Supplementary Data Acid sphingomyelinase is a critical regulator of cytotoxic granule secretion by primary T lymphocytes Jasmin Herz, Julian Pardo, Hamid Kashkar, Michael Schramm, Elza Kuzmenkina, Erik Bos, Katja Wiegmann, Reinhard Wallich, Peter J. Peters, Stefan Herzig, Elmon Schmelzer, Martin Krönke, Markus M. Simon, and Olaf Utermöhlen
Fig. S1 pm m m n m pm 5nm Fig. S1. Immuno-electronmicroscopic analysis of cytotoxic granules in and CD8 + T lymphocytes. LCMV-specific CD8 + T cells were immunomagnetically enriched from splenic single cell suspensions of and mice on day 8 after I.v. infection with LCMV. The cells were labeled with rabbit anti mouse gzmb-specific serum and protein A-gold (1nm). (Arrow: granule; Arrowhead: gzmb immuno-gold label; m: mitochondrion; n: nucleus; pm: plasma membrane)
Fig. S2 a GzmA + granules per cell 6 5 4 3 2 1 b Cells (%) 2 15 1 5 1 2 3 4 5 6 7 8 9 1 11 12 Granules per cell Fig. S2. Characterization secretory lysosomes in and CD8 + T lymphocytes. (a, b) CD8 + T cells were intracellularly stained on day 8 after infection with unlabeled rabbit anti-mouse gzma immune serum and goat anti-rabbit serum conjugated with FITC and embedded in ProLong Gold Antifade Reagent (Invitrogen). Using an Olympus IX81 fluorescence microscope, individual cells were photographed in the focus plane with the highest fluorescence intensity. GzmA + granules were counted in 167 cells of each genotype. Shown are the (a) mean ± standard error and (b) the frequency distribution of the number of granules per CTL as cumulative data from 2 experiments.
Fig. S3 a IFN-γ (pg / ml 1 6 cells 24h) 4 3 2 1 * P<.5 8 9 Time after infection (d) b IFN-γ secreting cells per 1 5 CD8 + T cells 4 3 2 1 8 9 Time after infection (d) c d IFN-γ + CD8 + cells (%) 14 12 1 8 6 4 2 8 9 Time after infection (d) RANTES (pg / ml 1 6 cells 24h) 6 4 2 8 9 Time after infection (d) Fig. S3. Selective secretory defects of CD8 + T cells from LCMV-infected mice. Mice were i.v. infected with 1 5 IU of LCMV. On day 8 and 9, CD8 + T cells were prepared from single cell suspensions and were stimulated with 1-6 M GP 33-41. After 24h IFN-γ (a) and RANTES (d) were measured in the supernatants by ELISA. Shown are the means and standard errors of four independent experiments. (b) The frequency of CD8 + T cells secreting IFN-γ was analyzed by ELISpot assay at an effector:target ratio of 1:1 in response to B6SV fibroblasts loaded with the epitope GP 33-41. Shown are the means and standard errors of four independent experiments. (c) The percentage of CD8 + CTL intracellularly accumulating IFN-γ was analyzed by flow cytometry. Shown are the means and standard errors of cumulative data from 4 (day 8) or 3 (day 9) independent experiments.
Fig. S4 Talin GzmA Merge #1 #2 #1 #2 Fig. S4. Formation of the outer ring of adhesion molecules at the immunological synapse between CD8 + T cells and target cells. and CTL were conjugated with L121 target cells in the presence of anti-cd3 at an effector:target ratio of 1:1 after 1 min of co-culture. Cells were stained with antibodies against talin (goat anti-talin (Santa Cruz Biotechnology) followed by goat anti-mouse 594 (Molecular Probes)) and with gzma-specific immune serum as described in methods. Shown are representative examples from two independent experiments.
Fig. S5 CTL conjugated to target cells (%) 6 5 4 3 2 1 5 1 2 3 Time after target contact (min) 4.5% 28.2% 31.1% 31.4% 38.2% CD8 4.4% 29.7% 27.6% 3.% 33.4% CD19 Fig. S5. Formation of conjugates between CD8 + T cells and target cells. and CD8 + T cells were incubated with L121 target cells in the presence of 3 µg/ml anti-cd3 at an effector:target ratio of 1:1 at 37 C. After the indicated times, conjugates were fixed, stained and analyzed by flow cytometry. CD8 + T cells were stained with APC-conjugated CD8- and targets were stained with PE-conjugated CD19-specific antibody. Indicated is the percentage of CD8 + T cells conjugated to target cells. Shown are means ± S.E. cumulated from four independent experiments (above) and individual dot plots from one of these experiments (below).
Fig. S6 PE PS PC SM LPC Origin Fig. S6. Lipid composition of plasma membranes of CD8 + T cells from and mice. Phospholipids were extracted from CD8 + T cells and analysed by TLC as described previously 45. On the left, the positions of phospho- or sphingolipid standards are indicated: PE, phosphatidylethanolamine; PS, phosphatidylserine; PC, phosphatidylcholine; SM, sphingomyelin; LPC, lysophosphatidylcholine.
