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.
Pay Per Article - You may access this article (from the computer you are currently using) for 1 day for US$37.50
Regain Access - You can regain access to a recent Pay per Article purchase if your access period has not yet expired.