Research ArticleT CELLS

Interfacial actin protrusions mechanically enhance killing by cytotoxic T cells

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Science Immunology  22 Mar 2019:
Vol. 4, Issue 33, eaav5445
DOI: 10.1126/sciimmunol.aav5445
  • Fig. 1 CTLs form protrusions on stimulatory micropillars.

    (A) Schematic diagram of thin micropillars used for force measurements (left) and thicker micropillars used for imaging protrusions (right). (B to E) OT1 CTLs were imaged by confocal microscopy on micropillars bearing H2-Kb-OVA and ICAM1. z-projection images (top views) are shown above with sagittal views below. Dashed lines [cyan in (B), (D), and (E); white in (C)] denote the slicing plane used for the sagittal images. (B) Time-lapse montage of a representative CTL expressing Lifeact-GFP, with micropillars shown in red. (C) Fixed image of a representative CTL stained with phalloidin (to visualize F-actin) and antitubulin antibodies. Micropillars are shown in gray in the left images. (D) Above, time-lapse montage of a representative CTL expressing Lifeact-mApple and Lamp1-GFP. Below left, mean distance between the lytic granule cloud and the cell front, graphed against time. Below right, centralization factor analysis of Lamp1-GFP. In both graphs, time 0 denotes initial contact with the pillars and error bars indicate SEM. n = 10. (E) Fixed image of a representative CTL stained with anti-LFA1 antibodies, with micropillars shown in red. All scale bars, 2 μm. In (B) and (D), time in minutes:seconds is indicated in the upper left corner of each top-view image.

  • Fig. 2 Granule fusion occurs at the base of synaptic protrusions.

    (A) Schematic diagram showing lytic granule fusion (visualized by pHluorin-Lamp1) on micropillar arrays. (B to E) OT1 CTLs expressing pHluorin-Lamp1 were imaged by confocal (C) or lattice light-sheet (B, D, and E) microscopy on micropillars bearing H2-Kb-OVA and ICAM1. (B) Time-lapse montage of a representative CTL expressing Lifeact-mRuby2 and pHluorin-Lamp1, with micropillars shown in gray. z-projection images (top views) are shown above with sagittal views below. The white dashed line denotes the slicing plane used for the sagittal images. Yellow arrowheads indicate the fusion event. Time in minutes:seconds is indicated in the upper left corner of each top-view image. Scale bars, 2 μm. (C) Graph showing the vertical displacements of fusion events (Fus., cyan) relative to the plane of the pillar tops (dashed gray line) along with the corresponding position of the cell front (red, visualized with a fluorescent Fab fragment against CD45). n = 11 events. P values were calculated from two-tailed paired Student’s t test. (D) F-actin accumulation in the region of granule fusion in z-projection images of CTLs expressing Lifeact-mRuby2 and pHluorin-Lamp1. Graph shows the average Lifeact-mRuby2 intensity within a 1-μm-diameter circle centered on the fusion site, starting two time points before the fusion event and ending two time points after. (E) Below, mean normalized (Norm.) Lifeact-mRuby2 intensity derived from linescans vertically bisecting the midpoint of the granule fusion site. The dotted black line denotes the plane of the pillar tops. Above, a representative image used for the analysis, with the linescan region indicated by the dashed white line. Error bars in (D) and (E) denote SEM. n = 18 events.

  • Fig. 3 CK666 blocks protrusion formation.

    (A) TCR induction of actin polymerization through NPFs and Arp2/3. (B and C) OT1 CTLs expressing Lifeact-GFP were imaged by confocal microscopy on fluorescent micropillars bearing H2-Kb-OVA and ICAM1 in the presence of the indicated concentrations of CK666. (B) Time-lapse montages of representative CTLs, with micropillars shown in red. z-projection images (top views) are shown above with sagittal views below. Cyan dashed lines denote the slicing planes used for the sagittal images. Time in minutes:seconds is indicated in the upper right corner of each top-view image. Scale bars, 2 μm. (C) Lifeact-GFP enrichment in protrusions was quantified over time, with time 0 indicating initial contact with the pillars. n = 5 cells for each condition, with error bars denoting SEM. P values were calculated by two-tailed Student’s t test comparing each CK666 condition against vehicle control. *P < 0.05 and **P < 0.01, colored to match the appropriate CK666 concentration. DMSO, dimethyl sulfoxide.

  • Fig. 4 CK666 inhibits force exertion and cytotoxicity.

