CONTROLLING RECEPTOR-LIGAND CONTACT TO EXAMINE KINETICS OF T-CELL ACTIVATION

A method for controlling the contact of cell-surface receptors with im mobilized ligands has been developed. Cells are trapped in an asymmetr ic liquid film that can be quantitatively thinned by reducing the film 's capillary pressure. Ligands adsorbed to the liquid-solid interface are forced into increasingly tighter contact with the cells as the air -liquid interface is drawn down. Controlling the degree of thinning al lows study of repulsive forces, and controlling its time course produc es a definite time 0 for analyzing signal transduction. This system wa s tested by examining the time course of calcium mobilization in T cel ls upon activation with anti-CD3 antibody at different dilutions and i onic strengths. The averaged calcium transient of the responding cells was essentially the same for each condition. However, the fraction of responding cells decreased with anti-CD3 dilution, and indicated that the critical ligand density for T cell activation lies between simila r to 35 and 70 molecules of anti-CD3 per mu m(2). Decreasing the mediu m's ionic strength from the normal value of 157 mM to 57 mM did not af fect either the average calcium response profile or the fraction of re sponding cells, but strongly affected receptor-ligand contact, decreas ing the percent of spontaneous activation from 38% to 5%. Such an impo sed decrease sets the stage for film thinning to impose much greater c ontrol of receptor-ligand contact.