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Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis
Published on: June 27, 2020
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ARTC2.2/P2RX7 Signaling during Cell Isolation Distorts Function and Quantification of Tissue-Resident CD8+ T Cell and
Henrique Borges da Silva1, Haiguang Wang1, Lily J Qian1
1Center for Immunology, Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN 55455.
Journal of Immunology (Baltimore, Md. : 1950)
|February 20, 2019
Summary
Blocking the ARTC2.2/P2RX7 pathway improves recovery of invariant NKT (iNKT) cells and CD8+ tissue-resident memory T cells (T cells) from mouse tissues. This blockade is crucial for accurate analysis and functional studies of these immune cells.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Invariant NKT (iNKT) cells and CD8+ tissue-resident memory T (TRM) cells express P2RX7, a receptor sensitive to extracellular ATP.
- High ATP or NAD levels during tissue preparation can trigger P2RX7-mediated cell death, potentially skewing analysis of these T cell populations.
- The expression patterns of ARTC2.2 and P2RX7 on different iNKT subsets and TRM, and the impact on recovery from various tissues, were not fully understood.
Purpose of the Study:
- To comprehensively analyze ARTC2.2 and P2RX7 expression in iNKT and CD8+ T cells across diverse tissues.
- To investigate the effect of ARTC2.2 blockade on the recovery and viability of iNKT and TRM cells during tissue preparation.
- To determine the necessity of ARTC2.2/P2RX7 axis blockade for preserving the functionality and proliferation of these T cell populations.
Main Methods:
- Flow cytometry analysis of ARTC2.2 and P2RX7 expression on iNKT subsets and CD8+ T cells in various tissues.
- Utilizing nanobody-mediated ARTC2.2 antagonism to block ARTC2.2 activity.
- Employing short-term P2RX7 blockade as a comparative method.
- Assessing cell recovery, viability, and proliferation post-blockade.
Main Results:
- NKT1 cells and CD8+ TRM showed higher ARTC2.2 and P2RX7 expression compared to NKT2, NKT17, and circulating memory subsets.
- ARTC2.2 blockade significantly enhanced the recovery of viable NKT1 and TRM cells from nonlymphoid tissues.
- Blockade of the ARTC2.2/P2RX7 pathway was essential for maintaining the functionality, viability, and proliferative capacity of these T cells.
- Direct P2RX7 blockade also improved TRM recovery, though to a lesser extent than ARTC2.2 antagonism.
Conclusions:
- Short-term in vivo blockade of the ARTC2.2/P2RX7 axis substantially improves the flow cytometry-based phenotyping and enumeration of murine iNKT and TRM cells from nonlymphoid tissues.
- This blockade strategy is a critical advancement for accurate functional studies of iNKT and TRM populations.
- The findings provide a vital methodological improvement for immunological research involving these specific T cell subsets.
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