An In Vitro Technique to Study T Cell Interaction with Supported Planar Lipid Bilayers

Take a flow slide containing a supported lipid bilayer, or SLB.

The SLB is conjugated to specific ligands — intercellular adhesion molecule 1 or ICAM-1 and anti-CD3  antibodies — labeled with fluorophores.

Add T cells and fluorophore-tagged anti-CD107a Fab — the antigen-binding fragment of an antibody against the degranulation marker.

The T cell receptor-CD3 or TCR-CD3 complex binds to the anti-CD3 antibody, triggering the TCR.

Triggering-induced inside-out signaling activates integrins on the surface, which bind to the immobilized ICAM-1.

The conjugation induces cytoskeletal reorganization — microtubule re-arrangement, and actin polymerization that leads to lateral spreading of the cell on the SLB surface.

The spreading allows more ligand-receptor interactions to form a supramolecular activation cluster or SMAC, creating an immune synapse.

T cell lytic granules move along microtubules and fuse with the plasma membrane, exposing degranulation markers on the cell surface, which bind to the anti-CD107a Fab.

Using a fluorescence microscope, visualize the immune synapse enriched in TCR-CD3 complexes, integrins, and degranulation markers.