Daan Pieren

69 Compromised DNA repair promotes the accumulation of regulatory T cells Statistics Statistical analyses were performed using GraphPad Prism 7 software (La Jolla, CA, USA). The appropriate parametric or non-parametric tests were used based on the tested normality of distribution of the data by Kolmogrov-Smirnov test and Shapiro-Wilk test. Dependent on the number of comparisons and the normality of distribution of the data, unpaired Student t Test, Mann-Whitney U test, or One-way ANOVA test followed by Holm-Sidak multiple comparisons test were performed as indicated in the figure legends. Data presented in bar graphs are expressed as the mean ± standard deviation (SD). For all analyses, p values<0.05 were considered statistically significant. RESULTS Compromised DNA repair contributes to increased proportions of memory T cells. With age, the total T-cell population among lymphocytes decreases, whereas the proportion of memory cells within the T-cell population increases [3,10,33]. We first assessed whether compromised DNA repair contributes to these aging- related changes. We compared the T-cell populations in spleens of accelerated aging Ercc1 -/ Δ 7 mice (n=7, 4 months of age) with those in spleens of littermate control Ercc1 +/+ mice (n=6, 4 months of age) ( Figure 1A-C ). As a reference we did similar analyses in WT young (n=6, 2 months of age) and WT aged (n=6, 22 months of age) mice ( Figure 1D-F ). Ercc1- deficient mice did not significantly show a decrease of CD3 + T-cell frequency among live spleen lymphocytes ( Figure 1A ) that occured in normal WT aging ( Figure 1D ). Subsequently, we analyzed the frequency of memory cells by expression of the memory marker CD44 among CD4 + T cells and CD4 - T cells. Of note, CD3 + CD4 - T cells can be considered CD8 + T cells as this subset mainly comprises CD8 + T cells and a very low proportion of CD4 - CD8 - cells in Ercc1 -/ Δ 7 mice that we analysed in an additional data set (3% on average) ( Supplementary Figure 1 ), similar to previous observations in WT mice (4% on average) [3]. Although Ercc1 -/ Δ 7 mice did not reach the major increase of memory CD4 + and CD4 - T cell frequencies found in WT aged mice ( Figure 1E,F ), the proportion of memory T cells was higher in Ercc1 -/ Δ 7 mice compared to control Ercc1 +/+ mice ( Figure 1B,C ). Thus, our data indicate that the rise in proportion of memory T cells among CD4 + and CD4 - T cells found in WT aged mice can in part be attributed to compromised DNA repair. 3