Research ArticleImmunology

A nanoscale reorganization of the IL-15 receptor is triggered by NKG2D in a ligand-dependent manner

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Science Signaling  10 Apr 2018:
Vol. 11, Issue 525, eaal3606
DOI: 10.1126/scisignal.aal3606
  • Fig. 1 Ligand-dependent reorganization of NKG2D nanoclusters at the surface of pNK cells.

    (A) Primary natural killer (pNK) cells were incubated for 24 hours in wells coated with poly-l-lysine (PLL), MHC (major histocompatibility complex) class I–related protein A (MICA), MICA and intercellular adhesion molecule–1 (ICAM-1), ULBP2, or ULBP2 and ICAM-1. The amounts of interferon-γ (IFN-γ) (left), CCL1 (middle), and tumor necrosis factor–α (TNF-α) (right) released by the cells were assessed by ELISA (enzyme-linked immunosorbent assay). Data are means ± SD from four to nine donors. Each color represents one donor. (B) Representative total internal reflection fluorescence (TIRF) and direct stochastic optical reconstruction microscopy (dSTORM) images of natural killer group 2D (NKG2D) on pNK cells incubated for 10 min on slides that were coated as described in (A) and then stained with a fluorescently labeled specific monoclonal antibody (mAb) against NKG2D. Scale bars, 4 μm. Regions outlined in white are magnified (zoom) and shown with corresponding density images according to the pseudocolor scale bar, binary maps, and Ripley’s K analysis. Scale bars, 1 μm. L(r)-r, degree of clustering relative to simulated random distributions; r, radial scale. CI, confidence interval. (C) Nanocluster areas (left), nanocluster density (middle), and percentage of localizations in nanoclusters (right) for NKG2D from the data shown in (B). Each symbol represents the median of several 5 μm × 5 μm regions from one cell. Lines represent means ± SD. Data are from a minimum of 40 cells from a minimum of three independent donors. Each color represents one donor. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by one-way analysis of variance (ANOVA) with Tukey’s post hoc test. Nonsignificant differences are not indicated.

  • Fig. 2 Blockade of NKG2D ligands prevents the reorganization of NKG2D nanoclusters.

    (A) Representative TIRF and dSTORM images of NKG2D on pNK cells incubated for 10 min on slides coated with PLL, MICA and ICAM-1, or ULBP2 and ICAM-1, blocked with specific antibodies against MICA or ULBP2 as appropriate, and then stained with fluorescently labeled specific mAb against NKG2D. Scale bars, 4 μm. Regions outlined in white are magnified and shown with corresponding density images according to the pseudocolor scale bar, thresholded binary maps, and Ripley’s K analysis. Scale bars, 1 μm. (B) Nanocluster areas (left), nanocluster density (middle), and percentage of localizations in nanoclusters (right) for NKG2D from the data shown in (A). Each symbol represents the median of several 5 μm × 5 μm regions from one cell. Lines represent means ± SD. Data are from a minimum of 12 cells from two independent donors. Each color represents one donor. *P < 0.05 and **P < 0.01 by one-way ANOVA with Tukey’s post hoc test. Nonsignificant differences are not indicated.

  • Fig. 3 Activation of NKG2D with ULBP2, but not MICA, induces the reorganization of IL-2/IL-15Rβ nanoclusters.

    (A) Representative TIRF and dSTORM images of IL-2/IL-15Rβ at the surface of pNK cells incubated for 10 min on slides coated with PLL (nonactivated), MICA, MICA and ICAM-1, ULBP2, or ULBP2 and ICAM-1 and stained with a fluorescently labeled specific mAb against NKG2D. Scale bars, 4 μm. Regions outlined by the white squares are magnified and shown with corresponding density images according to the pseudocolor scale bar, thresholded binary maps, and Ripley’s K analysis. Scale bars, 1 μm. (B) Nanocluster areas (left), nanocluster density (middle), and percentage of localizations in nanoclusters (right) for IL-2/IL-15Rβ from the data shown in (A) were calculated by subjecting dSTORM data to spatial point pattern analysis and thresholding. Each symbol represents the median of several 5 μm × 5 μm regions from the same cell. Horizontal lines and errors represent means ± SD. Data are from a minimum of 85 cells from a minimum of three independent donors. Each color represents one donor. *P < 0.05, ***P < 0.001, and ****P < 0.0001 by one-way ANOVA with Tukey’s post hoc test (left) and Kruskal-Wallis with Dunn’s post hoc test (middle and right). Nonsignificant differences are not indicated.

