Daan Pieren

42 Chapter 2 Therefore, ageing-related changes may be found beyond the shift from naive towards memory cell pools. Increased variation in the responsiveness of T-cell subsets of aged mice We next investigated the phenotypical changes that occur within Th, Tc, and Treg cell subsets after exposure to intermediate strength of stimulation ( Figure 6A, B ). viSNE analysis of Th, Tc, and Treg cell subsets before stimulation indicated ageing-related differences similar to analyses presented in Figure 5 : Cells of aged mice showed increased expression of PD-1, presence of CD122 + PD-1 + Tc cells, and diminished CD25 expression by Tregs (Day 0, Figure 6A, C, D, Supplementary Figure 7 ). After stimulation, the number of phenotypically distinct clusters identified in Th cells and Tc cells was higher among cells from aged mice (six Th cell clusters, nine Tc cell clusters) compared to the number of clusters found in Th cells and Tc cells from young mice (four Th clusters, four Tc clusters) ( Figure 6B ). The number of Treg cell clusters was comparable between young and aged mice (six clusters) ( Figure 6B ). Differences within each T cell subset of young mice were mainly found in the expression levels of CD69, CD25, CD122, PD-1, and CTLA-4 after stimulation ( Figure 6C, Supplementary Figure 7 ). Th, Tc, and Treg cells of young mice showed activation-induced PD-1 and CD25 expression which remained present over time, while their CTLA-4, GARP, CD69 expression was high at day two and had declined by day four ( Figure 6C, Supplementary Figure 7 ). Compared to young mice, Th and Tc clusters of aged mice showed greater phenotypical diversity. Almost all Tc cells of aged mice constitutively expressed CD122 but lost this expression after stimulation, while CD122 on Tc cells of young mice increased over time after stimulation. Moreover, among both Th and Tc-cell subsets, distinct activated (CD25 + CD69 + ) and non-activated (CD25 - CD69 - ) clusters were identified. Distinct activated cell clusters (CD25 + CD69 + ) expressing PD-1 were identified within both the Th and Tc subsets of aged mice after stimulation (Th and Tc, Figure 6C , blue arrows; Figure 6D , blue bars). In contrast, also CD25 - CD69 - clusters of PD-1 + Th and Tc cells were present (Th and Tc, Figure 6C , red arrows; Figure 6D , red bars). For Treg cells, aged mice also showed a cluster of CD25 - CD69 - cells expressing PD-1 after two days of stimulation, but this cluster was not identified at day four (Treg, Figure 6C ). These data demonstrate that the group of aged mice shows more variation in response to stimulation compared to the group of young mice. These differences are not likely to be due to differences in abundance of naive and memory cells, as PD-1 expression increased with age within both these compartments.