Supplementary Figure 2: Costimulation effects and control gene selection in early CD8⁺ T cell activation.

a, OT-I CD8⁺ T cells were stimulated as in Fig. 2 with pure peptide in the presence and absence of exogenous murine IL-2. After 6 hours, surface protein expression was examined by flow cytometry. Plots depict results from 3 separate mice; line depicts the mean. Stimulation in the presence of IL-2 primarily affected expression of the high affinity receptor CD25 in the first 6 hours, with minimal impact on other activation readouts. b, Cells were stained with a proliferation dye before stimulation as in a. After 2 days, proliferation was examined by flow cytometry, confirming previous reports that IL-2 is important for proliferation under low potency activation conditions. Results are representative of 3 separate mice. c, The Rpl39 control gene for RNA flow cytometry was identified in single-cell RNA-seq data as having low variance and minimal condition-dependence. Plots show expression in two independent scRNA-seq data sets and ANOVA p values without multiple testing correction; left, n = 46 cells for N4 6h, 47 for T4 6h, 46 for G4 6h, 46 for NP68 6h, 44 for unstimulated, 51 for N4 1h, and 64 for N4 3h; right, n = 45 cells for N4, 44 for T4, 48 for G4, and 47 for NP68. Box plots show the median, boxed interquartile range, and whiskers extending to the most extreme point up to 1.5 x the interquartile range. d, Control gene expression in an RNA flow cytometry experiment as depicted in Fig. 2. Histograms are representative of 3 independent experiments.