Signaling by Fyn-ADAP via the Carma1-Bcl-10-MAP3K7 signalosome exclusively regulates inflammatory cytokine production in NK cells - PubMed (original) (raw)

. 2013 Nov;14(11):1127-36.

doi: 10.1038/ni.2708. Epub 2013 Sep 15.

Affiliations

Signaling by Fyn-ADAP via the Carma1-Bcl-10-MAP3K7 signalosome exclusively regulates inflammatory cytokine production in NK cells

Kamalakannan Rajasekaran et al. Nat Immunol. 2013 Nov.

Erratum in

Abstract

Inflammation is a critical component of the immune response. However, acute or chronic inflammation can be highly destructive. Uncontrolled inflammation forms the basis for allergy, asthma and various autoimmune disorders. Here we identified a signaling pathway that was exclusively responsible for the production of inflammatory cytokines but not for cytotoxicity. Recognition of tumor cells expressing the NK cell-activatory ligands H60 or CD137L by mouse natural killer (NK) cells led to efficient cytotoxicity and the production of inflammatory cytokines. Both of those effector functions required the kinases Lck, Fyn and PI(3)K (subunits p85α and p110δ) and the signaling protein PLC-γ2. However, a complex of Fyn and the adaptor ADAP exclusively regulated the production of inflammatory cytokines but not cytotoxicity in NK cells. That unique function of ADAP required a Carma1-Bcl-10-MAP3K7 signaling axis. Our results have identified molecules that can be targeted to regulate inflammation without compromising NK cell cytotoxicity.

PubMed Disclaimer

Conflict of interest statement

COMPETING FINANCIAL INTEREST The authors declare no competing financial interest.

Figures

Figure 1

Figure 1. CD137 functions as an independent activation receptor in NK cells

(a) Flow cytometry analyses of H60 (top) and CD137L (bottom) expression in stably-transfected EL4 cells. Open histogram: background expression of H60 or CD137L in parental EL4 cells. Grey histogram: ligand expression in EL4-H60lo or EL4-CD137Llo. Black histogram: ligand expression in EL4-H60hi or EL4-CD137Lhi. (b) Mean percent cytotoxicity with standard deviation of IL-2-cultured WT NK cells following co-culture with indicated target cells. E/T denotes Effector to Target ratio. (c) Intracellular IFN-γ staining in IL-2-cultured CD3+NK1.1− T and CD3−NK1.1+ NK cells from WT mice either left unstimulated or stimulated with plate-bound anti-CD137, anti-CD3 mAb alone or in combination for 12 h. (d) Quantitative analyses of IFN-γ, GM-CSF, CCL3, CCL4, and CCL5 from WT NK cells following 18 h co-culture with indicated target cells using bioplex assays. *P<0.05, **P<0.01, ***P<0.001; NS, not significant (Student’s t-test; two-tailed, two sample equal variance (b, d). Data in a is a representative of three independent experiments. Data in b and d are from five and three WT mice, respectively (mean ± s.d. in b and d).

Figure 2

Figure 2. Lck is crucial in CD137-mediated signaling

(a) Immunoblot analyses of Lck and Fyn following immunoprecipitation of NKG2D and CD137 receptors in unstimulated, IL-2-expanded WT NK cells. (b) Quantitative analysis of IFN-γ production from NK cells after pre-incubation with Lck inhibitor, C8863. Open squares: C8863-treated NK cells with anti-NKG2D mAb activation; filled circles: C8863-treated NK cells with anti-CD137 mAb activation; open circles: C8863-treated NK cells with IL-12 and IL-18 activation. Cytokine production in the vehicle (DMSO)-treated NK cells in response to anti-NKG2D, anti-CD137 mAbs or IL-12 and IL-18 are indicated as closed or half-filled squares. (c) Immunoblot for Lck in NK cells following mock, Lck-specific and scrambled siRNA transfections. Fold change in Lck expression was determined by densitometry following normalization with actin. (d) Bar diagram represents the mean percent cytotoxicity of NK cells transfected with mock (open), Lck-specific siRNA (black) or scrambled siRNA (grey) against indicated target cells. E/T denotes Effector to Target ratio. (e) Quantitative analyses of cytokine and chemokine production, following activation with anti-NKG2D (left), anti-CD137 (middle) mAbs, and IL-12 and IL-18 (right) in WT NK cells transfected with mock, Lck-specific or scrambled siRNA. *P<0.05, **P<0.01, ***P<0.001; NS, not significant (Student’s t-test; two-tailed, two sample equal variance (b); two-tailed, paired (d, e). Data in a and c are representative of three independent experiments. Data in b, d, and e are mean values from three independent experiments consisting of three mice in each (mean ± s.d. in b, d, e).

