a, OT-I CD8+ T cells were stimulated with high potency ovalbumin peptide
(N4) for 0, 1, 3, or 6 hours before sorting for scRNA-seq by FACS. Protein
expression flow cytometry measurements are representative of at least 2
independent experiments. b, Principal components analysis of
scRNA-seq of cells sorted in a. c, Diffusion
pseudotime analysis of sequenced cells: cells are plotted by diffusion
components (DCs) 1 and 2, with a black line delineating the pseudotime
trajectory. d, Plot in c is colored by clusters in
diffusion pseudotime. e, The top 20 genes transcriptionally
upregulated in the early activation cluster versus the resting and late
activation clusters are depicted in a heatmap with genes clustered by Pearson
correlation. Blue (TR) indicates transcriptional regulatory genes.
f, Expression of selected transcription factors from
e is shown with a box plot indicating the median, boxed
interquartile range, and whiskers extending to the most extreme point up to 1.5
x the interquartile range. (b-f) n = 44 cells for 0h, 51 for 1h, 64
for 3h, and 46 for 6h.