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