Daan Pieren

66 Chapter 3 that enables investigation of the impact of compromised DNA repair on T-cell phenotype and responsiveness. Another important driver of cellular aging is the activation of the mammalian target of rapamycin (mTOR) [25]. mTOR is a well-known target in anti-aging research as inhibition of mTOR by rapamycin has been widely reported to slow down the process of aging in terms of life- and health span [26-28]. Moreover, dietary supplementation of rapamycin in an encapsulated form (eRapa) to aged mice has been shown to reduce age-related changes observed in T-cells [29]. Interestingly, activation of the mTOR kinase mTORC1 has been reported to impair DNA damage response signaling, leading to accumulation of unrepaired DNA lesions [30]. Moreover, mTORC1 activation may negatively interfere with the ataxia telangiectasia mutated (ATM) checkpoint that promotes DNA repair [31]. We therefore hypothesized that changes to the T-cell compartment induced by compromised DNA repair may be slowed down by in vivo blocking of mTOR by eRapa. In this study, we defined the impact of compromised DNA repair on T-cell phenotype and T-cell responsiveness in Ercc1 -/ Δ 7 mice. Additionally, we assessed whether the impact of compromised DNA repair on T cells could be avoided by inhibition of mTOR by in vivo dietary treatment with eRapa. We present evidence suggesting that compromised DNA repair promotes the aging-related accumulation of Tregs and reduced T-cell responsiveness that we find in wild- type (WT) aged mice, which appears to be independent of mTOR activation. MATERIALS AND METHODS Mice Young (2 months of age) and aged (22 months of age) wild type C57BL/6 female mice were purchased from Envigo (Venray, Limburg, The Netherlands). Young and aged mice were maintained at the animal facilities of the Institute for Translational Vaccinology (Bilthoven, Utrecht, The Netherlands). Generation and characterization of Ercc1 -/ Δ 7 mice has been previously described [24]. Breeding stocks of the parental strains, i.e. Ercc1 +/- mice in a pure C57BL6J background and Ercc1 +/ ∆ 7 mice in a pure FVB background were generated and maintened as described [23,24]. Genetically uniform F1-hybrid C57BL6-FVB Ercc1 -/ ∆ 7 mice were generated by combining both parental strains. Typical unfavorable characteristics, such as blindness in the FVB background or deafness in the C57BL6J background, do not occur in this hybrid background. Animals were housed in individual ventilated cages under specific pathogen-free conditions (20–22°C; 12 hr. light: 12 hr. dark cycle) and provided food and water ad libitum .