Daan Pieren

40 Chapter 2 quickly upregulated CD69 with a stimulatory dose-dependent increase, but never reached the maximum CD69 + cell frequencies of young cells. In contrast, Treg cells of old mice expressed similar or higher frequencies of CD69 + cells than Tregs of young mice after stimulation. Moreover, after one to two days of culture, all three subsets of young mice showed a decline in the frequency of CD69 + cells. This decline was less pronounced on the T cells of aged mice, as shown by persistent CD69 + cell frequencies, leaving stimulation-induced frequencies of CD69 + cells observed at later time points of the culture higher in the aged mice than in the young mice. Thus, these data indicate that activation kinetics of T cells at old age can in part be improved by increasing the stimulatory strength. However, a substantial fraction of the Th and Tc cells of aged mice still refrain from expressing CD25 or CD69. T cells of aged mice do not proliferate in response to exogenous IL-2. T cells of aged mice showed diminished proliferation, lower IL-2 secretion, and altered CD25 expression kinetics in response to stimulation ( Figure 2, 3, and 4 ). As IL-2 is an important driver of T cell proliferation, we hypothesized that the deficiency of IL-2 in culture may contribute to the diminished T cell proliferation observed in aged mice. Therefore, we assessed whether exogenous IL-2 could overcome the IL-2 deficiency and partially restore defective proliferation of T cells at old age. We stimulated cells of young and aged mice with a low stimulatory strength of anti-CD3 +/- exogenous recombinant murine IL-2 and monitored Th, Tc, and Treg cell proliferation ( Supplementary Figure 3 ). Addition of exogenous IL-2 did not enhance proliferation in T cell subsets of aged mice, whereas it enhanced proliferation of Th and Tc cells of young mice ( Supplementary Figure 3 ). Ageing influences naive and memory CD4 + and CD4 - T-cell heterogeneity by enrichment for regulatory cell types Age-related differences found in T cell responses have largely been ascribed to the shifted balance of naive T cells towards memory T cells during ageing rather than an ageing-related effect within these cell subsets [2]. We and others observed changes in the composition of the T cell pool at old age other than only a shifted naive/memory balance, such as increase of the Treg cell frequency [40] ( Figure 1D ). We therefore assessed if the impact of ageing may be reflected beyond the mere shift in balance of naive/memory T cells and may be further defined by changes of the heterogeneity within naive and memory T-cell populations. To address this question, we first investigated the proportion of naive and memory cells in young and aged mice based on expression of