Fig 2: In vivo imaging of allorejection in the anterior chamber of the eye (ACE) visualized by CTLs from GzmB-TFP-KI mice.(A) Binocular image of a mouse eye with islets of Langerhans from DBA/2 mice transplanted into the anterior chamber of the eye (ACE) 4 days prior to the image. Islets can be identified as small white spots in the range between 100–300 µm diameter located on top of the iris. Inset shows cultured islets before transplantation. (B) Confocal images of a pancreatic islet inside the ACE fixed and sliced at POD14. The outline defines the boundary of the islet of Langerhans localize on the iris. Green cells are cytotoxic T lymphocytes marked by endogenously expressed GzmB-mTFP having infiltrated the islet during the immune response. Antibody staining of CD8+ (blue) and CD4+ (magenta) T cells are shown. (C) In vivo confocal microscopy of an islet during allorejection in the ACE (POD14). White line marks the boundary of pancreatic islet toward the ACE lumen. Faint green (excitation 458 nm wavelength) and red (excitation 561 nm wavelength) signals are autofluorescence. CTLs marked by GzmB-mTFP (green, excitation 488 nm) infiltrated the islet during the immune response. (D) Velocity of 81 migrating CTLs tracked from panel (C) after imaging for >30 min. Mean is shown as a plus symbol, median as a line. See also Figure 5—video 1. (E) Two-photon image of GzmB-mTFP positive cells from the corneal area. Individual vesicles inside the CTL are easily identified (inset). See also Figure 5—video 2.

Fig 3: Islets of Langerhans were incubated with CTLs derived from GzmB-mTFP KI (left) and GzmB-mTFP KI/Perforin KO mice (middle) and imaged by confocal microscopy.CTLs started to infiltrate the islets and kill them, which resulted in a detachment of islets from the culture dish. Quantification of the detachment at day 1 and day 2 of co-culture (right panel) shows a clear killing deficit in GzmB-mTFP KI/Perforin KO compared to GzmB-mTFP KI alone.

Fig 4: Immuno-isolation of two classes of cytotoxic granules from CTLs using an anti-synaptobrevin2 (Syb2) antibody.a Scheme of Syb2-mRFP positive cytotoxic granules bound to the antibody-conjugated Dynabeads Protein G following immunoprecipitation (IP). b Representative western blots of IP6 and IP8 with input (IN, 2%), supernatant (SN, 2%) and IP (40%) volume. Protein markers for plasma membrane (Na+/K+-ATPase), cytotoxic granules (Syb2-mRFP, GzmB) and cytoskeleton (ß-actin) were used (NIP = 4). c 500 µl of sucrose fraction 6 and 8 from Syb2/GzmB DKI CTLs were pooled and centrifuged on glass coverslip (Ngradient = 1, nimages = 3). The confocal images are shown as single channel (green and magenta) and merge image. d Correlative light and electron microscopy (CLEM) images of immuno-isolated organelles of Syb2/GzmB DKI CTLs (NIP = 1, nimages = 7 and 4 for IP6 and IP8, respectively). Left to right: SIM fluorescence images of IPs are shown for Syb2 (magenta) and GzmB (green), and the corresponding organelle is represented in the TEM images. Upper and lower panel represent IP6 and IP8, respectively. Source data are provided as a Source data file.

Fig 5: Multi core granules contain supramolecular attack particles (SMAPs).a SIM images of WT (left) and GzmB KI (right) cells on supported lipid bilayers (SLB) represented in 3D (top) and 2D (bottom). Cells were stimulated on anti-CD3 antibody coated lipids for 90 min and stained with anti-GzmB (red) antibody after fixation. White arrows point to secreted SMAPs. Nmouse = 1; nimages = 3 and 6 for WT and GzmB KI, respectively. b SIM images of one resting WT CTL expressing GzmB-mCherry and Thrombospondin 1 (TSP-1)-GFPspark seeded on poly-ornithine coated coverslip. Nmouse = 1; ncells = 14. c SIM images of secreted SMAPs on SLB from GzmB KI CTLs pre-incubated with wheat germ agglutinin (WGA)-647 to label intracellular, glycoprotein-rich compartments. Secreted SMAPs were stained with anti-TSP-1 antibody. Nmouse = 1, nimages = 16. d Analysis of SCG and MCG percentage in resting cells (left) and stimulated cells at the IS (right). To trigger synapse formation CTLs were seeded on anti-CD3 coated coverslips. GzmB single positive granules were identified as SCGs, while GzmB and WGA double positive granules as MCGs. Black lines represent mean ± sem; ncells = 16; entire cells and ncells = 35; IS. Student’s t-test was used to compare values; *** Two-tailed P-value < 0.001. e Line profile (top) of one representative MCG (left) and SCG (right) acquired with SIM to depict granule diameter measurement method. Inset: SCG contained only GzmB (green) while the MCG contained WGA-647 and GzmB (yellow). Scale bar, 0.2 µm. Bottom scatter dot plot of granule diameter measured as the full width at half maximum in the line plots. Black line represents mean and gray line median. Nmice = 3; ncells = 20; ngranules = 275 and 156 for MCG and SCG, respectively. Mann–Whitney U test was used to compare values; ***p < 0.001. f CLEM images of a representative stimulated CTL as in (b) pre-incubated with WGA-647 and stained with DAPI. Shown are SIM images and its corresponding TEM overlaid image. In the enlarged TEM image, arrows point to different fluorescent proteins using the color-code of the upper panels. White arrow indicate the MCG marked in all three channels. Nmouse = 1; ncells = 18. Source data are provided as a Source data file.