Fig. S7 a b Indo bound / Indo unbound 2 1 α-cd3 α-higg 6 4 2 Thapsigargin 24 36 Time (sec) 12 24 36 Time (sec) Fig. S7. Release of intracellular Ca 2+ in and CD8 + T cells. 1 1 7 splenocytes were labelled with PE-conjugated anti-cd8 and subsequently with the calcium sensitive Fluorescence dye Indo-1 according to the instructions of the manufacturer (Molecular Probes) in the presence of Pluronic Acid for 45 minutes. After that calcium variation was analysed in a LSRII FACS (BD) by continuous measurement for 1 minutes at 37 C and data analysed with the FACSDiva software (BD). (a) TCR was ligated by CD3-specific mab 145.2C11 (α CD3) and cross-linked by anti hamster IgG (α higg). (b) To assess the overall contents of intracellular Ca 2+ stores thapsigargin was added at a concentration of 1µM.
Fig. S8 a ASMase activity (pmol/µg protein) 5 4 3 2 1 * ** ** *** * * ** *** b Specific lysis (%) 5 4 3 2 1 control 5 µm 25µM 5µM n.s. P<.1 Time (min): 5 136 5 136 5 136 5 136 Imipramine: -- 5µM 25µM 5µM 2 4 6 8 1 Effector : target ratio Specific lysis (%) c 3 2 1 6 min 3 min 1 min 1 min min * * d GzmA activity (munits / h) 2 175 15 125 1 75 5 25 Control 5 µm 25 µm 5 µm * ** e Hexosaminidase activity (relative fluorescence units) 4 3 2 1 p<.1 p<.1 2 4 6 8 1 Effector : target ratio.15.3.6 1.2 2.5 5. Cell equivalents ( 1 5 ) Imipramine (µm): 5 25 5 2µg/ml α-cd3 f g h 5 CD17a + CD8 + T cells (%) 4 3 2 1 Control Imipramine 25µM Time (h): 1 2 5 1 2 5 1 2 5 1 2 5 Antibody: None α-cd3 IFN-γ (pg / ml 1 6 cells 24h) 5 4 3 2 1 5 25 5 Imipramine (µm ) RANTES (pg / ml 1 6 cells 24 h) 8 6 4 2 5 25 5 Imipramine (µm ) Fig. S8. CD8 + T cells treated with imipramine resemble the phenotype of CD8 + CTL continued on the following page
Legend to Fig. S8, continued CD8 + T cells treated with imipramine resemble the phenotype of CD8 + CTL. CD8 + CTL were prepared from mice on day 8 after i.v. infection with 1 5 IU of LCMV. (a) Treatment with imipramine results in CD8 + T cells in a time- and dose-dependent decrease of ASMase activity. The LCMV-specific cytotoxic activity of CD8 + CTL is reduced by imipramine in a dose- (b) and time-dependent fashion (c). Imipramine dose-dependently decreases the release of gzma- (d) and hexosaminidase-activity (e) by TCR-triggered wt CD8 + CTL. In (a, c, d) statistical significance between the treated sample and the respective untreated control is indicated by asterisks: *: P<.5; **: P<.1; ***: P<.1. (f) CD8 + CTL pretreated with imipramine were co-incubated with target cells and the kinetics of CD17a/Lamp1 cell surface exposure was analyzed by flow cytometry. CD8 + CTL were pretreated with imipramine and assayed for secretion of (g) IFN-γ and (h) RANTES. (f, g, h) show the representative results of each one out of three similar experiments.
Fig. S9 Diameter of gzma + vesicles (µm) 1.2 1..8.6.4.2. n.s. Height n.s. Width Fig. S9. Size of GzmA-positive granules in and CD8 + CTL before TCR-triggering. CD8 + T cells were prepared from or mice on day 8 after i.v. infection with 1 5 IU of LCMV. Cells were permeabilized, stained with rabbit anti-murine gzma serum followed by Alexa-594-coupled chicken Ab specific for rabbit IgG, and analysed by confocal microscopy. The size of the area of gzma-positive granular clusters at the immunologic synapse was measured with the Carl Zeiss Image Browser Version 3.5. Shown are cumulative data from three independent experiments for : n=57 and n=6 granules. Also indicated are means (red diamonds) ± S.E.
Fig. S1 #1 #2 #3 #4 target target GzmA Phase target CTL Phase GzmA target CTL Fig. S1. Polarization of gzma containing cytotoxic granules towards the immunological synapse in CD8 + CTL CD8 + T cells (indicated CTL ) were prepared from or mice on day 8 after i.v. infection with 1 5 IU of LCMV and co-incubated with L121 target cells (indicated target ) at an effector:target ratio of 1:1 in the presence of CD3-specific mab for 1 min. The cells were permeabilized and stained with gzma-specific antiserum and analysed by confocal microscopy. Four representative T cell-target cell conjugates with either or CD8 + CTL are shown. The vesicular or rimlike gzma-positive structures in cells are indicated by white solid arrows.
