Daan Pieren

72 Chapter 3 have a significant impact on expression of CD122, CD25, and PD-1 by the naive Th population and therefore might not explain the aging-related changes based on these markers within the naive Th-cell subset in WT aged mice. Compromised DNA repair promotes the aging-related phenotype of memory Th cells. Next, we assessed the impact of compromised DNA repair on the phenotype of memory (CD44 Hi ) Th cells. These cells were represented by the FoxP3-negative clusters 2 and 3 within the memory CD4 + T cells in our viSNE analyses ( Figure 2G,H ). Cluster 2 was more abundant in Ercc1 -/ Δ 7 compared to Ercc1 +/+ mice (40.4% versus 34.0%), which points towards more Ercc1 -/ Δ 7 memory Th cells expressing PD-1 as reported previously in WT aged mice [3]. Indeed, a trend towards higher PD-1 + memory Th-cell frequency and higher expression of PD-1 on Ercc1 -/ Δ 7 memory Th cells compared to Ercc1 +/+ cells was found ( Supplementary Figure 4 ). Further, we observed a lower proportion of cluster 3 (18.6% versus 29.2%) in Ercc1 -/ Δ 7 compared to Ercc1 +/+ mice ( Figure 2I ), which is reflected in lower CD122 expression and CD122 + memory Th-cell frequency in Ercc1 -/ Δ 7 mice ( Supplementary Figure 4 ) and corresponds to earlier findings in WT aged mice [3]. Together, these data indicate that Ercc1 -deficiency imposes an aging-related phenotype on memory Th cells that has been reported based on the expression of PD-1 and CD122. 3 Aging-related changes in naive and memory Tc cells in mice with compro- mised DNA repair. CD3 + cells that do not express CD4 can be considered CD8 + cytotoxic T cells (Tc cells), as this subset mainly comprises CD8 + T cells ([3], Supplementary Figure 1 ). Clustering of naive (CD44 Lo ) Tc cells by viSNE showed relatively comparable cell-density plots, expression of phenotypical markers, and cluster proportions ( Figure 3A-C ) between Ercc1 -/ Δ 7 and Ercc1 +/+ mice, which suggests a low impact of Ercc1 -deficiency on naive Tc cells. Indeed, we observed no phenotypical difference in expression and cell frequencies of naive Tc cells ( Figure 3D,E; Supplementary Figure 4 ) apart from a slight decrease in the frequency of PD-1 + Tc cells of Ercc1 -/ Δ 7 mice ( Figure 3F ). These findings are in contrast with increased PD-1 + cell-frequencies found among naive Tc cells of WT aged mice [3].