Controlling duration of contact between T cells and antigen-presenting cells

A new method which allows precise control of the duration of contact betwee n T cells and antigen-presenting cells (APCs) has been developed. A glass c overslip coated with poly-L-lysine, and then with T cells, was placed at th e base of a cylindrical well, and the well was filled with liquid medium. A round coverslip, on which APCs were adhered, was supported on the surface of the medium by surface tension, cell-side down. By withdrawing medium fro m four capillary holes neat. the base of the well, the coverslip could be l owered to initiate contact between T cells and APCs at a defined time zero. The contact was broken at desired time points by re-introducing medium int o the well in order to separate the two coverslips. Each cell type remained adherent to its original surface after separation for all contact times st udied. The T cells were monitored for intracellular calcium mobilization us ing the fluorescent dye, Fura-2. Contact durations of less than 1 min did n ot trigger calcium signals. Contact durations of 3 and 5 min induced strong calcium signals. Breaking the contact caused a rapid decrease in intracell ular calcium levels. This method of cell manipulation allows precise contro l of the duration of contact of T cells with APCs, while keeping the cells under continuous observation. The measurements so obtained provide a quanti tative understanding of the dynamics of early T cell activation. (C) 2001 E lsevier Science B.V. All rights reserved.