Figure 3

Figure 3. Fyn plays a critical role in mediating NK cell effector functions

(a) Immunoblot analyses of phosphorylated (pTyr530) and total Fyn in NK cells that were left unstimulated or stimulated with plate-bound anti-NKG2D or anti-CD137 mAbs. Fold change in tyrosine phosphorylation is shown. (b) Cytotoxicity of WT (open circles) and Fyn−/− (closed circles) NK cells to H60+ and CD137L+ target cells. E/T denotes Effector to Target ratio. (c) Quantitative analyses of cytokines and chemokines following activation with plate-bound anti-NKG2D or anti-CD137 mAbs or with a combination of IL-12 and IL-18 for 18 h. (d) Quantitative analyses of cytokines and chemokines from WT and Fyn−/− NK cells following co-culture with EL4, EL4-H60hi or EL4-CD137Lhi target cells for 18 h. *P<0.05, **P<0.01, ***P<0.001; NS, not significant (Student’s t-test; two-tailed, two sample equal variance (b–d). Data in a is a representative of three independent experiments. Data in b, c and d are from seven, five and two mice per genotype from four, four, and two independent experiments, respectively (mean ± s.d. in b, c and d).

Figure 4

Figure 4. PI(3)K-p85α-p110δ is essential for cytotoxicity and cytokine production in NK cells

(a) Whole cell lysates from WT NK cells, unstimulated or activated with plate-bound anti-NKG2D and anti-CD137 mAbs were immunoprecipitated for Lck or Fyn and probed with anti-PI(3)K-p85α antibody. Fyn and Lck were also analyzed following their respective immunoprecipitation. Fold induction was determined by densitometry following normalization to the respective protein that was immunoprecipitated. (b) Immunoblot analysis of phosphorylated and total PI(3)K-p85α following activation of WT NK cells with plate-bound anti-NKG2D or anti-CD137 mAb. Fold induction was determined by densitometry, following normalization to total PI(3)K-p85α. (c) Mean percent cytotoxicity of NK cells from WT (open) or PI(3)K-p110δ (D910A) (filled) against indicated target cells. E/T denotes Effector to Target ratio. (d) Quantitative analyses of cytokines and chemokines produced by WT (open) or PI(3)K-p110δ (D910A) (filled) NK cells following activation with plate-bound antibodies to NKG2D and CD137 receptors. Cytokines and chemokines produced by NK cells in response to IL-12 and IL-18 stimulation are shown (right). Immunoblot indicating the (e) phosphorylated and total PLC-γ2 or (f) phosphorylated PKC-θ following activation of WT NK cells. Fold induction in (e) and (f) was determined by densitometry, following normalization to total PLC-γ2 and actin, respectively. *P<0.05, **P<0.01, ***P<0.001; NS, not significant (Student’s t-test; two-tailed, two sample equal variance (c, d). Data in c and d are from four and five mice per genotype from three independent experiments, respectively (mean ± s.d. in c and d). Data in a, b, e, and f are representative of three independent experiments

Figure 5

Figure 5. ADAP is essential for cytokine production but not for cytotoxicity in NK cells

(a) Mean percent cytotoxicity with standard deviation of NK cells from WT (open circles) or Adap−/− (filled circles) mice. E/T denotes Effector to Target ratio. (b) Quantitative analyses of cytokines and chemokines produced by NK cells obtained from WT (open) or Adap−/− (filled) mice following activation. Cytokines and chemokines produced by NK cells in response to IL-12 and IL-18 stimulation are also shown. (c) Relative expression of _Ifng-_encoding mRNA compared to Gapdh in WT (Open) and Adap−/− (filled) NK cells. (d) Immunoblot analyses of c-Fos, c-Jun, and NF-κB p65 in the nuclear extracts isolated from WT and Adap−/− NK cells stimulated under indicated conditions. YY1 and a non-specific band obtained in the immunoblot when probed for c-Fos are shown as loading controls. Fold change in the nuclear c-Fos, c-Jun, and NF-κB p65 in the WT and Adap−/− NK cells following activation were calculated against their respective unstimulated controls. *P<0.05, **P<0.01, ***P<0.001; NS, not significant (Student’s t-test; two-tailed, two sample equal variance (b–d). Data in a–c are from four, five and three mice per genotype from three, four, and three independent experiments, respectively (mean ± s.d. in a, b, and c). Data in d is a representative of three independent experiments.

Figure 6

Figure 6. Carma1 is essential for cytotoxicity and cytokine production in NK cells

