Research ArticleImmunology

Genetic diversity affects the nanoscale membrane organization and signaling of natural killer cell receptors

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Science Signaling  17 Dec 2019:
Vol. 12, Issue 612, eaaw9252
DOI: 10.1126/scisignal.aaw9252
  • Fig. 1 Inhibitory receptors encoded by different genes have distinct nanometer-scale arrangements.

    (A) Representative TIRF microscopy and STORM images of pNK cell clones (scale bar, 3 μm). STORM panels show the Gaussian-rendered image of coordinate data. The 2 μm × 2 μm regions (red boxes in the STORM images) are magnified, and the corresponding scatter plots, density maps (G&F analysis), and binary maps are shown (scale bar, 1 μm). The Ripley’s H function is plotted for a 3 μm × 3 μm region containing the enlarged region. CI, confidence interval of randomized control. (B) Quantitative analysis of binary maps. Each dot represents the mean value for a cell, and different colors represent different donors. Black bars show the median and interquartile range (IQR). Kruskal-Wallis test with Dunn’s multiple comparisons: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; KIR2DL1 (27 cells, three donors), KIR2DL3 (19 cells, three donors), KIR3DL1 (29 cells, three donors), NKG2A (19 cells, three donors), and LILRB1 (65 cells, three donors). (C) Representative PALM images of YTS cells expressing low and high amounts of KIR2DL1 tagged with a photoactivatable protein (mEos2). PALM images (scale bar, 5 μm) contain 2 μm × 2 μm regions (red boxes) that are magnified and shown as scatter plots (scale bar, 1 μm). (D) The normalized density within clusters [according to (24); see Materials and Methods] was plotted against the relative clustered area for three different receptors. Akaike’s information criteria (AICc) were used to compare theoretical curves for randomly organized events (gray lines) with the best-fit curves (red lines). KIR2DL1 (73 cells, five experiments), KIR2DL3 (11 cells, two experiments), and LILRB1 (18 cells, three experiments). See also figs. S1 to S3 for details on how NK clones were selected, various imaging control experiments, and how alternative methods for image analysis led to similar conclusions.

  • Fig. 2 KIR2DL1 allotypes differ in their nanoscale organization.

    (A) Flow cytometry analysis of representative pNK cell clones from a single donor that expressed different KIR2DL1 allotypes, stained for KIR2DL1 (gray) or with an isotype-matched control (white). (B) Representative TIRF microscopy and Gaussian-rendered STORM images of pNK cell clones with different KIR2DL1 allotypes (scale bar, 3 μm). The 2 μm × 2 μm regions (red boxes in the STORM images) are magnified, and the corresponding scatter plots, density maps, and binary maps are shown (scale bar, 1 μm). The Ripley’s H function is plotted for a 3 μm × 3 μm region containing the enlarged region. (C) Quantitative analysis of the binary maps. Each dot represents the mean value for a cell. Red bars represent the median and IQR. Mann-Whitney test: KIR2DL1*004 (13 cells, one clone, and one donor) and KIR2DL1*001 (11 cells, three clones, and one donor). **P < 0.01. See also fig. S4 for analogous data with KIR3DL1 and KIR2DL3.

  • Fig. 3 Allotypic differences do not affect the nanoscale organization of receptors when their abundance is controlled.

