Research ArticleImmunology

Educated natural killer cells show dynamic movement of the activating receptor NKp46 and confinement of the inhibitory receptor Ly49A

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Science Signaling  13 Feb 2018:
Vol. 11, Issue 517, eaai9200
DOI: 10.1126/scisignal.aai9200
  • Fig. 1 Dynamic confinement of NKp46 and Ly49A at the NK cell surface.

    (A to D) Representative fluorescence images showing transfected GFP-tagged Ly49A (Ly49A-GFP) in CHO cells. The images were merged from 6000 frames acquired at a 36 ms frame-to-frame rate. (A and B) Transfected Ly49A-GFP without antibody labeling. (C) Transfected Ly49A-GFP incubated with an antibody that recognizes Ly49A (clone JR9.318 conjugated to Abberior STAR 594). (D) JR9.318–Abberior STAR 594 labeling in the same cell as in (C). (E to J) SPT in freshly isolated H-2Dd compared to MHC−/− murine Ly49A+ NK cells captured at a 20-ms frame-to-frame rate. (E to H) Representative single-particle tracks of NKp46 (E and F) and Ly49A (G and H) in H-2Dd and MHC−/− NK cells. (I and J) The degree of anomalous diffusion (Hurst parameter) for NKp46 (median H-2Dd = 0.037 and MHC−/− = 0.049) (I) and Ly49A (median H-2Dd = 0.023 and MHC−/− = 0.037) (J) was calculated on 100-frame segments of trajectories. All the values calculated for a single trajectory were averaged. Each dot represents a single trajectory (H-2Dd NKp46, n = 264; MHC−/− NKp46, n = 94; H-2Dd Ly49A, n = 521; and MHC−/− Ly49A, n = 116). Data were pooled from 7 to 27 cells per group, from three to six independent experiments. The boxes represent the median (line inside the box), the notches represent the 95% confidence intervals, the ends of the boxes represent the first and third quartiles, and the whiskers represent 1.5 times the interquartile ranges. ***P ≤ 0.001 (two-sample Wilkoxon signed-rank test). Scale bars, 5 μm (A to D) and 2 μm (E to H).

  • Fig. 2 NKp46 diffuses faster and covers larger areas on educated NK cells, whereas Ly49A diffuses slower and covers smaller areas in educated NK cells.

    SPT trajectories in freshly isolated H-2Dd compared to MHC−/− murine Ly49A+ NK cells captured at a 20 ms frame-to-frame rate. Each dot represents a single trajectory. (A and B) NKp46 microdomain median width: H-2Dd = 0.21 μm and MHC−/− = 0.18 μm (A); Ly49A microdomain median width: H-2Dd = 0.19 μm and MHC−/− = 0.24 μm (B). (C and D) NKp46 short-range diffusion coefficient D1–10 median: H-2Dd = 1.1 × 10−3 μm2/s and MHC−/− = 0.89 × 10−3 μm2/s (C); Ly49A short-range diffusion coefficient D1–10 median: H-2Dd = 0.93 × 10−3 μm2/s and MHC−/− = 1.5 × 10−3 μm2/s (D). The tracks are the same as those presented in Fig. 1. The boxes represent the median (line inside the box), the notches represent the 95% confidence intervals, the ends of the boxes represent the first and third quartiles, and the whiskers represent 1.5 times the interquartile ranges. **P ≤ 0.01, ***P ≤ 0.001 (two-sample Wilcoxon signed-rank test).

  • Fig. 3 iMSD carpet analysis reveals a smaller Ly49A domain size and shorter average microdomain dwell times for NKp46 and Ly49A in educated NK cells compared to hyporesponsive NK cells.

