This study investigates the formation of immunological synapses between activated human antigen-presenting cells (APCs) and helper T lymphocytes. The process involves the interaction of receptors and the exchange of signaling molecules, crucial for T cell activation.
Immune cells communicate with each other via the interaction of multiple receptors, forming a specialized junction—an immunological synapse. This synapse serves as a site for exchanging signaling molecules essential for an immune response.
To study immunological synapse formation in vitro, begin with a basement matrix-coated microwell slide containing an adherent culture of activated human antigen-presenting cells, APCs.
The activated APCs present antigens bound to major histocompatibility complex or MHC-II molecules on their surface, essential for T cell activation. These cells are labeled with a blue fluorescent dye for easy identification.
Treat the wells with recombinant human helper T lymphocytes. These helper T lymphocytes express green fluorescent protein-tagged membrane proteins — CD63 found in secretory vesicles. This enables the tracking of secretory vesicles.
Lymphocytes' T cell receptors interact with MHC-antigen complexes on the APCs, bringing the helper T lymphocytes near the antigen-presenting cells, resulting in conjugation.
Upon conjugation, co-stimulatory signaling molecules on the APCs interact with lymphocyte receptors—forming a cluster around the conjugation site, creating an immunological synapse.
Synapse formation triggers the reorganization of lymphocyte cytoskeleton, involving the repositioning of cytoskeletal elements—actin filaments and microtubules. This reorganization facilitates the movement of vesicles toward the synapse near the APCs.
Observe the cells under a fluorescent microscope. Record the movement of green vesicles within the helper T cell toward the blue APCs, confirming immunological synapse formation.
Retrieve the chamber slides containing the cell tracker blue-labeled Staphylococcus Enterotoxin E-pulsed adhered Raji cells from the incubator. Carefully aspirate the culture medium from each well, one by one, with a pipette placed in a corner of the well.
Immediately replace the medium with 200 microliters of the resuspended Jurkat cells in cell culture medium. If a time-lapse is being performed, quickly proceed to the microscope.
Locate the chamber slide on the microscope stage incubator and select some appropriate fields.
Prepare the microscope in incubation chamber prior to imaging. After the Jurkat cells have been added to each well containing the Raji cells, quickly locate the microwell chamber slide on the preheated microscope stage incubator, and select some XY positions.
If only an end-point experiment is planned, incubate the chamber slide at 37 degrees Celsius with 5% carbon dioxide for 1 to 2 hours. After the culture period, check for conjugate formation and subsequently fix the conjugates, as outlined in the text protocol.
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