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Updated: May 29, 2025
Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry
Published on: January 7, 2019
A comprehensive guide to study the immunological synapse using imaging flow cytometry
Andrea Michela Biolato1, Liza Filali2, Max Krecke1
1Cytoskeleton and Cancer Progression, Department of Cancer Research, Luxembourg Institute of Health, Luxembourg, Luxembourg; Faculty of Science, Technology and Medicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg.
Insights
Cytotoxic lymphocytes form immunological synapses with cancer cells to eliminate them. New imaging flow cytometry methods quantify subcellular changes within these synapses, aiding cancer research.
Area of Science:
- Immunology
- Cell Biology
- Cancer Research
Background:
- Cytotoxic lymphocytes, including T cells and NK cells, eliminate malignant cells via direct contact.
- The lytic immunological synapse is crucial for signal integration and directed cytotoxicity.
- Cancer cells develop defenses to evade lymphocyte-mediated killing.
Purpose of the Study:
- To present protocols for examining subcellular processes within the immunological synapse using imaging flow cytometry.
- To quantify spatial redistribution of key cellular components during lymphocyte-cancer cell interactions.
- To establish methods for generating and analyzing specific effector-target cell conjugates.
Main Methods:
- Utilized imaging flow cytometry to analyze subcellular events in immunological synapses.
- Developed protocols for forming conjugates between cancer cell lines (MDA-MB-231, K-562) and NK cells (NK-92MI, primary human NK cells).
- Detailed procedures for evaluating synaptic polarization of actin cytoskeleton, CD63+ vesicles, MHC class I, and MTOC.
Main Results:
- Established quantitative imaging flow cytometry protocols for analyzing immunological synapses.
- Demonstrated the ability to assess subcellular dynamics within effector-target cell conjugates.
- Provided a framework for dissecting synaptic defense mechanisms employed by cancer cells.
Conclusions:
- Imaging flow cytometry offers a powerful approach to study the dynamics of the immunological synapse.
- These methods facilitate the quantitative analysis of cellular interactions critical for cancer immunity.
- The described protocols can advance understanding of lymphocyte cytotoxicity and cancer immune evasion strategies.
Abstract:
Cytotoxic lymphocytes, such as cytotoxic T cells and natural killer (NK) cells, are instrumental in the recognition and eradication of pathogenic cells, notably those undergoing malignant transformation. Cytotoxic lymphocytes establish direct contact with cancer cells via the formation of a specialized cell-cell junction known as the lytic immunological synapse. This structure serves as a critical platform for lymphocytes to integrate surface signals from potential cancer cells and to direct their cytolytic apparatus toward the confirmed targets. Conversely, cancer cells evolve synaptic defense strategies to evade lymphocyte cytotoxicity. This chapter delineates protocols using imaging flow cytometry to examine and quantify important subcellular processes occurring within cytotoxic lymphocytes and cancer cells engaged into an immunological synapse. These processes encompass the spatial redistribution of cytoskeletal components, vesicles, organelles and cell surface molecules. We specifically describe methods to generate and select conjugates between MDA-MB-231 breast cancer cells or K-562 leukemic cells and either the NK-92MI cell line or primary human NK cells. In addition, we detail procedures to evaluate the synaptic polarization of the actin cytoskeleton, CD63-positive vesicular compartments, MHC class I molecules, as well as the microtubule-organizing center in effector cells.
