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Multiple ITAM-coupled NK-cell receptors engage the Bcl10/Malt1 complex via Carma1 for NF-κB and MAPK activation to selectively control cytokine production

Olaf Gross, Christina Grupp, Christian Steinberg, Stephanie Zimmermann, Dominikus Strasser, Nicole Hannesschläger, Wolfgang Reindl, Helena Jonsson, Hairong Huo, Dan R. Littman, Christian Peschel, Wayne M. Yokoyama, Anne Krug and Jürgen Ruland

Article Figures & Data

Figures

  • Figure 1

    Bcl10 deficiency does not interfere with NK cell development. Splenocytes (A), bone marrow cells (B), and liver lymphocytes (C) from Bcl10-deficient and wild-type control animals were stained for expression of CD49b (DX5), NK1.1, and Ly49D NK-cell surface markers. The percentages of NK cells with respect to total cell numbers are indicated. Data are representative of 4 mice per group.

  • Figure 2

    Impaired cytokine production in NK cells from Bcl10- and Malt1-deficient NK cells upon NK-cell receptor stimulation. (A) Total splenocytes from control or Bcl10-deficient mice were stimulated with plate-bound anti-NK1.1, anti-Ly49D, or anti-NKG2D, stained for CD49b (DX5) and CD3 and intracellular cytokine production, and analyzed by flow cytometry. Cells were gated for DX5+CD3 cells. The percentages of IFN-γ– or TNF-α–positive cells with respect to total NK cells are indicated. (B) IL-15–expanded splenic NK cells from Bcl10- and Malt1-deficient mice were stimulated with plate-bound antibodies directed against various activating NK- cell receptors or PMA and ionomycin (PMA/Iono) as indicated for 24 hours. (C) NK cells from 2 wild-type and 2 Bcl10-deficient mice were prepared as in panel B and stimulated with 10 ng/mL IL-12 and/or 25 ng/mL IL-18 for 24 hours. Cytokine release was measured by ELISA. Data are means plus or minus SD of triplicate samples and representative of 3 independent experiments.

  • Figure 3

    Bcl10 is dispensable for NK-cell receptor–induced killing. NK cells were isolated from the spleens of WT and Bcl10−/− mice 36 hours after infection with 5 × 105 pfu MCMV. NK-cell cytotoxicity was assessed in a 5-hour 51Cr release assay using YAC-1 (A) and CHO (B) cells as target cells at the indicated effector to target ratios. Data are representative of 3 independent experiments with a total of 6 mice per group. (C) IL-15–expanded NK cells were stimulated with YAC-1 or CHO cells at a 1:1 ratio and IFN-γ production was quantified by ELISA. Data are representative of 2 independent experiments.

  • Figure 4

    ITAM-coupled NK-cell receptors activate NF-κB, JNK, and p38 via Bcl10 and Malt1. IL-15–expanded NK cells were stimulated with plate-bound anti-NK1.1 and anti-Ly49D antibodies or PMA/Iono for 20 and 60 minutes. Degradation of IκB-α (A) and phosphorylation of ERK, JNK, and p38 (B) was monitored by Western blot. (C) Activation of NF-κB was quantified with a NF-κB p65 transcription factor assay kit using nuclear protein lysates. (D) IFN-γ mRNA levels were quantified by real-time PCR after 24 hours of stimulation as indicated. (E,F) NK cells were stimulated as in Figure 2B,C with anti-NK1.1 and IL-18 either alone or in combination. Cytokine release was measured by ELISA. Data are means plus or minus SD of triplicate samples (where applicable) and representative of 3 independent experiments.

  • Figure 5

    Carma1, but not Card9, is required for NK-cell receptor signaling. IL-15–expanded NK cells from Card9-deficient (A) or IL-15–expanded NK cells from Carma1-deficient (B) or control mice were stimulated with plate-bound anti–NK-cell receptor antibodies or PMA/Iono as indicated for 24 hours. Cytokine release was measured by ELISA. Data are means plus or minus SD of triplicate samples and representative of 3 independent experiments.

  • Figure 6

    Bcl10 deficiency does not affect NK-cell proliferation and early control of viral replication in MCMV infection. (A) Proliferation of NK1.1+ NK cells upon intraperitoneal infection with 1.25 × 105 pfu MCMV was assessed on day 3 and day 6 after infection by BrdU staining. Percentages of proliferating BrdU+ NK cells are indicated. Data are representative of 4 mice per group. (B) Viral load of spleen and liver was quantified 6 days after intraperitoneal infection with 1.25 × 105 pfu MCMV using quantitative real-time PCR. Results are indicated as MCMV-DNA copies per 5 ng total purified organ DNA. Shown are values of single mice and of 4 mice per group.