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Updated: Jun 22, 2026
Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry
Published on: January 7, 2019
Numbers matter: quantitative and dynamic analysis of the formation of an immunological synapse using imaging flow
Fariyal Ahmed1, Sherree Friend, Thaddeus C George
1Immune Disease Institute, Harvard Medical School, 200 Longwood Avenue, Boston, Massachusetts 02115, USA.
Insights
Multispectral imaging flow cytometry visualized immune synapse formation. This technology revealed that CD3epsilon molecules stay longer at the synapse than Lck signaling molecules after T cell activation.
Area of Science:
- Immunology
- Cell Biology
- Biophysics
Background:
- T lymphocyte activation by antigen-presenting cells (APCs) leads to the formation of an immunological synapse.
- Key signaling and adhesion molecules redistribute to the T cell-APC interface during synapse evolution.
- Understanding the dynamics of molecular recruitment is crucial for deciphering immune synapse function.
Purpose of the Study:
- To visualize and quantify the recruitment dynamics of CD3epsilon and Lck signaling molecules during immune synapse formation.
- To utilize multispectral imaging flow cytometry for high-throughput analysis of T cell-APC interactions.
- To compare the residence times of CD3epsilon and Lck at the immunological synapse.
Main Methods:
- Multispectral imaging flow cytometry was employed to combine flow cytometry with imaging capabilities.
- Thousands of T cell/macrophage conjugates were analyzed at various experimental time points.
- Two independent methods were used to analyze the recruitment dynamics of Lck and CD3epsilon.
Main Results:
- The recruitment dynamics of Lck and CD3epsilon to the immune synapse were comparable following calcium-triggered T cell activation.
- CD3epsilon demonstrated significantly longer residence times at the synapse compared to Lck.
- CD3epsilon exhibited residence times exceeding 8 minutes at the synapse.
Conclusions:
- Multispectral imaging flow cytometry is a powerful tool for studying immune synapse dynamics.
- CD3epsilon and Lck exhibit distinct residence times at the immunological synapse, suggesting differential roles in signaling and stability.
- The findings provide insights into the temporal regulation of molecular events within the immune synapse.
Abstract:
Activation of T lymphocytes by antigen-presenting cells (APC) results in the formation of an immunological synapse. Following contact with the target cell, key signaling and adhesion molecules polarize within minutes to hours to the T cell-APC interface. Multispectral imaging flow cytometry, a new technology which combines flow cytometry with imaging, was used to visualize and quantify the recruitment of the CD3epsilon and Lck signaling molecules during the evolution of an immune synapse. Using this technology, thousands of T cell/macrophage conjugates could be analyzed for each experimental time point. Following Ca++ triggered T cell activation, the dynamics of Lck and CD3epsilon recruitment to the synapse, analyzed by two independent methods, were comparable. However, CD3epsilon exhibited longer residence times (>8 min) at the synapse than Lck.
