Blood, Vol. 114, Issue 17, 3693-3706, October 22, 2009

Cytolytic T cells induce ceramide-rich platforms in target cell membranes to initiate graft-versus-host disease
Blood Rotolo et al. 114: 3693

Supplemental materials for: Rotolo et al

Proliferation assays
To measure proliferation in response to mitogen, splenocytes harvested from asmase+∕+ or asmase−∕− hosts 21 days post transplant were treated with RBC lysis buffer (PBS + 0.15 M NH4Cl +10 mM KHCO3 + 0.1 M Na2EDTA) for 5 min, and incubated in quadruplicate in 96-well plates at 2 × 105 cells per well in 200 µL RPMI cell culture medium supplemented with 10% FBS ± 2.5 µg/mL conA for 72 hrs. For the final 20 hrs of incubation, 1 µCi/well of 3Hthymidine (NEN, Boston, MA) was added. Cells were harvested with a Filtermate 196 harvester (Packard, Meriden, CT), fixed with 70% ethanol, and 3Hthymidine incorporation measured on a Topcount NXT microscintillation counter (Packard, Meriden, CT). To measure proliferation in response to alloantigen, effector T cells were prepared as described above and incubated for 96 hrs in the presence of allogeneic (B6)-, syngeneic (LP/J)-, or third-party (Balb/c)-irradiated (2000 cGy) RBC-lysed splenocytes at an effector-to-target ratio of 2:1.

To measure the ability of asmase−∕− splenocytes to induce Balb/c T cells to proliferate, nylon wool-enriched, RBC-lysed Balb/c splenocytes (1 × 105) were incubated for 5 days at a 1:1 ratio with 20Gy irradiated C57BL/6 asmase+∕+ or asmase−∕− splenocytes. On day 5, 3Hthymidine was added and proliferation assessed as above.

Isolation of sphingolipid microdomains
Membrane sphingolipid microdomains were isolated by discontinuous sucrose density gradient centrifugation as described by Lisanti et al.1 Briefly, 0.8–1.5 × 107 Jurkat cells were washed with H/S, and treated with 1 µg/ml CH-11 a-Fas antibody for 2 minutes as described.2 Cells were lysed with 2 ml of MES-buffered saline containing 1% TX-100 using a cell homogenizer. Lystates were overlaid with 2 ml of 90% sucrose in MES-buffered saline, followed by 4 ml of 30% sucrose and 4 ml of 5% sucrose. The gradient was centrifuged at 200,000× g using a Beckman SW-40 rotor for 16 hrs, and 1 ml fractions were collected from the top of the gradient. 30–40 µl aliquots of each gradient were separated on a 12% SDS-PAGE gel. Proteins were transferred onto a nitrocellulose membrane, blocked with 3% non-fat milk for 60 min at room temperature. Membranes were blotted with α-Flotillin-1 (1:250 dilution) or α-Fas Apo-1–3 (1:500 dilution) antibodies for 60 min at room temperature, washed with TBST buffer containing 0.1% Tween-20, and thereafter incubated with HRP-conjugated α-mouse antibody for 60 min. Blots were washed for 30 min in TBST and developed using the ECL chemiluminescence kit (Amersham Pharmacia).