(a) Cytotoxic potentials of NK cells from WT (open circles), Carma1(ΔCARD) (grey circles), or Carma1−/− (black circles) mice. E/T denotes Effector to Target ratio. (b) Quantitative analyses of cytokines and chemokines produced by NK cells obtained from WT (open), Carma1(ΔCARD) (grey), or Carma1−/− (black) mice following activation. Cytokines and chemokines produced by NK cells in response to IL-12 and IL-18-mediated stimulation are shown. (c) Relative expression of _Ifng-_encoding mRNA compared to Gapdh in WT (Open), Carma1(ΔCARD) (grey), or Carma1−/− (black) NK cells left unstimulated or following activation. (d) Percent IFN-γ+ fresh NK cells (top panel) or IL-2-cultured NK cells (bottom panel) in WT (CD45.1+, open) and Carma1−/− (CD45.2+, filled) mice obtained from the spleen of irradiated and reconstituted Rag2−/−Il2rg−/− mice, one month after adoptive transfer of an equal mixture of WT and Carma1−/− bone marrow cells. *P<0.05, **P<0.01, ***P<0.001; NS, not significant versus WT (Student’s t-test; two-tailed, two sample unequal variance (a, b); two-tailed, two sample equal variance (c, d). Data in a is from five WT, three _Carma1_−/− and five Carma1(ΔCARD) mice and is from two independent experiments (mean ± s.d.). Data in b is from seven WT, three _Carma1_−/− and seven Carma1(ΔCARD) mice and is from three independent experiments (mean ± s.d.). Data in c and d are from four and three mice per genotype from three independent experiments, respectively (mean ± s.d.).

Figure 7

Figure 7. MAP3K7 links Carma1 to the CD137-mediated effector functions in NK cells

(a) Immunoblot showing the MAP3K7 expression in IL-2-cultured NK cells obtained from poly (I:C)-treated floxed Map3k7fx/fx and Mx1-Cre+Map3k7fx/fx mice. Fold change in MAP3K7 expression was determined by densitometry following normalization with actin. (b) Mean percent cytotoxicity with standard deviation of NK cells obtained from poly I:C-treated _Map3k7_fx/fx mice (open circle) or from poly (I:C) untreated (open square) and treated (filled square) Mx1-Cre+Map3k7fx/fx mice against the indicated targets. E/T denotes Effector to Target ratio. (c) Quantitative analyses of cytokines and chemokines produced by NK cells derived from poly (I:C)-treated Map3k7fx/fx (open) and Mx1-Cre+Map3k7fx/fx (filled) mice following 18 h of activation with plate-bound anti-CD137 mAb (left) or with IL-12 and IL-18 (right). *P<0.05, **P<0.01, ***P<0.001; NS, not significant versus WT (Student’s t-test; two-tailed, two sample unequal variance versus NK cells from Mx1-Cre+Map3k7fx/fx mice that were untreated with poly (I:C) (b); two-tailed, two sample equal variance versus NK cells from Map3k7fx/fx mice that were treated with poly (I:C) (c). Data in a is a representative of three independent experiments. Data in b, c are from three mice per genotype from three independent experiments (mean ± s.d.).

Figure 8

Figure 8. ADAP is essential for cytokine production but not for cytotoxicity in human NK cells

(a) Flow cytometry indicating the percentage CD3−CD56+ NK cells in total human PBMC (top) and in the cell fraction (bottom) obtained following negative selection for CD56+ NK cells. Purified CD56+ human NK cells were transfected with scrambled or ADAP-specific siRNA. (b) Immunoblot for ADAP in human NK cells that were transfected with scrambled or ADAP-specific siRNA. Fold change in ADAP expression was determined by densitometry following normalization with actin. (c) Dot plot represents the mean and percent cytotoxicity against K562 (left panels) and percent CD56+CD107a+ NK cells following plate-bound anti-NKG2D-mediated activation (right) of scrambled or ADAP-specific siRNA-transfected human NK cells. E/T denotes Effector to Target ratio. (d) Quantitative analyses of cytokines and chemokines produced by scrambled or ADAP-specific siRNA-transfected human NK cells following anti-NKG2D mAb-mediated activation. *P<0.05; NS, not significant versus NK cells treated with scrambled siRNA (Student’s t-test; two-tailed, paired (c, d). Data in a and b are representative of three independent experiments. Data in c and d are from eight and seven different human NK cells from five independent experiments. Horizontal bar in c and d represent the mean of the representative data set.

Comment in

Similar articles

Cited by

References

    1. Billadeau DD, Upshaw JL, Schoon RA, Dick CJ, Leibson PJ. NKG2D-DAP10 triggers human NK cell-mediated killing via a Syk-independent regulatory pathway. Nat Immunol. 2003;4:557–564. - PubMed
    1. Wu J, Cherwinski H, Spies T, Phillips JH, Lanier LL. DAP10 and DAP12 form distinct, but functionally cooperative, receptor complexes in natural killer cells. J Exp Med. 2000;192:1059–1068. - PMC - PubMed
    1. Zompi S, et al. NKG2D triggers cytotoxicity in mouse NK cells lacking DAP12 or Syk family kinases. Nat Immunol. 2003;4:565–572. - PubMed
    1. Myers LM, Vella AT. Interfacing T-cell effector and regulatory function through CD137 (4-1BB) co-stimulation. Trends Immunol. 2005;26:440–446. - PubMed
    1. Wilcox RA, Tamada K, Strome SE, Chen L. Signaling through NK cell-associated CD137 promotes both helper function for CD8+ cytolytic T cells and responsiveness to IL-2 but not cytolytic activity. J Immunol. 2002;169:4230–4236. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources