Role of V-ATPase a3-Subunit in Mouse CTL Function
Praneeth Chitirala1, Keerthana Ravichandran1, Claudia Schirra1
1Cellular Neurophysiology, Center for Integrative Physiology and Molecular Medicine, Saarland University, 66421 Homburg, Germany.
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.
Insights
The vacuolar adenosine triphosphatase a3-subunit acidifies cytotoxic granules (CG) in T cells. This acidification is crucial for CG maturation, transport to the immune synapse, and effective target cell killing.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Cytotoxic T lymphocytes (CTLs) kill target cells by releasing cytotoxic proteins from cytotoxic granules (CG).
- CGs have an acidic internal pH essential for processing cytotoxic proteins like granzymes and perforin.
- The precise role of this acidic pH in CG maturation, transport, and fusion remains unclear.
Purpose of the Study:
- To investigate the role of the vacuolar-type (H+) -adenosine triphosphatase a3-subunit in cytotoxic granule (CG) acidification.
- To determine the impact of a3-subunit function on CG maturation, transport, and fusion with the target cell.
Main Methods:
- Utilized primary murine CTLs and a novel CG-specific pH indicator, ClopHensorN(Q69M), to measure luminal pH.
- Employed knockdown of the a3-subunit to assess its functional importance.
- Applied total internal reflection fluorescence microscopy and superresolution microscopy to analyze CG fusion and interaction with microtubules.
- Used electron and structured illumination microscopy to examine CG morphology.
Main Results:
- The a3-subunit of vacuolar-type (H+) -adenosine triphosphatase is essential for maintaining an acidic pH (6.1) within CGs.
- Knockdown of the a3-subunit significantly reduced target cell killing and CG fusion (>50%).
- Reduced CGs at the immune synapse and impaired interaction with the microtubule network were observed upon a3-subunit knockdown.
- CG morphology, including diameter and density, was altered, though the total number of CGs per CTL remained unchanged.
Conclusions:
- The a3-subunit is critical for cytotoxic granule acidification.
- Acidification by the a3-subunit is vital for proper CG maturation and efficient transport to the immune synapse.
- This process is essential for effective CTL-mediated cytotoxicity.
