Daan Pieren

41 Novel features of ageing in murine T cells CD44 ( Figure 5A ). Aged mice indeed showed decreased naive and increased memory proportions in the CD4 + and CD4 - T-cell compartments ( Figure 5B ). We next aimed to assess T-cell heterogeneity within naive and memory T-cell subsets. We characterized phenotypically distinct clusters of T cells within the naive and memory cells among the CD4 + and CD4 - compartments of the T-cell pool by application of dimensionality reduction (viSNE) ( Supplementary Figure 4 ). This panel included the ageing-related markers PD-1 and CTLA-4, as well as the activation markers CD25, CD69, CD122, and Glycoprotein A repetitions predominant (GARP). Cluster analysis of CD4 + and CD4 - cells revealed heterogeneity within naive and memory cells, which differed between young and aged mice ( Figure 5C-F ). FoxP3, PD-1, CD25, and CD122 accounted for the heterogeneity within these subsets. Cluster 1 of naive and memory CD4 + T cells shows high expression of FoxP3, indicating that this cluster is highly enriched for Treg cells ( Figure 5C, D ). The proportion of cluster 1 in naive CD4 + T cells is larger in aged mice (40.9%) compared to young mice (10.4%) ( Figure 5C ), while in memory CD4 + T cells the proportion of cluster 1 is comparable between young (22.0%) and aged mice (24.9%) ( Figure 5D ). The increased proportion of CD4 + Treg cells among the naive CD4 + cells is reflected in individual aged mice ( Figure 5G ), while there is no ageing-related difference in Treg cell proportions among the memory T-cell pool. Previous studies have described a population of regulatory Tc cells (Tc reg cells) expressing CD122 and PD-1 [41,42]. Interestingly, cluster 1 of naive CD4 - Tc cells is CD122 + PD-1 + with a higher proportion of this cluster in the pool of aged mice ( Figure 5E ), which is reflected in individual mice ( Figure 5G, Supplementary Figure 5 ). The proportion of these Tc reg cells among the memory CD4 - pool is comparable between young and aged mice ( Figure 5F, G ). Finally, expression of PD-1 contributed to heterogeneity within naive and memory cell subsets ( Figure 5C-F ). This heterogeneity differed with age as aged mice showed higher frequencies of PD-1 + in naive Th, Tc, and Treg cells, and in memory Th and Treg cell subsets compared to young mice ( Supplementary Figure 6 ). Additionally, aged mice showed lower frequencies of CD25 + naive Treg cells and lower frequencies of GARP + naive and memory Treg cells ( Supplementary Figure 6 ). Thus, naive and memory T cells both are highly heterogeneous and this heterogeneity is changed by ageing. Importantly, ageing influences constitution of these subsets by increasing the expression of PD-1 and by enriching naive CD4 + and CD4 - cell subsets with cells expressing a regulatory phenotype. 2