    (A) The three-dimensional (3D) STICS series was calculated from a recorded image time series, followed by conversion into a 2D carpet by combining the spatial lags, ξ and ψ, into a single radial component, δ. (B) The data were fitted with four models representing different types of particle dynamics. Panes show the fitted models of a sample data set. (C) Residuals of the fit of each model for the sample data set in (B). The color scale in (B) and (C) represents the correlation amplitude. (D to I) TIRF imaging and fitting of the confinement iMSD carpet model to freshly isolated Ly49A+ NK cells. The graphs show analyses of microdomain widths of NKp46 (D), Ly49A (labeled with antibody YE1/48) (E), and Ly49A (labeled with antibody JR9.318) (F). (G to I) Receptor dwell times within individual domains of NKp46 (G), Ly49A (labeled with antibody YE/148) (H), and Ly49A (labeled with antibody JR9.318) (I). Each dot represents one cell. n = 67 NKp46 in H-2Dd cells, 72 NKp46 in MHC−/− cells, 53 Ly49A in H-2Dd cells, 68 Ly49A in MHC−/− cells (JR9.318), 35 Ly49A in H-2Dd cells, and 37 Ly49A in MHC−/− cells (YE/148). Data were pooled from six (NKp46 antibody and JR9.318) or three (YE1/48) independent experiments. No more than one outlier per sample was excluded before analysis, using Grubbs’ test. The boxes represent the median and the first and third quartiles, and the notches represent the 95% confidence intervals. The whiskers show the 95% ranges. *P ≤ 0.05, **P ≤ 0.01 (Mann-Whitney test). Comparisons that are not marked were not statistically significantly different.

  • Fig. 4 Actin depolymerization reduces NKp46 confinement and disrupts activating signaling.

    (A to D) TIRF imaging and fitting of the confinement iMSD carpet model to freshly isolated Ly49A+ NK cells. The graphs show analyses of NKp46 microdomain width (A) and microdomain dwell time (B) in NK cells treated with the actin polymerization inhibitor LatB, and NKp46 microdomain width (C) and microdomain dwell time (D) in H-2Dd NK cells treated with the myosin V inhibitor Myovin1 (50 or 200 μg/ml) (Myo 50 and Myo 200). MFI, mean fluorescence intensity. Each dot represents one cell. n = 34 H-2Dd control, 39 H-2Dd LatB, 32 MHC−/− control, and 34 MHC−/− LatB (A and B); n = 42 H-2Dd control, 45 Myo 200, 29 Myo 50, 42 MHC−/− control, 46 Myo 200, and 29 Myo 50 (C and D). Data were pooled from three independent experiments. No more than one outlier per group was detected and removed using Grubbs’ test. The boxes represent the median and the first and third quartiles, and the notches represent the 95% confidence intervals. The whiskers show the 95% range. *P ≤ 0.05 (Mann-Whitney test). (E to H) Representative Fluo-4 fluorescence time traces indicating Ca2+ release in H-2Dd and MHC−/− NK cells after treatment with ionomycin stimulation (E and G) or cross-linking of NKp46 (F and H), normalized to median baseline by subtraction, in H-2Dd and MHC−/− NK cells as indicated. (I) Peak difference in Ca2+ flux in resting NK cells after NKp46 cross-linking with or without pretreatment with LatB in the indicated NK cells. For the negative control, NKp46 was not cross-linked. Bars show the mean with SD. Data were pooled from eight independent experiments. **P < 0.01 (Mann-Whitney test). Statistical tests were performed between control groups and treatment within each mouse from which the cells were harvested (H-2Dd control versus H-2Dd–treated and MHC−/− control versus MHC−/−–treated); comparisons that are not marked were not statistically significantly different.

  • Fig. 5 The role of cholesterol in receptor dynamics and signal initiation.