    (A and B) OT1 CTLs were labeled with a fluorescent anti-CD45 Fab, incubated with 150 μm of CK666 or vehicle control (DMSO), and imaged on narrow fluorescent micropillars coated with H2-Kb-OVA and ICAM1. (A) Time-lapse montages of representative CTLs showing pillar deflection. Time in minutes:seconds is indicated in the upper left corner of each image. Scale bars, 2 μm. (B) Total force exertion against pillar arrays was graphed versus time. Color bar above the graph indicates the P value for each time point (two-tailed Student’s t test). n = 9 for DMSO, n = 10 for CK666. (C to E) RMA-s target cells were loaded with increasing concentrations of OVA and mixed with OT1 CTLs in the presence or absence of CK666 as indicated. (C) Specific lysis of RMA-s cells. (D) Lytic granule fusion measured by surface exposure of Lamp1 (gMFI, geometric mean fluorescence intensity). (E) CTL–target cell conjugate formation measured by flow cytometry. All error bars denote SEM. In (C) to (E), P values were calculated by two-tailed Student’s t test comparing each CK666 condition against vehicle control. *P < 0.05 and **P < 0.01, colored to match the appropriate CK666 concentration.

  • Fig. 5 WASP and WAVE2 control distinct subsets of protrusions.

    (A and B) OT1 CTLs expressing WASP-GFP or WAVE2-GFP were imaged by confocal microscopy on fluorescent micropillars bearing H2-Kb-OVA and ICAM1. (A) Time-lapse montages of representative CTLs, with micropillars shown in red. z-projection images (top views) are shown above with sagittal views below. Cyan dashed lines denote the slicing planes used for the sagittal images. Magenta arrowheads indicate representative lateral accumulations of WAVE2-GFP. (B) Centralization factor analysis of WASP-GFP and WAVE2-GFP, with time 0 denoting initial contact with the pillars. n = 6 for each cell type. (C and D) NT-CR, WASP-CR, and WAVE2-CR CTLs expressing Lifeact-GFP were imaged by confocal microscopy on fluorescent micropillars bearing H2-Kb-OVA and ICAM1. (C) Time-lapse montages of representative CTLs, with micropillars shown in red. z-projection images (top views) are shown above with sagittal views below. Cyan dashed lines denote the slicing planes used for the sagittal images. (D) Centralization factor analysis of Lifeact-GFP in NT-CR, WASP-CR, and WAVE2-CR OT1 CTLs, with time 0 denoting initial contact with the pillars. n = 6 for each cell type. In all montages, time in minutes:seconds is indicated in the upper left corner of each top-view image. Scale bars, 2 μm. In graphs, *P < 0.05 and **P < 0.01, calculated by two-tailed Student’s t test comparing WASP-GFP to WAVE2-GFP (B) or WASP-CR (red) and WAVE2-CR (green) to NT-CR (C). Error bars denote SEM.

  • Fig. 6 WASP and WAVE2 depletion induce distinct functional phenotypes.

    (A and B) NT-CR, WASP-CR, and WAVE2-CR OT1 CTLs were labeled with a fluorescent anti-CD45 Fab and imaged on narrow fluorescent micropillars coated with H2-Kb-OVA and ICAM1. (A) Time-lapse montages of representative CTLs showing pillar deflection. Time in minutes:seconds is indicated in the upper left corner of each top-view image. Scale bars, 2 μm. (B) Total force exertion against pillar arrays was graphed versus time. Color bar above each graph indicates the P value for each time point (two-tailed Student’s t test). (C) Histogram showing the distribution of strong deflections as a function of radial distance from the center of the IS. n = 10 for each cell type in (B) and (C). (D to F) RMA-s target cells were loaded with increasing concentrations of OVA and mixed with NT-CR, WASP-CR, or WAVE2-CR OT1 CTLs. (D) Specific lysis of RMA-s cells. (E) Lytic granule fusion measured by surface exposure of Lamp1. (F) CTL–target cell conjugate formation measured by flow cytometry. All error bars denote SEM. In (D) to (F), *P < 0.05 and **P < 0.01, calculated by two-tailed Student’s t test comparing WASP-CR (red) and WAVE2-CR (green) to NT-CR.

  • Fig. 7 WASP controls target cell deformation at the IS.

    (A) Schematic diagram of a CTL deforming an adherent target cell. (B and C) NT-CR, WASP-CR, and WAVE2-CR OT1 CTLs expressing Lifeact-GFP were applied to cultures of OVA-loaded endothelial target cells expressing iRFP670 and imaged using lattice light-sheet microscopy. (B) Left: Time-lapse montages of representative “vertically” oriented synapses, with z-projection images (top views) shown above and sagittal views below. Cyan dashed lines denote the slicing planes used for the sagittal images. In z-projection images, target cells are visualized by surface representation. Two z-projections are shown for each time point; Lifeact-GFP is shown on the left and the outline of the CTL of interest is shown on the right. Time in minutes:seconds is indicated in the upper left corner of each sagittal image. Scale bars, 2 μm. Yellow arrowheads denote protrusive structures in the NT-CR CTL that invade the target cell space. Right: Target IS volume graphed against time, with time 0 denoting IS initiation. Each line corresponds to one CTL–target cell conjugate. (C) Graph of minimum target IS volume values achieved during the first 400 s of conjugate formation. n = 7 for each cell type. Error bars denote SEM. P value was calculated by two-tailed Student’s t test.