    (A) Flow cytometry analysis of YTS cells expressing different KIR2DL1 allotypes or KIR2DL1*003 with a premature stop codon (KIR2DL1 p.K250X) stained for KIR2DL1 (gray) or with an isotype-matched control (white). (B) Lysis of 721.221 cells expressing a ligand for KIR2DL1 (HLA-C*0602) by YTS transfectants in the presence of a mAb that blocked KIR or an isotype-matched control. One of the two representative experiments is shown, measured in triplicate, showing means ± SEM. (C) Representative TIRF microscopy and Gaussian-rendered STORM images of YTS transfectants (scale bar, 5 μm). The 2 μm × 2 μm regions (red boxes) are magnified, and the corresponding scatter plots, density maps, and binary maps are shown (scale bar, 1 μm). The Ripley’s H function is plotted for a 3 μm × 3 μm region containing the enlarged region. (D) Quantitative analysis of the binary maps. Each dot represents the mean value for a cell, and the red bars show the median and IQR. Kruskal-Wallis test with Dunn’s multiple comparisons: *P < 0.05. KIR2DL1*001 (32 cells, four experiments), KIR2DL1*003 (31 cells, four experiments), KIR2DL1*004 (22 cells, four experiments), KIR2DL1*006 (44 cells, five experiments), and KIR2DL1 p.K250X (23 cells, four experiments). See also fig. S5 for analogous data for KIR3DL1.

  • Fig. 4 The abundance of KIR2DL1 determines its nanoscale organization.

    (A and B) The KIR2DL1+ pNK cell clones analyzed in Fig. 1 are presented, stratified according to receptor abundance. (A) Representative TIRF microscopy and Gaussian-rendered STORM images (scale bar, 3 μm) are shown. The 2 μm × 2 μm regions (red boxes) are magnified, and the corresponding scatter plots, density maps, and binary maps are shown (scale bar, 1 μm). The Ripley’s H function is plotted for a 3 μm × 3 μm region containing the enlarged region. (B) Quantitative analysis of the binary maps. Each dot represents the mean value for a cell, and different colors represent different donors. Black bars show the median and IQR. Kruskal-Wallis test with Dunn’s multiple comparisons: *P < 0.05, ***P < 0.001, ****P < 0.0001; nine cells per tertile; three experiments. (C and D) The YTS cells expressing KIR2DL1-mEos2 analyzed in Fig. 1 were stratified according to receptor abundance. (C) Representative Gaussian-rendered PALM images (scale bar, 5 μm) contain 2 μm × 2 μm regions (red boxes) that are magnified, and the corresponding scatter plots, density maps, and binary maps are shown (scale bar, 1 μm). The Ripley’s H function is plotted for a 3 μm × 3 μm region containing the enlarged region. (D) Quantitative analysis of the binary maps. Each dot represents the mean value for a cell, and the red bars show the median and IQR. Kruskal-Wallis test with Dunn’s multiple comparisons: *P < 0.05, **P < 0.01, ****P < 0.0001; 20 to 21 cells per tertile, five experiments. See also figs. S6 and S7 for analogous data for KIR3DL1 and for KIR2DL1 as assessed by an alternative imaging method.

  • Fig. 5 Ligation reorganizes KIRs.

    (A) Representative TIRF microscopy and Gaussian-rendered STORM images of YTS transfectants expressing KIR2DL1-FLAG cells on surfaces coated with mAbs against CD28, LFA-1, and an isotype-matched control antibody (activating conditions) or with mAbs against CD28, LFA-1, and FLAG (inhibitory conditions). (B) Quantitative analysis of the binary maps. Each dot represents the mean value for a cell, and the red bars represent the median and IQR. Mann-Whitney tests: *P < 0.05, ***P < 0.001. Activation conditions: 48 cells from five experiments; inhibitory conditions (FLAG ligation): 38 cells from five experiments. (C) Representative Gaussian-rendered PALM images of YTS transfectants expressing KIR2DL1-mEos2 on surfaces coated with mAbs against NKp30 and LFA-1, with HLA-C*0401 (“HLA-C2”) or a control protein, BSA. (D) Quantitative analysis of the binary maps depicted in (C). Each dot represents the mean value for a cell, and the red bars represent the median and IQR. Mann-Whitney tests: *P < 0.05, **P < 0.01. Activation conditions: 21 cells from three experiments; HLA-C2 ligation conditions: 22 cells from three experiments. (E) Representative TIRF microscopy and Gaussian-rendered STORM images of mAb 177407 staining of KIR3DL1+ YTS cells interacting with HLA-B*5701 (“HLA-Bw4”) or control protein, IgG2a. (F) Quantitative analysis of the binary maps of KIR3DL1. No ligation: 24 cells from three experiments; HLA-Bw4 ligation: 49 cells from six experiments. In images of whole cells (scale bars, 5 μm), 2 μm × 2 μm regions (red boxes) are magnified, and the corresponding scatter plots, density maps, and binary maps are shown alongside (scale bar: 1 μm). In each case, the Ripley’s H function is plotted for a 3 μm × 3 μm region containing the enlarged region. Each dot represents the mean value for a cell, and the red bars represent the median and IQR. Mann-Whitney tests: ****P < 0.0001.