REFERENCES

1. Lisanti MP, Sargiacomo M, Scherer PE. Purification of caveolae-derived membrane microdomains containing lipid-anchored signaling molecules, such as GPI-anchored proteins, H-Ras, Src-family tyrosine kinases, eNOS, and G-protein alpha-, beta-, and gamma-subunits. Methods Mol Biol. 1999; 11651–60.
2. Cremesti A, Paris F, Grassme H, et al. Ceramide enables fas to cap and kill. J Biol Chem. 2001; 276(26):23954–23961.
3. Rotolo JA, Zhang J, Donepudi M, Lee H, Fuks Z, Kolesnick R. Caspase-dependent and -independent activation of acid sphingomyelinase signaling. J Biol Chem. 2005; 280(28):26425–26434.
4. Grassme H, Cremesti A, Kolesnick R, Gulbins E. Ceramide-mediated clustering is required for CD95-DISC formation. Oncogene. 2003; 22(35):5457–5470.
5. Grassme H, Schwarz H, Gulbins E. Molecular mechanisms of ceramide-mediated CD95 clustering. Biochem Biophys Res Commun. 2001; 284(4):1016–1030.
6. Grassme H, Jekle A, Riehle A, et al. CD95 signaling via ceramide-rich membrane rafts. J Biol Chem. 2001; 276(23):20589–20596.
7. Fanzo JC, Lynch MP, Phee H, et al. CD95 rapidly clusters in cells of diverse origins. Cancer Biol Ther. 2003; 2(4):392–395.
8. Gajate C, Mollinedo F. The antitumor ether lipid ET-18-OCH(3) induces apoptosis through translocation and capping of Fas/CD95 into membrane rafts in human leukemic cells. Blood. 2001; 98(13):3860–3863.
9. Delmas D, Rebe C, Micheau O, et al. Redistribution of CD95, DR4 and DR5 in rafts accounts for the synergistic toxicity of resveratrol and death receptor ligands in colon carcinoma cells. Oncogene. 2004; 23(55):8979–8986.
10. Eramo A, Sargiacomo M, Ricci-Vitiani L, et al. CD95 death-inducing signaling complex formation and internalization occur in lipid rafts of type I and type II cells. Eur J Immunol. 2004; 34(7):1930–1940.
11. Hueber AO, Bernard AM, Herincs Z, Couzinet A, He HT. An essential role for membrane rafts in the initiation of Fas/CD95-triggered cell death in mouse thymocytes. EMBO Rep. 2002; 3(2):190–196.
12. Lacour S, Hammann A, Grazide S, et al. Cisplatin-induced CD95 redistribution into membrane lipid rafts of HT29 human colon cancer cells. Cancer Res. 2004; 64(10):3593–3598.
13. Scheel-Toellner D, Wang K, Singh R, et al. The death-inducing signalling complex is recruited to lipid rafts in Fas-induced apoptosis. Biochem Biophys Res Commun. 2002; 297(4):876–879.
14. Muppidi JR, Siegel RM. Ligand-independent redistribution of Fas (CD95) into lipid rafts mediates clonotypic T-cell death. Nat Immunol. 2004; 5(2):182–189.
15. Chamberlain LH. Detergents as tools for the purification and classification of lipid rafts. FEBS Lett. 2004; 559(1–3):1–5.
16. Lisanti MP, Sargiacomo M, Scherer PE. Purification of caveolae-derived membrane microdomains containing lipid-anchored signaling molecules, such as GPI-anchored proteins, H-Ras, Src-family tyrosine kinases, eNOS, and G-protein alpha-, beta-, and gamma-subunits. Methods Mol Biol. 1999; 11651–60.

Files in this Data Supplement:

  • Table S1. Necropsy results of SCID-asmase+∕+ and SCID-asmase−∕− animals receiving allogeneic BM and T cells (PDF, 39.7 KB) -
    SCID mice administered LP BM and T cells were sacrificed when exhibiting reduced motility and an agonal breathing pattern. Spinal cord, brain, lung, liver and small intestines were harvested and fixed in paraformaldehyde (Kaplan-Meier survival curves of these animals are shown in Fig. 1D). Note that the SCID-asmase−∕− mice displayed evidence of advanced Nieman-Pick disease (NPD) in spinal cord, brain, and lung samples, including Purkinje cell loss and pneumonia assessed by the Laboratory of Comparative Pathology, an MSKCC core-facility. Onset of NPD occurs typically after 6–9 months in single-mutant asmase−∕− mice. Liver and small intestines were scored for GvHD, including lymphoid infiltration into the small intestines and liver, intestinal crypt apoptosis, and hepatic venular and bile duct injury by a clinical pathologist.

  • Table S2. Serum hematologic recovery is not affected by recipient asmase genotype (PDF, 49.8 KB) -
    Hematologic profile of serum harvested from asmase+∕+ and asmase−∕− C57BL/6 recipients 21 days following LP/J BM and T cells was performed using a Hemavet950 Multi-species Hematology Analyzer (Drew Scientific, Oxford CT). wbc, white blood cells; ne, neutrophils; ly, lymphocytes; mo, monocytes; eo, eosinophils; ba, basophils; rbc, red blood cells; Hb, hemoglobin; mcv, mean cell volume; mch, mean cell hemoglobin; mchc, mean cell hemoglobin concentration; plt, platelets; mpv, mean platelet volume. Data (meanSEM) represent 3–8 determinations from two independent experiments.

  • Figure S1. T-cell proliferative capacity remains intact in asmase−∕− hosts (JPG, 244 KB) -
    Thymidine incorporation assay measuring proliferation of splenic T cells harvested from C57BL/6asmase+∕+ or C57BL/6asmase−∕− recipients of donor LP BM and T cells in response to syngeneic (LP), or allogeneic (Balb/c) splenocytes, or mitogen (ConA). Data (mean±SEM) represent triplicate determinations from three independent experiments.





  • Figure S2. Concanamycin A depletes allogeneic activated T-cell perforin (JPG, 158 KB) -
    Flow cytometry of intracellular perforin expression in CD3+ T cells harvested from a C57BL/6 recipient of LP/J BM and T cells, using PE-conjugated anti-mouse perforin eBioMAK-D (eBioscience). Green: 0 ng/ml CMA; Blue: 200 ng/ml CMA. Data are representative of three independent experiments.





  • Figure S3. Host ASMase expression does not impact the ability to present antigen or activate donor CTLs ex vivo (JPG, 366 KB) -
    (A) Irradiated (20 Gy) asmase+∕+ or asmase−∕− splenocytes were cultured with nylon wool-purified BALB/c T cells for 5 days at a stimulator:target ratio of 1:1. On day 5, T cells were labeled with 3Hthymidine as described in Materials & Methods, and proliferation was assessed 4 hr thereafter. (B) Wild-type hepatocytes isolated as described in Fig. 4 were coincubated in complete medium for 16 hr with 2 × 106 in vivo activated CTLs harvested from lethally-irradiated asmase+∕+ or asmase−∕− C57BL/6 recipients 14–21 days following transplantation of LP BM + T cells. Apoptosis was quantified as in Fig. 4C. Data (mean±SEM) represent triplicate determinations from three independent experiments.





