Fig. 2: SN-ROP reveals remodeling of ROS signaling networks in CD8⁺ T cells.

a UMAP projection of SN-ROP from pooled CD8⁺ T cells from OT-I mice at days 0-5 of OVA stimulation colored to show the distribution of cells across different time points. b Box plots displaying 99^th percentile normalized SN-ROP expression profiles for each CD8⁺ T cell activation time point (n = 3 independent samples). Each box represents the distribution of single-cell expression values. Box plots show the median (center line), interquartile range (IQR; box limits), and whiskers extending to 1.5×IQR; outliers beyond this range are shown as individual points. The plots are colored based on protein function or subcellular localization. No statistical comparisons were performed. c Pseudotime values calculated using the SCORPIUS package plotted in a heatmap along with the 99^th percentile normalized data, which was smoothed using a window size of 100 (n = 3 independent samples, data from one representative sample shown). d Slope (first derivative) heatmap of protein expression across pseudotime. The vertical dashed lines indicate significant inflection points (n = 3 independent samples, data from one representative sample shown). e Examples of expression of SN-ROP markers in organelles as a function of pseudotime. Data shown are the 99^th percentile normalized values for a representative sample (n = 3) smoothed using a window size of 1000. The vertical dashed lines indicate inflection times. f ASINH transformed data of CD8⁺ T cells from OT-I mice annotated with GO biological processes. Red represents a positive correlation, and blue represents a negative correlation (n = 3 independent samples, data from one representative sample shown). The features are grouped into four categories based on their functional roles: kinase signaling (EOMES, TIM3, oxPTP, pERK); protein synthesis/translation (HSP70, TCF1/7, REF/APE1, and GR); DNA damage/peroxidation (NNT, 53bp1, AQP8, PD1, CD137, Catalase, and ACOX3); and anti-oxidation (CD62L, oxDJ1, pNFκB, ERO1B, CTLA4, QSOX1, LAG3, KEAP1, and GPX4). g Pseudotime heat map with biological processes of the ROS functions divided into six pathways based on membership in one or more functional GO modules or pathways. h The percentage of CD8⁺ T cells from MC38 tumors at in vivo day 7 (early, blue) and day 14 (late, gold) distributed across in vitro time points (n = 3 biologically independent MC38 tumor-bearing mice per group). Bars represent the mean, and error bars indicate ± standard error of the mean (SEM). Statistical significance was assessed using a two-sided permutation t-test. i Mean expression levels of five late-stage activation markers in CD8⁺ T cells from MC38 tumors projected onto the in vitro timeline (n = 3).