Daan Pieren

37 Novel features of ageing in murine T cells Ageing-related alterations in cytokine profiles depend on response kinetics and strength of stimulation Cytokine production by stimulated T cells changes with ageing, which has widely been shown by measurements of cytokines secreted in supernatants. However, such findings are highly ambiguous due to the lack of studying the kinetics of cytokine responses, as studies often address cytokine secretion with a single stimulatory dose and at one point in time [20]. We examined the impact of ageing on the induction of effector cytokines during the response of T cells to differing stimulatory strengths after two and four days of culturing ( Figure 3 ). Stimulation of T cells with a low concentration of anti-CD3 did not result in detectable cytokine levels after two and four days in both young and old mice (data not shown). Using intermediate or high strength of stimulation, cells of young mice produced significantly higher levels of IL-2 at day two compared to cells of aged mice ( Figure 3A ). Cells of aged mice showed stronger Th2- related responses, with significantly higher IL-4 ( Figure 3B ) and IL-5 ( Figure 3C ) production compared to cells of young mice four days after exposure to intermediate or high strength stimuli. In addition, secretion of IL-10 was found only at day four, and this cytokine was produced at higher levels by cells of aged mice ( Figure 3D ). Interestingly, cells of aged mice but not young mice, produced high amounts of IL-10 in response to intermediate strength of stimulation, which indicates that aged mice require less stimulation to trigger IL-10 production compared to young mice. Detection of ageing-related differences in secretion of pro-inflammatory cytokines IL-17, IFN- γ , and TNF- α also depended on the time and strength of stimulation. Aged mice showed a trend towards higher IL-17 secretion, but only after four days and by high strength stimulation ( Figure 3E ). Aged mice also showed higher IFN- γ secretion, which became apparent only after four days of stimulation with both intermediate and high strength stimulation ( Figure 3F ). TNF- α secretion by cells of aged mice was consistently lower compared to secretion by cells of young mice ( Figure 3G ). Taken together, these results indicate that both time and the stimulatory strength are important in assessing ageing-related cytokine profiles. Measuring cytokines in supernatant does not indicate which T cell subset accounts for the cytokines produced. Therefore, we investigated the maximum potential of CD4 + and CD8 + cells to intracellularly express Th1-related (IFN- γ and TNF- α ) and Th2-related (IL-4 and IL-5) cytokines during a four-hour stimulation with PMA/ionomycin ( Supplementary Figure 2 ). Aged mice showed significantly higher frequencies of IFN- γ -producing cells in both CD4 + and CD8 + 2