Mapping the sensitivity of T cells with an optical trap: polarity and minimal number of receptors for Ca(2+) signaling

Abstract

Contact with antigen-presenting cells (APCs) initiates an activation cascade within T lymphocytes, including a rise in cytosolic calcium, lymphokine production, and cell division. Although T cell-APC physical contact is required for an immune response, little is known about the patterns of cellular interactions and their relation to activation. Calcium imaging combined with an optical trap enabled the T cell contact requirements and polarity to be 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 cell, which has a polarized appearance defined by the shape and direction of crawling. T cells were 3-fold more sensitive to APC contact made at the leading edge of the T cell than with contact made at the tail. Anti-CD3 mAb-coated 6-micrometer beads induced calcium signaling with approximately 10-fold higher frequency and approximately 4-fold shorter latency on contact with the leading edge of the T cell than on contact with the trailing edge. Alterations in antibody density (2 to 500 per micrometer(2)) and bead size (1 to 6 micrometer in 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 (transient vs. sustained or oscillatory) depended on antibody density on the bead. The presence of approximately 170 anti-CD3 mAb within the contact area elicited a detectable T cell calcium response. We propose here that engagement of no more than 340 T cell receptors (approximately 1% of the total on the cell) is sufficient to initiate Ca(2+) signaling. The minimal contact area was approximately 3 micrometer(2).

Figure 1

Optical trapping of anti-CD3 mAb coated-bead identifies functional T cell polarity. (A) Bright field and fluorescence images are shown as an overlay. Acetoxymethyl esters of Oregon Green and Fura Red were preincubated with T cells for 90 min. Cells produced a true-color emission shift from red to green when [Ca²⁺]_i was elevated. Three photomultipliers collected green fluorescence, red fluorescence, and bright field images simultaneously. FITC-conjugated anti-CD3 mAb coated-beads (6.2 μm in diameter) are shown presented by the optical trap to either the tail (a) or leading edge (b) of the T cell. (B) Time course of [Ca²⁺]_i for cells shown in A. [Ca²⁺]_i was estimated every 4 s by dividing 488-nm-excited green and red fluorescence images pixel by pixel and applying the equation of Grynkiewicz et al. (11).

Figure 2

Quantification of immobilized FITC-conjugated anti-CD3 mAb density on polystyrene beads by FACS analysis. (A) Antibody coating on beads. The two-step antibody coating method was assumed to prevent potential steric hindrance and increase anti-CD3 mAb binding efficacy. Beads were initially coated with 100 μg/ml anti-hamster Fc IgG mAb (mAb 1′) and then with varying concentrations of FITC-conjugated hamster anti-mouse CD3 mAb (mAb 2′) of known fluorescein/protein ratio. mAb 1′ binds the Fc portion of mAb 2′, leaving the antigen-binding site of mAb 2′ free to engage with the TCR:CD3 complex. (B) FACS analysis of beads shown in A. The number of anti-CD3 mAb on single beads was quantified by using FACS analysis to compare FITC-conjugated mAb-coated beads with a standard curve of microbeads labeled with defined numbers of fluorescein molecules. (C) The resulting anti-CD3 mAb density on beads showing a 3-log linearity with incubating antibody concentration.

Figure 3

T cell response depends on anti-CD3 mAb density on beads and contact area between the T cell and the bead (n = 30 cells per point). (A) T cell response percentage to beads of varying size and antibody density. Contact areas were 9.5 μm², 4.9 μm², 3.0 μm², and 2.0 μm² for 5-μm, 2.5-μm, and 1.14-μm doublet beads and 1.14-μm singlet beads, respectively. (B) T cell response latencies corresponding to initiation of [Ca²⁺]_i signals in A. Measured response latencies (T) for 5-μm-diameter beads were fit to a pseudo-first-order rate expression with constant offset (T₂): T = K*ln[D/(D − A)] + T₂ (dashed line). T, latency time; D, anti-CD3 mAb density on beads; K, constant; A, anti-CD3 mAb threshold density; T₂, the minimal time for downstream signal transduction events leading to the [Ca²⁺]_i rise. The parameters obtained from the fit were A = 13 per μm² and T₂ = 23 s. Standard error bars are shown in the figure.