  • Fig. 6 KIRlow cells generate more pCrk than do KIRhigh cells.

    (A to E) KIR2DL1+ YTS cells were ligated with surfaces coated with mAbs against FLAG or an isotype-matched control. (A) Quantification of the intensity of pCrk clusters triggered by the ligation of KIR2DL1 or KIR2DL1-p.K250X as measured by STED microscopy. Kruskal-Wallis test with Dunn’s multiple comparisons: **P < 0.01, ****P < 0.0001. KIR2DL1 no ligation (57 cells, four experiments), KIR2DL1 FLAG ligation (98 cells, four experiments), KIR2DL1 p.K250X no ligation (7 cells, three experiments), KIR2DL1 p.K250X FLAG ligation (9 cells, three experiments). (B) Representative flow cytometry analysis of unstimulated YTS clones that expressed KIR2DL1*003 at high (KIRhigh; dark gray) or low (KIRlow; light gray) amounts stained for surface KIR2DL1, or intracellular CrkII or SHP-1, compared to an isotype-matched control mAb (white). (C to E) Quantification and representative images relating to STED microscopy analysis of KIRlow and KIRhigh cells on surfaces that ligated KIR2DL1. (C) Quantification of KIR2DL1 staining. Kruskal-Wallis test with Dunn’s multiple comparisons: *P < 0.05, ****P < 0.0001. KIRlow no ligation (32 cells, four experiments), KIRlow FLAG ligation (40 cells, four experiments), KIRhigh no ligation (39 cells, three experiments), and KIRhigh FLAG ligation (58 cells, three experiments). (D) Representative images of pCrk (scale bar, 5 μm) and outlines of the high-intensity regions of pCrk staining selected for analysis using a custom ImageJ script (masks). Red outlines show the high-intensity areas of pCrk staining, whereas yellow outlines show the cell area [from interference reflection microscopy (IRM) images]. (E) Quantitative analysis of pCrk nanoclusters. Kruskal-Wallis test with Dunn’s multiple comparisons: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. KIRlow no ligation (32 cells, four experiments), KIRlow FLAG ligation (40 cells, four experiments), KIRhigh no ligation (48 cells, four experiments), and KIRhigh FLAG ligation (74 cells, four experiments). (F) Flow cytometry analysis of representative unstimulated pNK cell clones expressing high (KIRhigh; dark gray) or low (KIRlow; light gray) amounts of KIR2DL1, stained for surface KIR2DL1, intracellular CrkII, or with an isotype-matched control (white). (G) The concentration of IFN-γ in supernatants from pNK clones stimulated with ICAM-1 and a combination of mAbs against activating receptors (NKp30) or inhibitory receptors (KIR2DL1) at a range of concentrations (1, 2, 5, and 7.5 μg/ml), and isotype-matched controls, was measured by ELISA. Graphs are shown for representative clones without KIR2DL1 (left, one clone) or expressing low (central, one clone) or high (right, three clones) amounts of KIR2DL1. Each line represents a different clone. Triplicate measurements were performed. (H) Representative STED microscopy images of pCrk in pNK clones stimulated with coated surfaces (scale bar, 3 μm). (I) Quantitative analysis of pCrk nanoclusters. Each dot represents the mean value for a cell. Red bars represent the median and IQR. Kruskal-Wallis test with Dunn’s multiple comparisons: *P < 0.05, **P < 0.01, ***P < 0.001. KIRlow no ligation (35 cells, four experiments), KIRlow KIR ligation (40 cells, four experiments), KIRhigh no ligation (20 cells, three experiments), and KIRhigh KIR ligation (28 cells, three experiments). A.U., arbitrary units.

