Daan Pieren

76 Chapter 3 eRapa increases the proportion of memory Tregs but does not reduce the accumulation of total Tregs eRapa did not prevent the rise in proportion of FoxP3 + Tregs within the overall CD3 + T-cell pool in Ercc1 -/ Δ 7 mice ( Figure 4D ). However, we observed that eRapa decreased the proportion of Tregs within the naive CD4 + T-cell pool of Ercc1 -/ Δ 7 mice and strongly increased the proportion of Tregs within the memory CD4 + T-cell pool of these mice ( Figure 4E,H ). eRapa did not prominently reverse the aging-related lower CD25 + and higher PD-1 + cell frequencies found among naive Tregs of Ercc1 -/ Δ 7 mice ( Figure 4F,G ). In contrast, eRapa highly increased the frequency of CD25 + and PD-1 + cells among memory Tregs, which was not observed in Ercc1 -/ Δ 7 mice without eRapa ( Figure 4I,J ). Together, these data suggest that accumulation of Tregs with an aging-related phenotype by compromised DNA repair may be independent of mTOR activation. Aging-related reduction of CD122 + memory Th-cell frequencies can be restored by eRapa. One of the few observations in the Th-cell subset of Ercc1 -/ Δ 7 mice that reflected findings in WT aged mice [3] was a decreased frequency of CD122 + cells among memory Th cells ( Supplementary Figure 4 ). eRapa restored the proportion of CD122 + memory Th cells to levels found in Ercc1 +/+ mice ( Supplementary Figure 6 ). This finding shows that eRapa may reverse this aging-related aspect of memory Th cells and suggests that CD122 expression by memory Th cells is dependent on mTOR activation. Compromised DNA repair limits T-cell receptor/Interleukin-2-mediated Treg proliferation and activation. Reduced proliferation and reduced upregulation of the activation marker CD25 in response to cellular stimulation are hallmarks of declined T-cell responsiveness at older age [3,37]. To investigate the consequences of compromised DNA repair on T-cell responses, we exposed total spleen cells of Ercc1 +/+ and Ercc1 -/ Δ 7 mice, or young and aged WT mice to anti-CD3 alone to mimic stimulation of the T-cell receptor (TCR), or in combination with anti-CD28 or interleukin-2 (IL-2) as a co-stimulator. After four days, we measured T-cell responsiveness by proliferation and upregulation of the activation marker CD25. Additionally, we stimulated T cells of eRapa-fed Ercc1 -/ Δ 7‑ mice to assess whether eRapa would prevent consequences of compromised DNA repair on T-cell responsiveness.