Daan Pieren

47 Novel features of ageing in murine T cells DISCUSSION In this study, we investigated the impact of ageing on T-cell response kinetics by studying the effect of the duration and strength of in vitro stimulation on T-cell activation markers, proliferation, and cytokine secretion in young and aged mice. Our study shows that T-cell response kinetics are a valuable tool to better understand the impact of ageing on T cells. Moreover, our findings indicate that PD-1 expression and impaired proliferation at old age may not imply unresponsive T cells per se. Decreased induction of CD69 and CD25 expression has been used as a measure for lower rate of activation in CD4 + and CD8 + T cells of aged humans and mice [7,10-12], but only few studies have addressed CD25 and CD69 expression kinetics [7]. The stimulation-induced frequency of CD25 + and CD69 + Th and Tc cells was lower in aged mice compared to young mice during the early phase of the response. Levels found in T cells of aged mice eventually reached levels close to those found in young mice. Moreover, the presence of CD69 + cells was more persistent in all T cell subsets of aged mice after their stimulation and became even higher than the declining number of CD69 + cells in the T-cell pool of young mice at later time points. These findings indicate that early activation of Th and Tc cells of aged mice is diminished and that subsequent downregulation of CD69 is delayed. Persistent CD69 + Tc cell frequencies after stimulation have previously been reported in aged mice [7], and we now show that Th and Treg cell subsets of aged mice also contain cells with persistent stimulation-induced CD69 expression. Persistent frequencies of CD69 + Tregs in aged mice may harbor a Treg subset with enhanced suppressor activity [43,44]. Whether such CD69 + Tregs may account for reduced Th and Tc cell activation at old age remains to be addressed in future studies. Treg cells of aged mice could still be adequately activated as they were capable of reaching CD25 + cell frequencies similar to those observed in young mice. However, these comparable levels only occurred after increasing the stimulatory strength. This finding may explain earlier reports showing unaltered suppressive functionality of Treg cells at old age despite their reduced CD25 expression at baseline [40,45,46]. Thus, ageing has a differential impact on CD25 kinetics of Treg cells compared to Th and Tc cells. We observed declined proliferative capacity of Th, Tc, and Tregs cells with progressing age, and increasing the stimulatory strength did not resolve diminished proliferation. Declined proliferation of T cells is a major hallmark of replicative senescence [6]. Moreover, expression of PD-1 has been postulated as a marker of cell populations with diminished proliferative capacity [16,47]. 2