Daan Pieren

73 Compromised DNA repair promotes the accumulation of regulatory T cells 1 2 3 4 1 2 3 4 Ercc1 +/+ Ercc1 -/ Δ 7 Naive CD4 + T cells 1 2 3 4 FoxP3 CD25 CD122 PD-1 0 10 20 30 Cluster 4 Cluster 3 Cluster 2 Cluster 1 Cluster proportion (%) Ercc1 +/+ Ercc1 -/ ∆ 7 A B C D E F 0 3 Expression 1 2 3 1 2 3 Ercc1 +/+ Ercc1 -/ Δ 7 FoxP3 CD25 CD122 PD-1 1 2 3 0 10 20 30 40 Cluster 3 Cluster 2 Cluster 1 Cluster proportion (%) Ercc1 +/+ Ercc1 -/ ∆ 7 Memory CD4 + T cells G H I J K L 0 3 Expression viSNE1 viSNE2 viSNE1 viSNE2 0 10 20 30 % Treg of naive CD4 + ** 70 80 90 100 % CD25 + of Treg ** 0 10 20 30 % PD-1 + of Treg * 0 20 40 60 % Treg of memory CD4 + ns 40 60 80 100 % CD25 + of Treg ns 0 20 40 60 80 % PD-1 + of Treg ns Figure 2. Compromised DNA repair promotes accumulation of FoxP3 + Tregs within the naive CD4 + T-cell subset. Naive (CD44 Lo ) and memory (CD44 Hi ) cells were identified within the CD4 + T-cell population . Cell density maps of dimensionality reduced single-cell data by viSNE show clustering within ( A ) naive CD4 + T cells and ( G ) memory CD4 + T cells of pooled flow cytometry datafiles of Ercc1 +/+ mice (n=6) and pooled datafiles of Ercc1 -/ Δ 7 mice (n=7). Numbers in the density maps correspond to the cluster numbers above the heat maps ( B,H ) . 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 ( D-F, J-L ) show the frequency of ( D ) naive and ( J ) memory FoxP3 + regulatory T cells in individual Ercc1 +/+ and Ercc1 -/ Δ 7 mice, as well as the frequency of ( E,K ) CD25 + and ( F,L ) PD-1 + cells within these naive and memory Treg subsets. 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