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Updated: May 29, 2025
Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions
Published on: June 29, 2016
Measuring interaction force between T lymphocytes and their target cells using live microscopy and laminar shear flow
Sophie Goyard1, Amandine Schneider2, Jerko Ljubetic2
1Institut Pasteur, Université Paris Cité, Diagnostic Test Innovation & Development Core Facility, Paris, France.
Insights
This study introduces a new method to measure the forces of immunological synapses, finding that adenomatous polyposis coli (APC) is crucial for T cell adhesion to tumor cells, impacting anti-tumor immunity.
Area of Science:
- Immunology
- Cell Biology
- Biophysics
Background:
- Understanding the immunological synapse is key to immune response.
- Existing methods for measuring cell-cell interaction forces are limited, often analyzing one pair at a time.
Purpose of the Study:
- To develop and validate a novel laminar shear flow-based method for measuring immunological synapse rupture forces.
- To investigate the role of adenomatous polyposis coli (APC) in the stability of cytotoxic T cell-tumor cell interactions.
Main Methods:
- A laminar shear flow system was used to apply controlled dragging forces to immobilized T cell-target cell conjugates.
- Synapse rupture forces were measured by monitoring cell detachment at increasing flow rates across a population of cell pairs.
- Cells were collected and sorted based on measured rupture forces.
Main Results:
- The method allows for the measurement of synapse rupture forces ranging from 10 pN to 20 nN for hundreds of cell pairs in a single experiment.
- Reduced expression of adenomatous polyposis coli (APC) was found to impair T cell adhesion to tumor target cells.
- This suggests a significant impact of APC mutations on anti-tumor immune defense.
Conclusions:
- The developed shear flow method provides a high-throughput approach to quantify immunological synapse stability.
- Adenomatous polyposis coli (APC) plays a critical role in maintaining the stability of cytotoxic T cell-tumor cell interactions.
- APC's function in T cell adhesion highlights its importance in effective anti-tumor immune responses.
Abstract:
Understanding the immunological synapse formation and dynamics can be enriched by measuring cell-cell interaction forces and their kinetics. Microscopy imaging reveals structural organization of the synapse, while physical methods detail its mechanical construction. Various techniques have been reported for measuring forces needed to rupture the interface between a T lymphocyte and its target cell but most of them measure one pair at a time. We describe here a laminar shear flow-based method that exerts dragging forces on T cell-target cells pairs immobilized on the surface of a flow chamber. Increasing flow rate allows us to observe the detachment of hundreds of cell conjugates on the wide field of a light transmission microscope. Monitoring precisely the flow rate gradient exerted on T cells readily yields synapse rupture measurements. Dragging forces are measured at the point of rupture as a linear function of the flow speed in minutes from 10pN to 20nN for each cell pair among a statistically representative cell population in the whole field of view of a single experiment. The output cells can be collected in multi-well plate sorted in the increasing order of rupture forces. We used this approach to unveil the involvement of the cytoskeleton regulator adenomatous polyposis coli (APC) in the stability of immunological synapses formed between human cytotoxic T cell and tumor target cells. APC is a polarity regulator and tumor suppressor associated with familial adenomatous polyposis and colorectal cancer. Reduced APC expression impairs T cell adhesion with tumor target cells suggesting an impact of APC mutation in anti-tumor immune defense.