  • Fig. 4 Blockade of NKG2D ligands prevents the reorganization of IL-2/IL-15Rβ nanoclusters.

    (A) Representative TIRF and dSTORM images of IL-2/IL-15Rβ on pNK cells incubated for 10 min on slides previously coated with PLL, MICA and ICAM-1, or ULBP2 and ICAM-1, blocked with specific antibodies against MICA or ULBP2 as appropriate, and stained with fluorescently labeled specific mAb against NKG2D. Scale bars, 4 μm. Regions outlined in white are magnified and shown with corresponding density images according to the pseudocolor scale bar, thresholded binary maps, and Ripley’s K analysis. Scale bars, 1 μm. (B) Nanocluster areas (left), nanocluster density (middle), and the percentage of localizations in nanoclusters (right) for IL-2/IL-15Rβ from the data shown in (A). Each symbol represents the median of several 5 μm × 5 μm regions from one cell. Lines represent means ± SD. Data are from a minimum of 12 cells from two independent donors. Each color represents one donor. One-way ANOVA with Tukey’s post hoc test was used. Nonsignificant differences are not indicated.

  • Fig. 5 Trans-presentation of IL-15 increases the density of IL-2/IL-15Rβ nanoclusters.

    (A to C) Representative TIRF and dSTORM images of NKG2D (A), IL-2/IL-15Rβ (B), and IL-2Rα (C) at the surface of pNK cells incubated for 10 min on slides coated with IL-15Rα unloaded or loaded with IL-15 and stained with fluorescently labeled NKG2D-specific mAbs. Scale bars, 4 μm. Regions outlined by the white squares are magnified and shown with corresponding density images according to the pseudocolor scale bar, thresholded binary maps, and Ripley’s K analysis. Scale bars, 1 μm. (D to F) Nanocluster area (D), nanocluster density (E), and percentage of localizations in nanoclusters (F) for NKG2D, IL-2/IL-15Rβ, and IL-2Rα from the data shown in (A) to (C) were calculated by subjecting dSTORM data to spatial point pattern analysis and thresholding. Each symbol represents the median of several 5 μm × 5 μm regions from the same cell. Horizontal lines and errors represent means ± SD. Data are from a minimum of 45 cells from a minimum of three independent donors. Each color represents one donor. *P < 0.05, **P < 0.01, and ****P < 0.0001 by two-tailed t test assuming unequal variance. Nonsignificant differences are not indicated.

  • Fig. 6 ULBP2 induces the association between NKG2D and IL-2/IL-15Rβ nanoclusters.

    (A) TIRF and dSTORM images showing NKG2D and IL-2/IL-15Rβ on pNK cells incubated for 10 min on slides coated with PLL, MICA and ICAM-1, ULBP2 and ICAM-1, or IL-15Rα–IL-15 and stained with anti–NKG2D-Atto488 and anti–IL-2/IL-15Rβ–AF647 mAbs. Regions outlined in white are magnified with relative fluorescence intensity profiles along the white lines. Scale bars, 4 and 1 μm (zoom). As a positive control, cells on PLL-coated slides were stained with anti–NKG2D-Atto488 and anti–DAP10-AF647 mAbs or were stained with anti–NKG2D-Atto488 mAb, followed by anti–mouse-IgG1-AF647 secondary antibody (PC). Colocalization between channels is shown in white. a.u., arbitrary units. (B) Coordinate-based colocalization (CBC) histograms of the single-molecule distributions of the colocalization parameter for NKG2D and IL-2/IL-15Rβ in cells incubated as in (A). Data are from a minimum of 10 cells from two independent donors. Bars represent means ± SD. (C) Nearest-neighbor distance (NND) analysis of the data shown in (A). Data are from a minimum of 30 cells from two independent donors; two donors for the positive controls. Symbol represents the median NND of all paired single-molecule localizations from one cell. Lines/errors represent means ± SD. *P < 0.05 and ***P < 0.001 by Kruskal-Wallis (Dunn’s post hoc test). (D) Histogram distributions of the NND between the centroids of nanoclusters from one channel and the centroid of their nearest neighbor (NN) from the second channel from cells incubated as described in (A). Nonsignificant differences are not indicated.

