Daan Pieren

79 Compromised DNA repair promotes the accumulation of regulatory T cells 0 20 40 60 80 100 % Proliferated Tregs *** *** *** ns aCD3 aCD28 IL-2 - - - + - - + + - + - + A C 0 50000 100000 150000 200000 250000 300000 CD25 expression Treg (MFI) ns **** ns ** aCD3 aCD28 IL-2 - - - + - - + + - + - + E D aCD3 aCD3 + aCD28 aCD3 + IL-2 Ercc1 -/ Δ 7 + Rapamycin Ercc1 -/ Δ 7 Ercc1 +/+ CellTrace (BV421) B Ercc1 +/+ Ercc1 -/ ∆ 7 Ercc1 -/ ∆ 7 + Rapamycin Ercc1 +/+ Ercc1 -/ ∆ 7 Ercc1 -/ ∆ 7 + Rapamycin 0 20 40 60 80 100 % Proliferated Tregs aCD3 aCD28 IL-2 - - - + - - + + - + - + ns 0.08 0.08 ns ns *** * ns 0 50000 100000 150000 200000 CD25 expression Treg (MFI) aCD3 aCD28 IL-2 - - - + - - + + - + - + 0.07 ns ns ns ns ns WT Young (2 months) WT Aged (22 months) Figure 5. Compromised DNA repair limits T-cell receptor/Interleukin-2-mediated Treg pro- liferation and activation. Total splenocytes of WT young (n=6) and aged (n=6) mice, and Ercc1 +/+ (n=5), Ercc1 -/ Δ 7 (n=7), and Ercc1 -/ Δ 7 mice treated with rapamycin (n=3-6) were exposed to anti-CD3 alone or in combination with anti-CD28 or IL-2 for four days. Treg proliferation was traced by CellTrace labeling ( A ); dilution of CellTrace label intensity indicates cellular proliferation. Bar graphs show ( B ) Treg proliferation of Ercc1 -/ Δ 7 and Ercc1 -/ Δ 7 mice and of ( C ) WT young and aged mice. Bar graphs show CD25 expression by Tregs of ( D ) Ercc1 -/ Δ 7 and Ercc1 -/ Δ 7 mice and ( E ) WT young and aged mice . Bar graphs show mean ± SD; * p < 0.05, ** p < 0.01, *** p <0.001, **** p < 0.0001 ns = not statistically significant for the difference between groups using parametric one-way ANOVA corrected with Holm-Sidak correction for multiple comparisons. Due to the low number of eRapa-fed Ercc1 -/ Δ 7 mice (n=3), statistical significance of this group was determined by non- parametric Mann-Whitney U Test. 3