  • Fig. 7 SHP-1 activity controls Crk phosphorylation and potentiates immune synapse formation.

    (A) Mean intensity of KIR2DL1 (FLAG), pCrk, and pSHP-1 in YTS KIRlow and KIRhigh cells ligated with HLA-C*0401 coated onto slides, as measured by confocal microscopy. Each dot represents the mean value for a cell. Mann-Whitney test: *P < 0.05, **P < 0.01, ****P < 0.0001. KIRlow KIR2DL1/pCrk (72 cells, three experiments), KIRhigh KIR2DL1/pCrk (66 cells, three experiments), KIRlow pSHP-1 (51 cells, three experiments), and KIRlow pSHP-1 (50 cells, three experiments). (B to D) Quantification of pCrk clusters in confocal images of YTS KIRlow and KIRhigh cells on surfaces coated with mAbs against (B) KIR2DL1 or (D) LFA-1 ± NKp30. YTS cells were pretreated with the SHP-1/2 inhibitor NSC87877 (empty) or were untreated (filled). Kruskal-Wallis test with Dunn’s multiple comparisons: *P < 0.05, **P < 0.01, ****P < 0.0001. KIRlow anti-KIR (61 cells, three experiments), KIRlow anti-KIR + NSC87877 (52 cells, three experiments), KIRhigh anti-KIR (50 cells, three experiments), KIRhigh anti-KIR + NSC87877 (47 cells, three experiments), KIRlow anti–LFA-1 (60 cells, three experiments), KIRhigh anti–LFA-1 (60 cells, three experiments), KIRlow anti–LFA-1 anti-NKp30 (59 cells, three experiments), KIRhigh anti–LFA-1 anti-NKp30 (60 cells, three experiments), KIRlow anti–LFA-1 + NSC87877 (57 cells, three experiments), KIRhigh anti–LFA-1 + NSC87877 (60 cells, three experiments), KIRlow anti–LFA-1 anti-NKp30 + NSC87877 (59 cells, three experiments), and KIRhigh anti–LFA-1 anti-NKp30 + NSC87877 (58 cells, three experiments). (C) Representative confocal images of pCrk (scale bar, 5 μm) relating to (B) and (D). (E) Representative F-actin and pCrk STED microscopy images of a KIR2DL1+ YTS cell on a surface ligating LFA-1 and NKp30 (scale bar, 5 μm). (F) Mander’s colocalization of pCrk clusters with the brightest spots of actin, compared to colocalization when the pCrk clusters are artificially placed at random within the cell region of interest. Mann-Whitney test: ****P < 0.0001 (38 cells, six experiments). (G) Representative F-actin, brightfield, and IRM images of YTS KIRlow and KIRhigh cells on surfaces coated with ICAM-1 ± mAb against NKp30 ± NSC87877 (scale bar, 20 μm). (H) Quantification of spread cell area from IRM images of the cells represented in (G). Kruskal-Wallis test with Dunn’s multiple comparisons: **P < 0.01, ****P < 0.0001. KIRlow ICAM-1 (731 cells, six experiments), KIRhigh ICAM-1 (711 cells, six experiments), KIRlow ICAM-1 anti–NKp30 (535 cells, six experiments), KIRhigh ICAM-1 anti-NKp30 (547 cells, six experiments), KIRlow ICAM-1 + NSC87877 (246 cells, six experiments), KIRhigh ICAM-1 + NSC87877 (259 cells, six experiments), KIRlow ICAM-1 anti-NKp30 + NSC87877 (390 cells, six experiments), and KIRhigh ICAM-1 anti-NKp30 + NSC87877 (459 cells, six experiments). (I and J) Quantification of the number of immune synapses (“contacts”) made by KIRlow and KIRhigh cells with 721.221 cells when suspended in a three-dimensional extracellular matrix and imaged by confocal microscopy for 8 hours. (I) Mean number of synapses made per YTS cell and (J) the number of synapses made by each YTS cell over 8 hours (KIRlow: 130 cells, three experiments; KIRhigh: 172 cells, three experiments). Mann-Whitney test: ***P < 0.001. (K) The amounts of IFN-γ secreted from NKG2A and NKG2A+ pNK clones stimulated with ICAM-1 and anti-NKp30 mAb were measured by ELISA. Each dot represents a clone. NKG2A (three clones, one donor) and NKG2A+ (three clones, one donor). (L) Quantitative analysis of pCrk nanoclusters in STED microscopy images of NKG2A and NKG2A+ pNK clones. Each dot represents the mean value for a cell. Mann-Whitney test: *P < 0.05. NKG2A (58 cells, 12 experiments, one donor) and NKG2A+ (17 cells, three experiments, one donor). In all graphs, bars represent the median and IQR.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/12/612/eaaw9252/DC1

