Daan Pieren

78 Chapter 3 We observed that compromised DNA repair in part explains diminished Treg proliferation in WT aged mice, as Tregs of Ercc1 -/ Δ 7 mice showed a trend towards reduced proliferation in response to anti-CD3 alone and significantly reduced proliferation in response to anti-CD3 with IL-2 ( Figure 5A,B ). However, Tregs of WT aged mice also showed reduced proliferation in response to anti- CD3 combined with co-stimulation by anti-CD28 ( Figure 5C ), whereas CD28- mediated co-stimulation appeared to remain intact in Tregs from Ercc1 -/ Δ 7 mice. eRapa did not restore the reduction of Treg proliferation observed in Ercc1 -/ Δ 7 mice ( Figure 5A,B ). Additionally, Tregs of Ercc1 -/ Δ 7 mice showed a trend towards reduced CD25 upregulation in response to anti-CD3 with IL-2, although the variation within the group of Ercc1 -/ Δ 7 mice was relatively high ( Figure 5D ). These findings in part reflect observations in WT aged mice, as WT aged mice also showed reduced CD25 expression in response to anti-CD3 in the absence of exogenous IL-2 ( Figure 5E ). eRapa did not restore Treg CD25 expression levels in Ercc1 -/ Δ 7 compared to Ercc1 +/+ mice ( Figure 5D ). It has to be noted that the number of eRapa-fed Ercc1 -/ Δ 7‑ mice stimulated with anti-CD3 with IL-2 was low (n=3) due to a limited number of splenocytes available for the different assays and these results should therefore be approached with caution. Together, our findings indicate that compromised DNA repair contributes to reduced Treg responsiveness to anti-CD3 and IL-2 observed with WT aging, which may be independent of mTOR activation. Compromised DNA repair limits T-cell receptor/Interleukin-2 mediated Th- and Tc-cell responsiveness. Th and Tc cells of Ercc1 -/ Δ 7 mice showed a trend towards reduced proliferation in response to anti-CD3 with IL-2, whereas Th and Tc cells of WT aged mice showed reduced proliferation also in response to anti-CD3 with anti-CD28 ( Supplementary Figure 7 ). Despite the small number of mice, eRapa did not appear to restore proliferation of Th and Tc cells from Ercc1 -/ Δ 7 mice in response to anti-CD3 with IL-2 ( Supplementary Figure 7 ). Tc cells but not Th cells of Ercc1 -/ Δ 7 mice showed reduced CD25 expression induced by response to anti- CD3 with IL-2, despite the individual response within each group being highly variable. In contrast, both cell types showed reduced CD25 induction in WT aged mice ( Supplementary Figure 7 ). In vivo treatment of Ercc1 -/ Δ 7 mice with eRapa did not affect stimulation-induced CD25 expression by their Th cells ( Supplementary Figure 7 ). Thus, compromised DNA repair may partly explain impaired Th- and Tc-cell responses found in WT aged mice.