  • Fig. 7 Trans-presentation of IL-15 by IL-15Rα augments the activation of NK cells by ULBP2, but not MICA.

    (A) Representative TIRF and dSTORM images of membrane-proximal F-actin in pNK cells incubated for 10 min at 37°C on slides coated with PLL, unloaded IL-15Rα, IL-15–loaded IL-15Rα, or MICA or ULBP2 in the presence of ICAM-1, with or without IL-15Rα–IL-15. Scale bars, 4 μm. Regions outlined by the white squares (middle row) are magnified (bottom row). Scale bars, 500 nm. An example of an actin mesh hole is indicated in red. (B) Histogram of the percentage of cells with dense peripheral actin rings. pNK cells were incubated as described in (A). F-actin was visualized using fluorescently labeled phalloidin, and the percentages of cells forming dense peripheral F-actin rings were quantified from confocal images. Data are from 100 to 500 cells from at least two independent donors. Bars represent means ± SD. (C) Flow cytometric analysis of CD107a surface abundance on pNK cells incubated for 5 hours at 37°C on slides coated as described in (A). Data are from one experiment and are representative of five experiments. (D) Percentage of pNK cells that degranulated as calculated from the flow cytometric data. Bars represent means ± SD from five donors. Each color represents one donor. *P < 0.05 and ****P < 0.0001 by one-way ANOVA with Tukey’s post hoc test. ns, not significant.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/525/eaal3606/DC1

    Fig. S1. Titration of MICA and ULBP2 concentrations for coating slides.

    Fig. S2. The binding of NKG2D to MICA or ULBP2 does not block the binding of the NKG2D mAb 1D11 to the receptor.

    Fig. S3. Label density variation analysis of NKG2D and IL-2/IL-15Rβ.

    Fig. S4. Ligand-dependent reorganization of NKG2D at different times.

    Fig. S5. Fold change in the nanoscale organization of NKG2D, IL-2/IL-15Rβ, and IL-2Rα nanoclusters.

    Fig. S6. Surface expression analysis of NKG2D and IL-2/IL-15Rβ.

    Fig. S7. Ligand-dependent reorganization of IL-2/IL-15Rβ at different times.

    Fig. S8. The nanoscale organization of IL-2Rα remains unaltered upon ligation of NKG2D.

    Fig. S9. Time-dependent association between NKG2D and IL-2/IL-15Rβ nanoclusters induced by ULBP2.

    Fig. S10. Isotype-matched control staining.

  • Supplementary Materials for:

    A nanoscale reorganization of the IL-15 receptor is triggered by NKG2D in a ligand-dependent manner

    Štefan Bálint, Filipa B. Lopes, Daniel M. Davis*

    *Corresponding author. Email: daniel.davis{at}manchester.ac.uk

    This PDF file includes:

    • Fig. S1. Titration of MICA and ULBP2 concentrations for coating slides.
    • Fig. S2. The binding of NKG2D to MICA or ULBP2 does not block the binding of the NKG2D mAb 1D11 to the receptor.
    • Fig. S3. Label density variation analysis of NKG2D and IL-2/IL-15Rβ.
    • Fig. S4. Ligand-dependent reorganization of NKG2D at different times.
      Fig. S5. Fold change in the nanoscale organization of NKG2D, IL-2/IL-15Rβ, and IL-2Rα nanoclusters.
    • Fig. S6. Surface expression analysis of NKG2D and IL-2/IL-15Rβ.
    • Fig. S7. Ligand-dependent reorganization of IL-2/IL-15Rβ at different times.
    • Fig. S8. The nanoscale organization of IL-2Rα remains unaltered upon ligation of NKG2D.
    • Fig. S9. Time-dependent association between NKG2D and IL-2/IL-15Rβ nanoclusters induced by ULBP2.
    • Fig. S10. Isotype-matched control staining.

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    © 2018 American Association for the Advancement of Science

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