  • Fig. 8 Cytolytic mechanopotentiation by WASP-dependent synaptic protrusions.

    Diagram of the cytolytic IS showing peripheral WAVE2-dependent protrusions and central WASP-dependent protrusions. Red arrows denote force exertion.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/33/eaav5445/DC1

    Materials and Methods

    Fig. S1. F-actin accumulates at the leading edges of synaptic protrusions.

    Fig. S2. Centralization factor analysis.

    Fig. S3. PTEN depletion enhances F-actin accumulation in protrusions.

    Fig. S4. Effects of CK666 on T cell activation.

    Fig. S5. WASP and WAVE2 depletion induce distinct functional phenotypes.

    Fig. S6. CTLs physically manipulate the target cell surface.

    Table S1. Data used to generate all graphs.

    Movie S1. CTLs form dynamic F-actin–rich protrusions on stimulatory micropillars.

    Movie S2. Granule fusion occurs at the base of synaptic protrusions.

    Movie S3. Protrusion formation by vehicle-treated CTLs.

    Movie S4. Protrusion formation by CK666-treated CTLs.

    Movie S5. Force exertion by vehicle-treated CTLs.

    Movie S6. Force exertion by CK666-treated CTLs.

    Movie S7. WAVE2-GFP accumulates in peripheral synaptic protrusions.

    Movie S8. WASP-GFP accumulates in central synaptic protrusions.

    Movie S9. Protrusive behavior of NT-CR CTLs.

    Movie S10. Protrusive behavior of WASP-CR CTLs.

    Movie S11. Protrusive behavior of WAVE2-CR CTLs.

    Movie S12. Force exertion by NT-CR CTLs.

    Movie S13. Force exertion by WASP-CR CTLs.

    Movie S14. Force exertion by WAVE2-CR CTLs.

    Movie S15. Target cell manipulation by NT-CR CTLs.

    Movie S16. Target cell manipulation by WASP-CR CTLs.

    Movie S17. Target cell manipulation by WAVE2-CR CTLs.

  • Supplementary Materials

    The PDF file includes:

    • Materials and Methods
    • Fig. S1. F-actin accumulates at the leading edges of synaptic protrusions.
    • Fig. S2. Centralization factor analysis.
    • Fig. S3. PTEN depletion enhances F-actin accumulation in protrusions.
    • Fig. S4. Effects of CK666 on T cell activation.
    • Fig. S5. WASP and WAVE2 depletion induce distinct functional phenotypes.
    • Fig. S6. CTLs physically manipulate the target cell surface.
    • Legends for movies S1 to S17

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Data used to generate all graphs.
    • Movie S1 (.mp4 format). CTLs form dynamic F-actin–rich protrusions on stimulatory micropillars.
    • Movie S2 (.mp4 format). Granule fusion occurs at the base of synaptic protrusions.
    • Movie S3 (.mp4 format). Protrusion formation by vehicle-treated CTLs.
    • Movie S4 (.mp4 format). Protrusion formation by CK666-treated CTLs.
    • Movie S5 (.mp4 format). Force exertion by vehicle-treated CTLs.
    • Movie S6 (.mp4 format). Force exertion by CK666-treated CTLs.
    • Movie S7 (.mp4 format). WAVE2-GFP accumulates in peripheral synaptic protrusions.
    • Movie S8 (.mp4 format). WASP-GFP accumulates in central synaptic protrusions.
    • Movie S9 (.mp4 format). Protrusive behavior of NT-CR CTLs.
    • Movie S10 (.mp4 format). Protrusive behavior of WASP-CR CTLs.
    • Movie S11 (.mp4 format). Protrusive behavior of WAVE2-CR CTLs.
    • Movie S12 (.mp4 format). Force exertion by NT-CR CTLs.
    • Movie S13 (.mp4 format). Force exertion by WASP-CR CTLs.
    • Movie S14 (.mp4 format). Force exertion by WAVE2-CR CTLs.
    • Movie S15 (.mp4 format). Target cell manipulation by NT-CR CTLs.
    • Movie S16 (.mp4 format). Target cell manipulation by WASP-CR CTLs.
    • Movie S17 (.mp4 format). Target cell manipulation by WAVE2-CR CTLs.

    Files in this Data Supplement:

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