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Role of V-ATPase a3-Subunit in Mouse CTL Function

Praneeth Chitirala, Keerthana Ravichandran, Claudia Schirra, Hsin-Fang Chang, Elmar Krause, Uli Kazmaier, Marcel A. Lauterbach and Jens Rettig
J Immunol May 15, 2020, 204 (10) 2818-2828; DOI: https://doi.org/10.4049/jimmunol.1901536
Praneeth Chitirala
*Cellular Neurophysiology, Center for Integrative Physiology and Molecular Medicine, Saarland University, 66421 Homburg, Germany;
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Keerthana Ravichandran
*Cellular Neurophysiology, Center for Integrative Physiology and Molecular Medicine, Saarland University, 66421 Homburg, Germany;
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Claudia Schirra
*Cellular Neurophysiology, Center for Integrative Physiology and Molecular Medicine, Saarland University, 66421 Homburg, Germany;
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Hsin-Fang Chang
*Cellular Neurophysiology, Center for Integrative Physiology and Molecular Medicine, Saarland University, 66421 Homburg, Germany;
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Elmar Krause
*Cellular Neurophysiology, Center for Integrative Physiology and Molecular Medicine, Saarland University, 66421 Homburg, Germany;
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Uli Kazmaier
†Organic Chemistry, Saarland University, 66123 Saarbrücken, Germany; and
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Marcel A. Lauterbach
‡Molecular Imaging, Center for Integrative Physiology and Molecular Medicine, Saarland University, 66421 Homburg, Germany
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Jens Rettig
*Cellular Neurophysiology, Center for Integrative Physiology and Molecular Medicine, Saarland University, 66421 Homburg, Germany;
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Key Points

  • The a3-subunit of V-ATPase acidifies cytotoxic granules in mouse CD8+ T lymphocytes.

  • Neutralization of luminal pH leads to altered morphology of cytotoxic granules.

  • Knockdown of a3-subunit disturbs trafficking of cytotoxic granules.

Abstract

CTLs release cytotoxic proteins such as granzymes and perforin through fusion of cytotoxic granules (CG) at the target cell interface, the immune synapse, to kill virus-infected and tumorigenic target cells. A characteristic feature of these granules is their acidic pH inside the granule lumen, which is required to process precursors of granzymes and perforin to their mature form. However, the role of acidic pH in CG maturation, transport, and fusion is not understood. We demonstrate in primary murine CTLs that the a3-subunit of the vacuolar-type (H+)–adenosine triphosphatase is required for establishing a luminal pH of 6.1 inside CG using ClopHensorN(Q69M), a newly generated CG-specific pH indicator. Knockdown of the a3-subunit resulted in a significantly reduced killing of target cells and a >50% reduction in CG fusion in total internal reflection fluorescence microscopy, which was caused by a reduced number of CG at the immune synapse. Superresolution microscopy revealed a reduced interaction of CG with the microtubule network upon a3-subunit knockdown. Finally, we find by electron and structured illumination microscopy that knockdown of the a3-subunit altered the diameter and density of individual CG, whereas the number of CG per CTL was unaffected. We conclude that the a3-subunit of the vacuolar adenosine triphosphatase is not only responsible for the acidification of CG, but also contributes to the maturation and efficient transport of the CG to the immune synapse.

Footnotes

  • This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 894 A10, SFB 894 A12, SFB 894 P1, and IRTG 1830) (to J.R.).

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    a.u.
    arbitrary unit
    CG
    cytotoxic granule
    GzmB
    granzyme B
    IS
    immune synapse
    KI
    knock-in
    mTFP
    monomeric teal fluorescent protein
    ns-siRNA
    nonsilencing small interfering RNA
    RT
    room temperature
    SIM
    structured illumination microscopy
    SiR
    silicon rhodamine
    siRNA
    small interfering RNA
    STED
    stimulated emission depletion
    SybKI
    synaptobrevin2-mRFP knock-in
    TIRFM
    total internal reflection fluorescence microscopy
    V-ATPase
    vacuolar-type (H+)–adenosine triphosphatase.

  • Received December 31, 2019.
  • Accepted February 26, 2020.
  • Copyright © 2020 by The American Association of Immunologists, Inc.
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The Journal of Immunology: 204 (10)
The Journal of Immunology
Vol. 204, Issue 10
15 May 2020
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Role of V-ATPase a3-Subunit in Mouse CTL Function
Praneeth Chitirala, Keerthana Ravichandran, Claudia Schirra, Hsin-Fang Chang, Elmar Krause, Uli Kazmaier, Marcel A. Lauterbach, Jens Rettig
The Journal of Immunology May 15, 2020, 204 (10) 2818-2828; DOI: 10.4049/jimmunol.1901536

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Role of V-ATPase a3-Subunit in Mouse CTL Function
Praneeth Chitirala, Keerthana Ravichandran, Claudia Schirra, Hsin-Fang Chang, Elmar Krause, Uli Kazmaier, Marcel A. Lauterbach, Jens Rettig
The Journal of Immunology May 15, 2020, 204 (10) 2818-2828; DOI: 10.4049/jimmunol.1901536
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