Daan Pieren

70 Chapter 3 CD3 + T cells Memory CD4 + cells Memory CD4 - cells WT Young (2 months) WT Aged (22 months) A B C D E F Ercc1 +/+ (4 months) Ercc1 -/ Δ 7 (4 months) 0 20 40 60 % CD3 + of viable lymphocytes ns 0 10 20 30 % CD44 Hi of CD4 + CD3 + ** 0 10 20 30 % CD44 Hi of CD4 - CD3 + ** 0 20 40 60 % CD3 + of viable lymphocytes ** 0 20 40 60 80 100 % CD44 Hi of CD4 - CD3 + *** 0 20 40 60 80 100 % CD44 Hi of CD4 + CD3 + *** Figure 1. Compromised DNA repair contributes to increased proportions of memory T cells. The frequencies of ( A ) total T cells (CD3 + cells of viable lymphocytes), ( B ) memory CD4 + T cells (CD44 Hi of CD4 + CD3 + cells), and ( C ) memory CD4 - T cells (CD44 Hi of CD4 - CD3 + cells) were determined in the spleen of Ercc1 +/+ (n=6, 4 months of age) and Ercc1 -/ Δ 7 (n=7, 4 months of age) mice, as well as in ( D-F ) WT young (n=6, 2 months old) and aged mice (n=6, 22 months old). Bar graphs show mean ± SD; ** p < 0.01, *** p < 0.001, ns = not statistically significant for the difference between groups using unpaired Student’s t test, two-tailed. Compromised DNA repair promotes accumulation of FoxP3 + Tregs within the naive CD4 + T-cell subset. We previously observed that naive CD4 + T cells of WT aged mice are enriched with regulatory cells, as defined by expression of Foxp3, that express an aging- related phenotype characterized by increased expression of PD-1 and lower expression of CD25 [3]. Here we asked whether this major hallmark of aging in WT aged mice can be attributed to compromised DNA repair. We applied dimensionality reduction (viSNE) to form phenotypically distinct clusters based on simultaneous expression of different aging-related molecules within CD4 + naive (CD44 Lo ) and memory (CD44 Hi ) T cells of Ercc1 -/ Δ 7 mice and Ercc1 +/+ mice ( Supplementary Figure 2 , gating strategy). Cluster formation was based on the combined expression of FoxP3, CD25, CD122, and PD-1 as aging-related markers previously used to reveal aging-related clusters of T cells [3]. viSNE analysis of T cells of Ercc1 +/+ (n=6 pooled) and Ercc1 -/ Δ 7 mice (n=7 pooled) showed four phenotypically distinct clusters within the naive CD4 + T-cell subset ( Figure 2A ). Cluster 1 highly expressed the Treg marker FoxP3 ( Figure 2B ) and the proportion of this cluster was higher in Ercc1 -/ Δ 7 mice compared to Ercc1 +/+ mice (16.2% versus 10.8%) ( Figure 2C ). Accumulation of Tregs within the naive CD4 + T-cell subset was indeed significantly higher in