Daan Pieren

38 Chapter 2 T cells, suggesting that Th and Tc cells both contribute to the higher IFN- γ we measured in supernatants ( Figure 3F ). In contrast to higher TNF- α in the supernatant ( Figure 3G ), CD4 + and CD8 + T cells of aged mice produced higher levels of TNF- α than those of young mice. This difference may be due to the different modes of stimulation or by different rates of TNF- α consumption. Additionally, the higher levels of IL-4 and IL-5 we detected in the supernatant of aged mice was paralleled by a significantly higher frequency of aged IL-4/ IL-5 + CD4 + T cells ( Supplementary Figure 2 ). This suggests that predominantly CD4 + T cells account for the elevated levels of IL-4/5 found in supernatants of spleen cells cultured from aged mice. Int High IL-2 IL-4 IL-5 IL-10 IL-17 IFN- γ TNF- α Time (Days) A B C D E F G Young (2 months) Aged (22-24 months) Int High Int High Int High Int High Int High Int High 2 4 2 4 0 500 1000 1500 2000 IL-2 (pg/mL) *** *** 2 4 2 4 0 100 200 300 400 IL-4 (pg/mL) * ** 2 4 2 4 0 500 1000 1500 IL-10 (pg/mL) *** 2 4 2 4 0 500 1000 1500 2000 IL-17 (pg/mL) p = 0.06 2 4 2 4 0 20 40 60 80 100 TNF- α (pg/mL) *** ** *** *** 2 4 2 4 0 200 400 600 800 IL-5 (pg/mL) ** * 2 4 2 4 0 2000 4000 6000 8000 IFN- γ (pg/ml) * ** Figure 3. Duration and strength of stimulation determine ageing-related cytokine profiles. Cytokine levels of IL-2 ( A ), IL-4 ( B ), IL-5 ( C ), IL-10 ( D ), IL-17 ( E ), IFN- γ ( F ), and TNF- α ( G ) were measured in supernatant of splenocytes exposed for two or four days to an intermediate (int) or high stimulatory strength. Data represent one experiment with young (n=6, 2 months old) and aged (n=6, 22-24 months old) mice. Mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001 for difference between young and old mice at time points indicated using Two-way ANOVA. Reduced induction of T cell activation in aged mice can partially be re- stored by stronger stimulation We next analyzed the expression of the classical activation markers CD69 and CD25 to investigate to what extent ageing influences the activation kinetics of T cells ( Figure 4 ). After low strength stimulation, the frequency of CD25 + Th and Tc cells of young mice increased and peaked at day one ( Figure 4A ). With higher strength of stimulation, the maximum frequencies of CD25 + cells induced were higher and maintained over time, irrespective of age. In Th and Tc cells of aged mice, maximum frequencies of CD25 + cells however never reached those of young mice. Treg cells of aged mice showed significantly