  • Figure S4. Fas colocalizes within ceramide-rich paltforms (JPG, 263 KB) -
    Representative confocal images of hepatocytes stimulated with alloactivated T cells for 10 min as described in Materials and Methods. Following stimulation, hepatocytes were fixed with 4% formalin-buffered phosphate, stained with anti-ceramide mAb and His6-tagged FasL, and detected using FITC–anti-mouse IgM and rabbit anti-His6 followed by Cy3–anti-rabbit IgM. Colocalization of red and green signal is depicted as yellow. Data are representative of three independent experiments.





  • Figure S5. GM1 colocalizes within ceramide-rich platforms during AICD (JPG, 70.2 KB) -
    Representative confocal images of asmase+∕+ and asmase−∕− splenocytes following 4hr induction of AICD with 10 ng/ml anti-CD3 as described in Materials and Methods. Cells were fixed with 4% formalin-buffered phosphate, and stained with DAPI, anti-ceramide mAb (detected using Cy3–anti-mouse IgM) and FITC-conjugated cholera toxin, and imaged as in Materials and Methods. Colocalization of red and green signal is depicted as yellow. Data are representative of three independent experiments.





  • Figure S6. Fas colocalizes within ceramide-rich platforms during AICD (JPG, 66.7 KB) -
    Representative confocal images of asmase+∕+ and asmase−∕− splenocytes were treated and imaged as in Fig. S5. Cells were stained with DAPI, anti-ceramide mAb and His6-tagged FasL, and detected using FITC–anti-mouse IgM and rabbit anti-His6 followed by Cy3–anti-rabbit IgM. Colocalization of red and green signal is depicted as yellow. Note that the yellow signal detected within the asmase−∕− splenocyte represents less than 5% of the yellow signal acquired from the asmase+∕+ splenocyte. Data are representative of three independent experiments.





  • Figure S7. Splenic T-cell Fas expression is independent of ASMase (JPG, 97.7 KB) -
    Flow cytometry of (A) Fas and (B) FasL expression in asmase+∕+ and asmase−∕− C57BL/6 splenic T cells induced to undergo AICD as described in Materials and Methods. Left panel, orange: activated asmase+∕+, blue: activated asmase−∕−; Right panel, red: control asmase+∕+, orange: activated asmase+∕+, blue: control asmase−∕−, green: activated asmase−∕−. Data are representative of three independent experiments.





  • Figure S8. Triton X-100 titration reveals Fas translocates into ceramide-rich platforms in type II Jurkat cells (JPG, 122 KB) -
    Divergent opinions have been published regarding the sub-cellular localization of Fas within plasma membrane GEMs. Whereas all groups find Fas within GEMs in type I cells, differences exist in the reported distribution in type II cells. Studies from our laboratory3–7 and others8–13 have demonstrated Fas localization to GEMs plays a critical role in Fas-induced apoptosis in all T lymphocytes, irrespective of their type I or II status, while Siegel et al.14 argue that Fas localizes to GEMs only in type I cells. Specifically, these authors reported that Fas resides constitutively in GEMs of type I SKW6.4 cells, while it is absent in GEMs isolated from unstimulated or anti-Fas activating antibody stimulated type II Jurkat cells. As published evidence indicates that excessive detergent may solubilize non-resident GEM proteins,15 potentially obscuring their in vivo locale, we titrated down the concentration of Triton X-100 used to isolate membrane GEMs. We confirm in (A) that Fas is constitutively present in GEMs of type I SKW6.4 cells. For these studies, 100 × 106 SKW6.4 cells were stimulated with 50 ng/ml anti-Fas CH-11 activating antibody or an irrelevant IgM, and GEMs were isolated in MBS buffer containing 0.3% Triton X-100 followed by sucrose density flotation, as described.16 Fas was enriched in low-density, flotillin-containing GEM fractions 4–6 of SKW6.4 cells irrespective of anti-Fas stimulation. In (B), we show that when 50 × 106 Jurkat cells were stimulated with 50 ng/ml anti-Fas CH-11 activating antibody or irrelevant IgM, and GEMs were isolated as in (A), localization depended on the concentration of Triton X-100 used to solubilize the plasma membrane. We replicate the data of Siegel et al.14 using 0.5% Triton X-100 (lower panel) showing that Fas fails to accumulate in GEMs at 2 min post-stimulation. However, when Triton X-100 was reduced to 0.3% (top panels), Fas translocation from bulk membrane fractions 9–12 to the flotillin-enriched light density fractions of type II Jurkat cells was detected. Data represent 1 of 2 studies in (A), and 1 of 3 studies in (B).