Daan Pieren

48 Chapter 2 We here showed that none of the PD-1 + or PD-1 - Tc cells of aged mice older than 24 months proliferated in response to stimulation. Since PD-1 - Tc cells do not proliferate at old age, the expression of PD-1 cannot be used as sole predictor for diminished proliferation of Tc cells. Moreover, replicative senescence does not imply that T cell subsets of aged mice are unresponsive, as T cells of aged mice did show upregulation of activation markers and cytokine secretion. Reports on the impact of ageing on secretion of cytokines are highly ambiguous due to the diversity in stimuli used and the lack of measurements on cytokine response kinetics [20]. Our data highlight the importance of studying both the time and strength of stimulation to address cytokine production. In addition, a shift from a Type-1 to a Type-2 cytokine response with ageing has been reported, but is still under debate [20,21,23,24,36,37]. Our data show no prominent shift from a Type-1 to a Type-2 cytokine profile as the Type-2 cytokines IL-4, IL-5, IL-10, but also Type-1 cytokine IFN- γ were more abundantly secreted by cells from aged mice compared to young mice after four days of culturing. Collectively, our data indicate that aged mice require lower strength of stimulation compared to young mice to produce Type-1 and Type-2 cytokines. This was found in supernatant for most cytokines, and our analyses of intracellular cytokines suggested increased numbers of T cells being able to produce both Th1- and Th2-related cytokines. In addition to its role as an exhaustion marker, PD-1 can also be considered as an activation marker since it can be induced upon stimulation [16,47,48]. Indeed, in T cells from young mice we found significant upregulation of PD-1 upon stimulation. In contrast to young mice, the frequency of PD-1 + cells did not increase in Tc cells of aged mice upon stimulation, which indicates that PD-1 on Tc cells may not be activation-induced at old age. Moreover, in parallel to the absence of significantly increased PD-1 expression upon stimulation, Tc cells did show induced expression of CD25 and CD69. Therefore, these findings may suggest that induced expression of CD69 and CD25 we found on PD-1 + Tc cells of aged mice represent activated PD-1 + Tc cells. The limited number of cells we obtained from our mice were all used for the assays presented and did not allow extensive sorting of different subsets. Conclusive evidence for the rate of activation of PD-1 + versus PD-1 - cells upon stimulation requires stimulation of sorted PD-1 + and PD-1 - Tc cells to provide insight into whether PD-1 + cells can be activated. Age-related differences found in T cells have largely been ascribed to the shifted balance of naive T cells towards memory T cells during ageing [2]. Therefore, many of the aging-related differences have been explained by differences between naïve versus memory cells rather than to aging-related