Fig. 5

2D kinetics and T-cell Ca²⁺ signaling. a 2D micropipette adhesion frequency assay. A micropipette-aspirated T cell (right) was driven by a piezoelectric translator to make controlled contact with an RBC coated with pMHC held by another pipette (left). The retraction of T cells to the starting position resulted in elongation of the RBC, enabling the visual detection of the TCR–pMHC bond. Also see Fig. S15 and Movie S6. Adhesion curves for 5C.C7 TCRs interacting with the K5 (b), MCC (c), and 102S (d) pMHCs measured by micropipette assay at 25 °C at the indicated pMHC site densities. Each cell pair was tested 50 times with a given contact duration to estimate the adhesion probability, and three cell pairs were tested for each contact duration to calculate the mean adhesion probability. The data (points) were fitted by a probabilistic kinetic model (curves) to determine the 2D binding kinetics. The data are summarized in Table 1 and Table S1. e Real-time single T-cell calcium signaling measured by fluorescence micropipette. A CH27 cell loaded with the K5 (top row) or null peptide (bottom row, control) was precisely controlled to make contact with a primary 5C.C7 T cell loaded with the Fluo-4 calcium indicator at 37 °C. The fluorescence signal was recorded in real time by time-lapsed microscopy, and the fold increase in Ca²⁺ signaling (F/F₀) is shown in pseudocolor. Representative Ca²⁺ imaging experiments for the K5 and null peptides consisting of 6–8 independent experiments for each peptide are shown. Also see Fig. S15c, d for the peptides MCC and 102S. See Movie S7 for more data. f Representative time trajectories for Ca²⁺ signaling stimulated by the K5, MCC, 102S, and null peptides. Fluorescence intensity values (F) at any given timepoint were divided by the initial fluorescence intensity value at time zero (F₀) to obtain the fold increase in Ca²⁺ signaling after cell contact. g The time course of Ca²⁺ signaling (colored curves) and TCR–CD3ζ conformational changes (black curves). h TCR–CD3ζ conformational changes were caused by a calcium signaling feedback loop. TCR–pMHC bond formation causes CD3ζ dissociation and initiates Ca²⁺ flux, which in turn promotes CD3ζ dissociation by neutralizing the negative charges of the anionic phospholipids in the T-cell membrane to fully expose the ITAMs on CD3ζ to allow phosphorylation