    Fig. S1. Selection of NK cell clones expressing receptors of interest for imaging.

    Fig. S2. Data processing controls for STORM data.

    Fig. S3. Inhibitory receptors are clustered according to STORM analysis methods.

    Fig. S4. The abundances of KIR2DL3 and KIR3DL1 correlate with their nanoscale organization.

    Fig. S5. Allotypic differences in KIR3DL1 do not affect its nanoscale organization when its abundance is controlled.

    Fig. S6. The abundance of KIR3DL1 determines its nanoscale organization.

    Fig. S7. The abundance of KIR2DL1 determines its nanoscale organization in YTS cells, as assessed by STORM.

    Fig. S8. In silico model of the effects of increasing KIR2DL1 density reveals that the receptors must be added into existing clusters to enlarge the cluster area.

    Fig. S9. YTS cells expressing different amounts of KIR2DL1 can be equally inhibited.

    Fig. S10. Inhibition of SHP-1/2 does not change the organization of KIR2DL1 in YTS-KIRlow or KIRhigh cells.

    Table S1. Primers for the cloning of inhibitory receptors.

    Table S2. Primers for the genomic investigation of KIR2DL1.

    Table S3. Primers for differentiating KIR2DL1 alleles expressed in pNK clones.

  • This PDF file includes:

    • Fig. S1. Selection of NK cell clones expressing receptors of interest for imaging.
    • Fig. S2. Data processing controls for STORM data.
    • Fig. S3. Inhibitory receptors are clustered according to STORM analysis methods.
    • Fig. S4. The abundances of KIR2DL3 and KIR3DL1 correlate with their nanoscale organization.
    • Fig. S5. Allotypic differences in KIR3DL1 do not affect its nanoscale organization when its abundance is controlled.
    • Fig. S6. The abundance of KIR3DL1 determines its nanoscale organization.
    • Fig. S7. The abundance of KIR2DL1 determines its nanoscale organization in YTS cells, as assessed by STORM.
    • Fig. S8. In silico model of the effects of increasing KIR2DL1 density reveals that the receptors must be added into existing clusters to enlarge the cluster area.
    • Fig. S9. YTS cells expressing different amounts of KIR2DL1 can be equally inhibited.
    • Fig. S10. Inhibition of SHP-1/2 does not change the organization of KIR2DL1 in YTS-KIRlow or KIRhigh cells.
    • Table S1. Primers for the cloning of inhibitory receptors.
    • Table S2. Primers for the genomic investigation of KIR2DL1.
    • Table S3. Primers for differentiating KIR2DL1 alleles expressed in pNK clones.

    [Download PDF]

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