Fig. S11a PMA+Iono: - + Time (min) 5 1 15 3 6 Extracellular GzmB Fig. S11 GzmB cannot be detected on the surface of CD8 + T cells during granule secretion. CD8 + T cells were immununomagnetically enriched from spleens of day 8-immune or mice and stimulated with PMA and ionomycin. (a) At the indicated times of stimulation, cells were stained for extracellular gzmb (red lines) with PE-conjugated mab GB11 (Caltag) or isotype control (black lines) and analyzed by flow cytometry. (b) An aliquot of the PMA +ionostimulated cells described in (a) was additionally stained by a PE-conjugated amplifyer Ab against mouse IgG 1 (Molecular Probes).
Fig. S11b Time (min) 5 1 15 3 6 Extracellular GzmB
Fig. S12a SRB FM1-43 Phase #1 #2 #3 #4 #1 #2 #3 #4 b Fig. S12. Influx of polar tracers from the surrounding medium into granules after their fusion with the plasma membrane in response to TCR-stimulation of CD8 + CTL. CD8 + T cells were immununomagnetically enriched after restimulation of spleen cell cultures of day 8-immune or mice with 1 units/ml recombinant IL-2 for 2 days. CD8 + T cells were immersed in medium containing the polar tracers sulforhodamin B (SRB) and FM1-43 in chamber slides coated with (A) CD3- and CD28-specific mab or (B) isotype controls. Influx of extracellular medium into cytotoxic granules after TCR-triggered fusion of the granule s membrane with the plasma membrane was observed via two-photon microscopy by staining of the lumen and the membrane of the granules with SRB and FM1-43, respectively. Four representative CD8 + T cells stimulated with CD3/CD28 for 2 min are shown.
Fig. S13 2.5 pre-fusion post fusion 2. Diameter (µm) 1.5 1..5. Height Width Height Width 2.5 pre-fusion post fusion 2. Diameter (µm) 1.5 1..5. Height Width Height Width Fig. S13. Correlation between width and height of individual cytotoxic granules in and CD8 + CTL prior to and after fusion with the plasma membrane. In CD8 + T cells prepared from or mice on day 8 after i.v. infection with 1 5 IU of the LCM virus, the width (largest diameter in the x/y-plane) and height (diameter perpendicular to width) of individual cytotoxic granules were determined prior to (42 granules per genotype) and after fusion (number of granules: : n=26; wt: n=31) as described in the legend of Fig. 8. The corresponding width and height for each individual granule are connected by a line.
Fig. S14 CTL Target cell Target ASMase Ceramide Sphingomyelin Effector molecules Microtubule Cytotoxic granule MTOC Fig. S14. Deficiency in ASMase impairs contraction of cytotoxic granules fused to the plasma membrane in CD8 + T cells.. continued on the following page
Legend to Fig. S14, continued. Deficiency in ASMase impairs contraction of cytotoxic granules fused to the plasma membrane in CD8 + T cells. Left, schematic representation of a CD8 + CTL contacting a target cell at the immunologic synapse. The CTL contains several cytotoxic granules attached to microtubules or to the microtubule organizing centre (MTOC) prior to fusion with the plasma membrane at the immunologic synapse. Right, higher magnification schematic of boxed areas in (a). Top, prior to fusion with the plasma membrane, cytotoxic granules in and CD8 + T lymphocytes are of identical size and cannot be distinguished morphologically. After fusion with the plasma membrane, the granules in CD8 + CTL shrink by more than 8% of their initial volume and expel cytolytic effector molecules into the immunological synapse. During fusion, the extreme bending at the junction of the luminal leaflet of the granule with the outer leaflet of the plasma membrane introduces lateral gaps in the organization of the lipid packing. This increases ASMase activity both by increasing its penetration into the bilayer and by facilitating better access to its substrate sphingomyelin. Sphingomyelin hydrolysis to ceramide reduces the surface area of the inner leaflet of the granule leading within of the granule. In T cells, ceramide is not produced. As a result the surface area of the inner leaflet is not reduced; only limited shrinkage occurs and thus the contents of the granule are largely retained.
Suppl. Table 1. Expression of mrna transcripts encoding cytotoxic effector molecules in CD8 + T cells 8 days after infection with LCMV Number of RNA copies per 1 4 CD8 + T cells GzmA GzmB Perforin 2.3 1 5 5.9 1 5 1.2 1 4 Wild-type 1.8 1 5 6.4 1 5 1.5 1 4 CD8 + T cells were immunomagnetically enriched from the spleens of mice infected i.v. with 1 5 IU of LCMV 8 days prior to the assay. Copy numbers of RNA coding for gzma, gzmb, and perforin were determined as described in ONLINE METHODS.