Fig. 4: ACE enables comprehensive TCR signaling network profiling at single-cell resolution.

a, Thirty key markers related to the TCR signaling network in the human Jurkat T cells were measured with or without ACE amplification and plotted as green or blue boxes, respectively. Mean ion counts for unamplified and linear amplified staining were indicated by the green and blue points with lines between them color coded according to amplification fold change. Boxplots present the first quartile (Q1), median and third quartile (Q3) in unamplified or linear amplified data. The IQR defines the distance between Q1 and Q3. The upper whisker extends from the hinge to the largest value no further than 1.5 × IQR from the hinge. The lower whisker extends from the hinge to the smallest value no further than 1.5 × IQR of the hinge. ACE enabled, on average, 17-fold and a maximum of 41-fold signal amplification. The signal from p-AKT (p-T308) was analyzed with an additional ACE branching amplification (shown as a red box) with 15-fold higher p-AKT (p-T308) counts after the branching amplification. b, Jurkat cells treated with anti-CD3 and anti-CD28 antibodies, and subsequently crosslinked with anti-mouse IgG antibody to activate TCR signaling. Cells were harvested at 0, 0.5, 2, 5, 10, 15, 30 and 60 min after TCR activation. Linear amplification allowed the quantification of key signaling nodes in the TCR network during the 1-h TCR stimulation timecourse. Negative control p-SMAD2 did not show differential phosphorylation levels as measured with ACE. c, Signal responses of p-AKT (p-T308) to TCR activation were analyzed without amplification (bottom), with linear amplification (middle) and with branching amplification (top). Fold changes in the p-AKT signal relative to the first timepoint (unstimulated) were calculated and showed that only with branching ACE could the signaling trajectory be captured. d, Circos plot shows 73 pairs of strong signaling relationships in the TCR signaling networks as detected with the BP-R² method on the TCR stimulation timecourse data measured by ACE (inner layer). The middle layers show the coefficients of variation of measured phosphorylation sites at all timepoints following TCR stimulation and the signal amplitude for each phosphorylation site. The outer layer demonstrates the signaling trajectory for each marker, characterized by normalized mean ion counts across all timepoints during the TCR stimulation timecourse. Cells used in these analyses were in G1 phase to avoid cell cycle confounding effects. Experiments were performed in biological replicates to confirm reproducibility.