Daan Pieren

75 Compromised DNA repair promotes the accumulation of regulatory T cells 1 2 3 1 2 3 Ercc1 +/+ Ercc1 -/ Δ 7 FoxP3 CD25 CD122 PD-1 1 2 3 0 20 40 60 Cluster 3 Cluster 2 Cluster 1 Cluster proportion (%) Ercc1 +/+ Ercc1 -/ ∆ 7 Memory Tc cells G H I J K L FoxP3 CD25 CD122 PD-1 1 23 1 23 1 2 3 0 20 40 60 Cluster 3 Cluster 2 Cluster 1 Cluster proportion (%) Ercc1 +/+ Ercc1 -/ Δ 7 Naive Tc cells Ercc1 +/+ Ercc1 -/ ∆ 7 A B C D E F 0 3 Expression 0 3 Expression viSNE1 viSNE2 viSNE1 viSNE2 0 2 4 6 8 10 % CD25 + of Tc ns 0 2 4 6 8 10 % CD122 + of Tc ns 0 2 4 6 8 10 % PD-1 + of Tc * 0 5 10 15 20 % CD25 + of Tc ns 0 20 40 60 80 100 % CD122 + of Tc ** 0 10 20 30 % PD-1 + of Tc ** Figure 3. Aging-related changes in naive and memory Tc cells in mice with compromised DNA repair. Naive (CD44 Lo ) and memory (CD44 Hi ) cells were identified within the CD4 - T-cell population (Tc cells) . Cell density maps of dimensionality reduced single-cell data by viSNE show clustering within ( A ) naive Tc cells and ( G ) memory Tc cells of pooled flow cytometry datafiles of Ercc1 +/+ mice (n=6) and pooled datafiles of Ercc1 -/ Δ 7 mice (n=7). Numbers in ( B,H ) the density maps correspond to the cluster numbers above the heat maps. These heat maps depict the Arcsinh-transformed median expression of the indicated markers. Bar graphs ( C,I ) indicate the proportion of each cluster within the total viSNE for Ercc1 +/+ (blue) and Ercc1 -/ Δ 7 (orange) mice. Bar graphs show the frequencies of ( D,J ) CD25 + , ( E,K ) CD122 + , and ( F,L ) PD-1 + cells among naive and memory Tc cells in individual Ercc1 +/+ and Ercc1 -/ Δ 7 mice. Bar graphs show mean ± SD; * p < 0.05, ** p < 0.01, ns = not statistically significant for the difference between Ercc1 +/+ and Ercc1 -/ Δ 7 mice using parametric unpaired Student’s t test or non-parametric Mann-Whitney test, two-tailed, dependent on the tested normality of distribution of the data. 3