    NKp46 microdomain dwell time (A) and Ly49A (antibody clone JR9) microdomain dwell time (B), as detected by antibodies, after adding cholesterol (chol), depleting cholesterol with MβCD, or in untreated control cells (ctrl). Each dot represents one cell. n = 27 H-2Dd ctrl, 21 H-2Dd chol, 29 H-2Dd MβCD, and 14 MHC−/− ctrl, 21 MHC−/− chol, 21 MHC−/− MβCD (A); n = 27 H-2Dd ctrl, 24 H-2Dd chol, 28 H-2Dd MβCD, and 14 MHC−/− ctrl, 23 MHC−/− chol, 25 MHC−/− MβCD (B). Data were pooled from three independent experiments. No more than one outlier per group was detected and removed using Grubbs’ test. The boxes represent the median and the first and third quartiles, and the notches represent the 95% confidence intervals. The whiskers show the 95% range. (C to F) Representative Fluo-4 fluorescence time traces indicating Ca2+ release in H-2Dd and MHC−/− NK cells after stimulation with ionomycin (C and E) or cross-linking of NKp46 (D and F), normalized to median baseline by subtraction, in the absence or presence of added cholesterol (+chol). (G) Peak Ca2+ release in H-2Dd and MHC−/− NK cells in the absence or presence of cholesterol. Data were pooled from eight independent experiments. *P ≤ 0.05, **P < 0.01 (Mann-Whitney test). Statistical tests were performed between control groups and treatment within each mouse (H-2Dd ctrl versus H-2Dd–treated and MHC−/− ctrl versus MHC−/−–treated); differences not marked in the figure were not statistically significantly different.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/517/eaai9200/DC1

    Fig. S1. Diffusion rate and cluster size at the cell surface are not affected by antibody binding.

    Fig. S2. Dynamic clustering of NKp46 and Ly49A at the NK cell surface.

    Fig. S3. Simulation of diffusion with different numbers of molecules and variance of the noise.

    Fig. S4. Fitting of simulated data sets of the different modes of movement.

    Fig. S5. Cluster dynamics of transfected Ly49A-GFP at the cell surface is not significantly affected by antibody binding.

    Fig. S6. Carpet iMSD analysis of Ly49A after LatB and Myovin1 treatment.

    Fig. S7. Carpet iMSD microdomain widths after cholesterol or MβCD treatment.

    Table S1. Simulation of diffusion with different numbers of molecules and variance of the noise.

    Table S2. R2 of fitting simulated data to the four different models, using iMSD carpet analysis.

    Table S3. Median R2 and percentage of cells with best fit for each model.

    Movie S1. Single-molecule tracking of NKp46 in H-2Dd NK cells.

    Movie S2. Single-molecule tracking of Ly49A in H-2Dd NK cells.

  • Supplementary Materials for:

    Educated natural killer cells show dynamic movement of the activating receptor NKp46 and confinement of the inhibitory receptor Ly49A

    Elina Staaf, Per Niklas Hedde, Sunitha Bagawath Singh, Joachim Piguet, Enrico Gratton, Sofia Johansson*

    *Corresponding author. Email: sofia.e.johansson{at}ki.se

    This PDF file includes:

    • Fig. S1. Diffusion rate and cluster size at the cell surface are not affected by antibody binding.
    • Fig. S2. Dynamic clustering of NKp46 and Ly49A at the NK cell surface.
    • Fig. S3. Simulation of diffusion with different numbers of molecules and variance of the noise.
    • Fig. S4. Fitting of simulated data sets of the different modes of movement.
    • Fig. S5. Cluster dynamics of transfected Ly49A-GFP at the cell surface is not significantly affected by antibody binding.
    • Fig. S6. Carpet iMSD analysis of Ly49A after LatB and Myovin1 treatment.
    • Fig. S7. Carpet iMSD microdomain widths after cholesterol or MβCD treatment.
    • Table S1. Simulation of diffusion with different numbers of molecules and variance of the noise.
    • Table S2. R2 of fitting simulated data to the four different models, using iMSD carpet analysis.
    • Table S3. Median R2 and percentage of cells with best fit for each model.
    • Legends for movies S1 and S2

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

    Size: 1.23 MB

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mov format). Single-molecule tracking of NKp46 in H-2Dd NK cells.
    • Movie S2 (.mov format). Single-molecule tracking of Ly49A in H-2Dd NK cells.

    Citation: E. Staaf, P. N. Hedde, S. Bagawath Singh, J. Piguet, E. Gratton, S. Johansson, Educated natural killer cells show dynamic movement of the activating receptor NKp46 and confinement of the inhibitory receptor Ly49A. Sci. Signal. 11, eaai9200 (2018).

    © 2018 American Association for the Advancement of Science

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