CALCIUM RESPONSE OF HELPER T-LYMPHOCYTES TO ANTIGEN-PRESENTING CELLS IN A SINGLE-CELL ASSAY

We developed a dynamic, single-cell assay involving alternating differential interference contrast and fluorescence microscopy, together with digital imaging, for both viewing the physical interaction of live helper T lymphocytes (Th cells) with antigen-presenting cells (APCs) and monitoring the increases in the intracellular free calcium concentration of the Th cell, an early event in Th cell activation. We obtained Th-APC conjugates by allowing the Th cells to migrate toward and interact with APCs that either settled nearby or had been micromanipulated in close proximity to the Th cells. Th cell motility played an important role in initiating Th-APC contacts but not in determining the Th cell calcium response. We found that the intracellular calcium responses of individual Th cells are heterogeneous and an all-or-none phenomenon, independent of antigen concentration. However, the fraction of Th-APC conjugates involving responding Th cells is an increasing function of the antigen concentration. Finally, we measured some characteristics of the developing Th-APC contact area. We used all of these data together with previously developed mathematical models to estimate that only 1 to 20 major histocompatibility class II-antigen complexes are required in the initial Th-APC contact area to elicit a Th cell calcium response.