Fig. 7: Both classes of CGs, MCG and SCG, fuse at the IS and are killing competent. | Nature Communications

Fig. 7: Both classes of CGs, MCG and SCG, fuse at the IS and are killing competent.

From: Identification of distinct cytotoxic granules as the origin of supramolecular attack particles in T lymphocytes

Fig. 7

a Total internal reflection fluorescence microscopy (TIRFM) snapshot images of GzmB KI (green) CTL stained with WGA-647 (magenta) on supported lipid bilayer. White solid arrows point to one SCG fusion event and white open arrows point to one MCG fusion event over time. Blue arrows indicate the time of fusion. Pictures in frame show the MCG prior to fusion (before) and releasing several SMAPs (after). Note Images were recorded at 5 Hz. Nmice = 3, ncells = 16. b Representative granule secretion profile of SCG and MCG from the cell shown in (a). The loss of fluorescence indicates the release of soluble GzmB and the remaining/increased WGA signal indicates the secreted SMAPs on the supported lipid bilayer. c Percent of secreted MCG and SCG from experiments as shown in (a); Nmice = 3, ncells = 25. d TIRFM snapshot images of a WT CTL expressing GzmB-mCherry (red) and TSP-1-GFPspark (green) on glass coverslip coated with an anti-CD3 antibody. White arrows point to one MCG secretion event, blue arrows indicate the time of secretion. Frame shows the MCG before and after fusion. Images were recorded at 10 Hz. Nmice = 2, ncells = 17. e Target cell killing assay. Confocal images of P815 target cells seeded coverslips covered with MCG (fraction 6) and SCG (fraction 8). Cells were stained with anti-cleaved caspase3 antibody (green) to determine cell apoptosis at two different time points. Cells seeded on poly-ornithine alone were used as control. DAPI staining (blue) was used to count the cells. f Time dependent analysis of the killing assay is shown as a violin plot with superimposed box plot and outliers. The boundaries of the box represent the 25th to 75th percentiles, while the whiskers are 10th/90th percentiles. The white dot corresponds to the median. Nmouse = 1, ncells = 300. N, n correspond to the number of independent experiments and analyzed cells, respectively. One-way ANOVA on rank was used to compare values within time point group; ***p < 0.001. Source data are provided as a Source data file. Scale bar in framed pictures are 0.5 µm.

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