Daan Pieren

71 Compromised DNA repair promotes the accumulation of regulatory T cells individual Ercc1 -/ Δ 7 mice compared to control Ercc1 +/+ mice ( Figure 2D ), which closely resembled findings in WT aged mice ( Supplementary Figure 3 ). Additionally, accumulated Tregs of Ercc1 -/ Δ 7 mice comprised significantly lower CD25 + ( Figure 2E ) and higher PD-1 + cell frequencies ( Figure 2F ) and expression levels per cell (MFI) ( Supplementary Figure 4 ) compared to Ercc1 +/+ mice, which is similar to observations in WT aged mice [3]. Thus, compromised DNA repair appears to contribute to the accumulation of Tregs within the naive CD4 + T-cell compartment with an aging-related phenotype that resembles findings in WT aged mice. FoxP3 + Tregs within the memory CD4 + T-cell subset in mice with compro- mised DNA repair. WT aged mice do not accumulate Tregs within the memory CD4 + T-cell pool ([3] and Supplementary Figure 3 ) and we assessed whether Ercc1 -/ Δ 7 mice resemble these findings. viSNE analysis of memory (CD44 Hi ) CD4 + T cells of Ercc1 -/ Δ 7 and Ercc1 +/+ mice showed three phenotypically distinct clusters ( Figure 2G ) with cluster 1 indicating FoxP3-expressing Tregs ( Figure 2H ). Whereas the proportion of cluster 1 containing Foxp3 + cells was higher in Ercc1 -/ Δ 7 mice compared to Ercc1 +/+ mice (37.4% versus 32.9%) ( Figure 2I ), the frequency of Foxp3 + Tregs within the memory CD4 + T-cell subset did not show a difference between individual Ercc1 -/ Δ 7 and Ercc1 +/+ mice ( Figure 2J ). In contrast to previous findings in WT aged mice [3], Ercc1 -deficiency did not show altered frequency and expression of CD25 and PD-1 on Tregs within the memory CD4 + T-cell subsets ( Figure 2K,L; Supplementary Figure 4 ). These findings suggest that compromised DNA repair may not significantly contribute to phenotypical changes of Tregs within the memory CD4 + T-cell pool. The aging-related phenotype of naive Th cells in mice with compromised DNA repair. We next investigated whether compromised DNA repair contributes to phenotypical changes within naive (CD44 Lo ) helper T cells (Th cells). viSNE- clusters 2, 3, and 4 identified within the naive CD4 + T-cell pool do not express FoxP3 ( Figure 2A,B ), indicating that these cells are naive Th cells. The minimal difference in proportion of clusters 2, 3, and 4 between Ercc1 -/ Δ 7 and Ercc1 +/+ mice ( Figure 2C ) indicates that Ercc1 -deficiency did not profoundly affect the phenotype of naive Th cells based on the markers we included. Indeed, Ercc1 -/ Δ 7 mice did not show the increased frequencies or expression of PD-1 + cells among naive Th cells ( Supplementary Figure 4 ) that we previously found in WT aged mice [3]. Thus, these findings suggest that Ercc1 -deficiency may not 3