Mapping the sensitivity of T cells with an optical trap: Polarity and minimal number of receptors for Ca2+ signaling

Contact with antigen-presenting cells (APCs) initiates an activation cascad e within T lymphocytes, including a rise in cytosolic calcium, lymphokine p roduction, and cell division. Although T cell-APC physical contact is requi red for an immune response, little is known about the patterns of cellular interactions and their relation to activation. Calcium imaging combined wit h an optical trap enabled the T cell contact requirements and polarity to b e investigated at the single-cell level. APCs or anti-CD3 mAb-coated beads were trapped with a laser and placed at different locations along the T cel l, which has a polarized appearance defined by the shape and direction of c rawling, T cells were 3-fold more sensitive to APC contact made at the lead ing edge of the T cell than with contact made at the tail. Anti-CD3 mAb-coa ted 6-mu m beads induced calcium signaling with approximate to 10-fold high er frequency and approximate to 4-fold shorter latency on contact with the leading edge of the T cell than on contact with the trailing edge. Alterati ons in antibody density (2 to 500 per mu m(2)) and bead size (1 to 6 mu m i n diameter) were used to determine the spatial requirements and the minimal number of receptors which must be engaged to transmit a positive signal. T cell response percentage, latency, and calcium-signaling pattern (transien t vs. sustained or oscillatory) depended on antibody density on the bead. T he presence of approximate to 170 anti-CD3 mAb within the contact area elic ited a detectable T cell calcium response. We propose here that engagement of no more than 340 T cell receptors (approximate to 1% of the total on the cell) is sufficient to initiate Ca2+ signaling. The minimal contact area w as approximate to 3 mu m(2).