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.

Begin by using polypropylene scissor-type forceps to submerge glass coverslips in freshly prepared acidic piranha solution for 25 to 30 minutes. At the end of the wash, rinse the coverslips seven times in a fresh volume of ultra-pure water for each wash, and set the wet coverslips aside to let the remaining water roll off the clean glass.

When the coverslips are dry, aliquot two microliters of the final liposome mixture, precisely, in the center of a self-adhesive slide channel within a sterile flow hood, and immediately but carefully, align one clean and dry coverslip with the slide to allow the coverslip to be gently lowered onto the sticky side of the slide, and use the outer ring of the polypropylene scissor-type forceps to apply gentle pressure to the peripheral contact of the coverslip with the slide, making sure that the slip is tightly attached to the slide to prevent leakage. Then, turn the slide over, and use a permanent marker to draw four dots around the bilayer on the external side of the slide assembly.

Before the first injection, designate one port of the channel as the entry port and the other as the exit port, maintaining this designation throughout the experiment. To avoid bubble formation, insert the end of the pipette tip directly into the entry port until you feel the rubber seal of the slide channel being penetrated with the tip.

Slowly, fill the channel with 50 microliters of warm assay buffer. Inject 200 microliters of nickel(II) chloride in casein-blocking solution into the entry port, and remove 50 microliters of the buffer from the exit port. After a 45-minute incubation, remove the excess casein solution from the exit port.

Inject 100 microliters of an ICAM-1 and streptavidin solution into the entry port for a 45-minute incubation at room temperature. At the end of the incubation, remove the excess protein solution from the exit port, and wash the bilayer two times with 100 microliters of assay buffer per wash. Then, label the bilayers with 100 microliters of fluorophore-conjugated anti-CD3 antibody for 45 minutes at room temperature, followed by two washes in 100 microliters of assay buffer per wash, as demonstrated.

To image T cell-bilayer interactions, place the slide on the 37-degree Celsius heated stage of a Total Internal Reflection Fluorescence, or TIRF, microscope, and adjust the stage using the ink marks to center the bilayer onto the 100 power objective.

For granule release imaging, add fluorescence-conjugated anti-CD107a antibody Fab fragments to the CD4 T cell suspension, and resuspend the labeled cells in 50 microliters of assay buffer for injection into the entry port of the slide channel. Then, select the appropriate number of experimental fields, and record images of each field once every minute for 30 minutes after the injection.