a significantly lower copy number of IFN-γ–encoding mRNA in both BM- and spleen-derived p110δD910A/D910A NK cells, indicating a defect at the transcriptional level. To determine whether this is generalized hyporesponsiveness or an exclusive defect associated with YINM- and ITAM-containing activation receptors, we stimulated the NK cells with IL-12, IL-18, or both in the presence of a suboptimal concentration of anti-NKG2D mAb (A10). Interestingly, both WT/WT and p110δD910A/D910A-derived NK cells responded equally well to distinct combinations of this activation (Fig. 8 b). Similar results were obtained using IL-12, IL-18, or both without anti-NKG2D mAb (unpublished data). This demonstrates that p110δD910A/D910A NK cells are fully capable of responding through their cytokine receptors.


The p110 delta of PI3K plays a critical role in NK cell terminal maturation and cytokine/chemokine generation.

Guo H, Samarakoon A, Vanhaesebroeck B, Malarkannan S - J. Exp. Med. (2008)

Defect in cytokine production occurs at the transcriptional level, and IL-12/IL-18–mediated generation of cytokine/chemokine is intact in p110δD910A/D910A NK cells. (a) IFN-γ–encoding mRNA levels were reduced in p110δD910A/D910A NK cells. NK cells were stimulated with anti-NKG2D mAb for 6 h, and mRNA was extracted and IFN-γ–encoding transcripts were quantified. (b) NK cell cytokine and chemokine production upon stimulation with a suboptimal concentration of anti-NKG2D mAbs in the presence or absence of IL-12, IL-18, or both. Data presented are means ± SD and are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2556795&req=5

fig8: Defect in cytokine production occurs at the transcriptional level, and IL-12/IL-18–mediated generation of cytokine/chemokine is intact in p110δD910A/D910A NK cells. (a) IFN-γ–encoding mRNA levels were reduced in p110δD910A/D910A NK cells. NK cells were stimulated with anti-NKG2D mAb for 6 h, and mRNA was extracted and IFN-γ–encoding transcripts were quantified. (b) NK cell cytokine and chemokine production upon stimulation with a suboptimal concentration of anti-NKG2D mAbs in the presence or absence of IL-12, IL-18, or both. Data presented are means ± SD and are representative of three independent experiments.
Mentions: NK cells can generate inflammatory cytokines and chemokines such as IFN-γ, GM-CSF, macrophage inflammatory protein (MIP) 1α, MIP-1β, and regulated upon activation, normal T cell expressed and secreted (RANTES) (46). Therefore, we tested CD3−NK1.1+ NK cells for cytokine generation. Freshly purified CD3−NK1.1+ NK cells from WT/WT spleens generated an optimal amount of cytokines and chemokines when activated by plate-bound anti-NKG2D, anti-Ly49D, and anti-NK1.1 mAbs. However, NK cells from p110δD910A/D910A mice were significantly impaired in generating these cytokines and chemokines (Fig. 6 a). Thus, the ex vivo–purified NK cells that use NKG2D–DAP10 complexes for signaling depend on p110δ to generate cytokine response. Next, we tested the role of p110δ in cytokine and chemokine generation by IL-2–activated splenic NK cells. As seen in Fig. 6 b, WT/WT NK cells produced a large amount of IFN-γ or GM-CSF when stimulated with anti-NKG2D, anti-Ly49D, and anti-NK1.1 mAbs. However, p110δD910A/D910A NK cells were severely impaired to produce these cytokines. Further, generation of the chemokines MIP1-α, MIP1-β, and RANTES from p110δD910A/D910A NK cells was also significantly reduced (Fig. 6 b). Similar reductions in the cytokines and chemokines were observed in the BM-derived p110δD910A/D910A NK cells (Fig. S7, available at http://www.jem.org/cgi/content/full/jem.20072327/DC1). This substantial reduction could be caused by the inability of the p110δD910A/D910A NK cells to produce cytokines or simply by a defect in cytokine secretion. To distinguish between these two possibilities, IFN-γ production in response to anti-NKG2D mAb was measured by intracellular staining. The percentage of IFN-γ–positive cells among gated NK1.1+ populations in p110δD910A/D910A NK cells was significantly lower compared with WT/WT NK cells (P = 0.017; Fig. 7 a) Inactivation of p110δ reduced the number and functions of NK cells, particularly, the Ly49C/I subsets. Therefore, to determine the cytokine defects in individual Ly49 subsets, we analyzed the levels of intracellular IFN-γ in each one of them. All of the subsets from p110δD910A/D910A mice generated a significantly less amount of intracellular IFN-γ (P < 0.007; Fig. 7 b), and we did not see any preferential reduction in Ly49C/I subsets. This indicates that although p110δ regulates both of the effector functions, it is critically required for cytokine generation. To further investigate whether the defect is at the transcriptional level, we quantified the amounts of IFN-γ–encoding mRNA before and after plate-bound anti-NKG2D mAb activation. Fig. 8 a demonstrates a significantly lower copy number of IFN-γ–encoding mRNA in both BM- and spleen-derived p110δD910A/D910A NK cells, indicating a defect at the transcriptional level. To determine whether this is generalized hyporesponsiveness or an exclusive defect associated with YINM- and ITAM-containing activation receptors, we stimulated the NK cells with IL-12, IL-18, or both in the presence of a suboptimal concentration of anti-NKG2D mAb (A10). Interestingly, both WT/WT and p110δD910A/D910A-derived NK cells responded equally well to distinct combinations of this activation (Fig. 8 b). Similar results were obtained using IL-12, IL-18, or both without anti-NKG2D mAb (unpublished data). This demonstrates that p110δD910A/D910A NK cells are fully capable of responding through their cytokine receptors.

Bottom Line: Further, p110 delta(D910A/D910A) NK cell-mediated antiviral responses through natural cytotoxicity receptor 1 were reduced.Analysis of signaling events demonstrates that p110 delta(D910A/D910A) NK cells had a reduced c-Jun N-terminal kinase 1/2 phosphorylation in response to NKG2D-mediated activation.These results reveal a previously unrecognized role of PI3K-p110 delta in NK cell development and effector functions.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Immunology, Blood Research Institute, Medical College of Wisconsin, Milwaukee, WI 53226, USA.

ABSTRACT
Phosphatidylinositol 3-kinases (PI3Ks) play a critical role in regulating B cell receptor- and T cell receptor-mediated signaling. However, their role in natural killer (NK) cell development and functions is not well understood. Using mice expressing p110 delta(D910A), a catalytically inactive p110 delta, we show that these mice had reduced NK cellularity, defective Ly49C and Ly49I NK subset maturation, and decreased CD27(High) NK numbers. p110 delta inactivation marginally impaired NK-mediated cytotoxicity against tumor cells in vitro and in vivo. However, NKG2D, Ly49D, and NK1.1 receptor-mediated cytokine and chemokine generation by NK cells was severely affected in these mice. Further, p110 delta(D910A/D910A) NK cell-mediated antiviral responses through natural cytotoxicity receptor 1 were reduced. Analysis of signaling events demonstrates that p110 delta(D910A/D910A) NK cells had a reduced c-Jun N-terminal kinase 1/2 phosphorylation in response to NKG2D-mediated activation. These results reveal a previously unrecognized role of PI3K-p110 delta in NK cell development and effector functions.

Show MeSH
Related in: MedlinePlus