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Acid sphingomyelinase is a key regulator of cytotoxic granule secretion by primary T lymphocytes

Nature Immunology volume 10, pages 761–768 (2009)Cite this article

Abstract

Granule-mediated cytotoxicity is the main effector mechanism of cytotoxic CD8+ T cells. We report that CD8+ T cells from acid sphingomyelinase (ASMase)-deficient (ASMase-KO) mice are defective in exocytosis of cytolytic effector molecules; this defect resulted in attenuated cytotoxic activity of ASMase-KO CD8+ T cells and delayed elimination of lymphocytic choriomeningitis virus from ASMase-KO mice. Cytolytic granules of ASMase-KO and wild-type CD8+ T cells were equally loaded with granzymes and perforin, and correctly directed to the immunological synapse. In wild-type CD8+ T cells, secretory granules underwent shrinkage by 82% after fusion with the plasma membrane. In ASMase-KO CD8+ T cells, the contraction of secretory granules was markedly impaired. Thus, ASMase is required for contraction of secretory granules and expulsion of cytotoxic effector molecules.

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Figure 1: Reduced T cell-mediated LCMV-specific immune responses in ASMase-KO mice.
Figure 2: ASMase-KO CD8+ T cell perforin-mediated cytotoxicity is impaired.
Figure 3: Reduced release of cytotoxic effector molecules by ASMase-KO CD8+ T cells.
Figure 4: Intracellular localization of ASMase, gzmA, Lamp1 and RANTES in LCMV-specific CD8+ CTLs.
Figure 5: Proper polarization and fusion of cytotoxic granules at the immunological synapse in ASMase-KO CD8+ CTLs.
Figure 6: Identical secretion of fluorescent dextran molecules from cytotoxic granules of ASMase-KO and wild-type (WT) CD8+ T lymphocytes.
Figure 7: Impaired exocytosis of cytotoxic granules by ASMase-KO CD8+ T cells.
Figure 8: Impaired contraction of cytotoxic granules fused to the plasma membrane in ASMase-KO CD8+ T cells.

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Acknowledgements

We thank R. Kolesnick (Memorial Sloan-Kettering Cancer Center) and D.R. Green and T. Lin (La Jolla Institute for Allergy and Immunology) for ASMase-KO mice; H. Hengartner (Institute for Experimental Immunology, University Hospital Zürich) for perforin-deficient mice; U. Karow, A. Ekiciler and B. Hub for expert technical assistance; and E.-M. Menke, D. Weßler and J. van de Burgwal for expert work at the animal facility. Supported by the Deutsche Forschungsgemeinschaft (SFB 670 and SPP1110 to O.U. and M.K.), an Alexander von Humboldt foundation fellowship (J.P.), Fundacion Aragon I+D (J.P.) and the Christiane Nüsslein-Volhard Foundation (E.K.).

Author information

Author notes

  1. Julian Pardo

    Present address: Present address: Departamento Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Spain.,

  2. Jasmin Herz and Julian Pardo: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute for Medical Microbiology, Immunology and Hygiene, Medical Center, University of Cologne, Germany

    Jasmin Herz, Hamid Kashkar, Michael Schramm, Katja Wiegmann, Martin Krönke & Olaf Utermöhlen

  2. Center for Molecular Medicine University of Cologne, Cologne, Germany

    Jasmin Herz, Hamid Kashkar, Michael Schramm, Stefan Herzig, Martin Krönke & Olaf Utermöhlen

  3. Metschnikoff Laboratory, Max Planck Institute for Immunobiology, Freiburg, Germany

    Julian Pardo & Markus M Simon

  4. Department of Pharmacology, University of Cologne, Cologne, Germany

    Elza Kuzmenkina & Stefan Herzig

  5. The Netherlands Cancer Institute, Amsterdam, The Netherlands

    Erik Bos & Peter J Peters

  6. Institute for Immunology, University of Heidelberg, Germany

    Reinhard Wallich

  7. Max Planck Institute for Plant Breeding Research, Cologne, Germany

    Elmon Schmelzer

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Contributions

J.H. performed and evaluated experiments and provided the respective probes to the other coauthors. J.P., R.W. and M.M.S. measured activities of perforin and granzymes A and B. H.K. and M.S. performed confocal microscopy. E.K and S.H. performed and evaluated the patch-clamp experiments. E.B. and P.J.P. performed the electron microscopy. K.W. analyzed the lipids and measured ASMase activity. E.S. performed the two-photon microscopy. M.K. and O.U. designed and coordinated the study, performed (O.U.) and evaluated experiments and wrote the manuscript.

Corresponding authors

Correspondence to Martin Krönke or Olaf Utermöhlen.

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Herz, J., Pardo, J., Kashkar, H. et al. Acid sphingomyelinase is a key regulator of cytotoxic granule secretion by primary T lymphocytes. Nat Immunol 10, 761–768 (2009). https://doi.org/10.1038/ni.1757

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