Fig. 5: Single-cell age estimation facilitates the discovery of molecular markers of peripheral influx T cells underlying the anti-PD1 response.

a, Schematic overview of the experiment. Biopsies were taken from patients with basal cell carcinoma before (pre) and after (post) anti-PD1 treatment and subjected to scATAC-seq. b, Cell number (above) and EpiTrace age (below) of T_ex cells, separated by treatment response (R: responder; NR: non-responder) and T cell phenotypic maturity (Immature/Interim/Mature). Sample numbers of independent biological cells: n = 322 (NR group, Immature cell, Pre-PD1); 596 (NR group, Immature cell, Post-PD1); 77 (NR group, Interim cell, Pre-PD1); 108 (NR group, Interim cell, Post-PD1); 97 (NR group, Mature cell, Pre-PD1); 14 (NR group, Mature cell, Post-PD1); 77 (R group, Immature cell, Pre-PD1); 237 (R group, Immature cell, Post-PD1); 222 (R group, Interim cell, Pre-PD1); 923 (R group, Interim cell, Post-PD1); 378 (R group, Mature cell, Pre-PD1); and 2,452 (R group, Mature cell, Post-PD1). c, Heatmap showing scATAC peak activity in pseudobulk single cells grouped by phenotype (R/NR), sampling time (pre-PD1 or post-anti-PD1) and EpiTrace age. Correlations between peak activity and EpiTrace age are shown on the left. Peaks were clustered according to their activity profile into response-specific, non-response-specific and age-associated clusters. d, Correlation coefficient between clusters of peaks and treatment (PD1: pre/post = 0/1), response (R: NR/R = 0/1) and cell age (Age). Non-significant correlations are labeled with ‘X’. e, GO enrichment of the C1 cluster peaks as in c. Enrichment was tested by one-sided Fisher’s exact test. −logP values were adjusted by multiple comparison. f, ChrAcc (top) and cross-correlation between peaks (bottom) of CD109 loci from pseudobulk single cells grouped with phenotype (R/NR), sample (pre-PD1 or post-anti-PD1) and EpiTrace age (Young/Interim/Mature). The association of the CD109 promoter ChrAcc across age is shown in the right panel. g, ChrAcc (top) and cross-correlation between peaks (bottom) of CHRNA1 loci from pseudobulk single cells grouped as in f. The association of the CHRNA1 promoter ChrAcc across age is shown in the right panel. Correlation test: Pearson’s. For box plots, the upper and lower bounds of boxes show 25% and 75% percentiles of the data. The median of data is shown as the horizontal line in the box. The distribution minima and maxima, defined as farthest data point distanced ≤1.5 IQR from the box bounds, are shown by the whiskers. The violin plot shows the empirically estimated density distribution of data. Corr.coef., correlation coefficient.