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Updated: Aug 15, 2026
A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins
Published on: March 22, 2012
Fluorogenic Granzyme A Substrates Enable Real-Time Imaging of Adaptive Immune Cell Activity
Zhiming Cheng1, Emily J Thompson1, Lorena Mendive-Tapia1
1Centre for Inflammation Research, The University of Edinburgh, Edinburgh, UK.
Abstract:
Cytotoxic immune cells, including T lymphocytes (CTLs) and natural killer (NK) cells, are essential components of the host response against tumors. CTLs and NK cells secrete granzyme A (GzmA) upon recognition of cancer cells; however, there are very few tools that can detect physiological levels of active GzmA with high spatiotemporal resolution. Herein, we report the rational design of the near-infrared fluorogenic substrates for human GzmA and mouse GzmA. These activity-based probes display very high catalytic efficiency and selectivity over other granzymes, as shown in tissue lysates from wild-type and GzmA knock-out mice. Furthermore, we demonstrate that the probes can image how adaptive immune cells respond to antigen-driven recognition of cancer cells in real time.
Insights
New near-infrared probes detect active granzyme A (GzmA) from cytotoxic immune cells like T lymphocytes and NK cells. These tools enable real-time imaging of anti-cancer immune responses.
Area of Science:
- Immunology
- Biochemistry
- Molecular Biology
Background:
- Cytotoxic immune cells, such as cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, are crucial for anti-tumor immunity.
- These cells release granzyme A (GzmA) upon encountering cancer cells.
- Existing methods lack the spatiotemporal resolution to detect active GzmA at physiological levels.
Purpose of the Study:
- To develop novel near-infrared (NIR) fluorogenic substrates for detecting active human and mouse granzyme A (GzmA).
- To create tools with high catalytic efficiency and selectivity for GzmA.
- To enable real-time imaging of immune cell activity in cancer contexts.
Main Methods:
- Rational design of NIR fluorogenic probes specific for GzmA.
- In vitro validation using tissue lysates from wild-type and GzmA knockout mice.
- Demonstration of probe utility in imaging adaptive immune cell responses to cancer cells.
Main Results:
- Successfully designed and synthesized highly efficient and selective NIR fluorogenic substrates for both human and mouse GzmA.
- Probes exhibited excellent selectivity, distinguishing GzmA activity from other granzymes.
- The probes enabled real-time visualization of adaptive immune cell responses during antigen-driven cancer cell recognition.
Conclusions:
- Developed novel activity-based probes for sensitive and specific detection of GzmA.
- These probes offer unprecedented spatiotemporal resolution for studying cytotoxic immune cell function.
- The technology provides a powerful new tool for investigating anti-tumor immunity and immune cell dynamics in real-time.
