Daan Pieren

43 Novel features of ageing in murine T cells Young (2 months) CD3 + T cells Aged (28 months) CD3 + T cells CD4 CD44 Naive Treg cells (CD4 + CD44 Lo FoxP3 + ) Memory Treg cells (CD4 + CD44 Hi FoxP3 + ) Naive Tc reg cells (CD4 - CD44 Lo CD122 + PD-1 + ) Memory Tc reg cells (CD4 - CD44 Hi CD122 + PD-1 + ) Young (2m) Aged (28m) Naive CD4 + cells (CD3 + CD4 + CD44 Lo ) 1 2 3 1 2 3 FoxP3 CD69 CD25 CD122 PD-1 CTLA-4 GARP 1 2 3 1 2 3 FoxP3 CD69 CD25 CD122 PD-1 CTLA-4 GARP Memory CD4 + cells (CD3 + CD4 + CD44 Hi ) 1 2 3 4 1 2 3 4 FoxP3 CD69 CD25 CD122 PD-1 CTLA-4 GARP 1 2 3 4 FoxP3 CD69 CD25 CD122 PD-1 CTLA-4 GARP 1 2 3 4 1 2 Naive CD4 - cells (CD3 + CD4 - CD44 Lo ) 2 1 FoxP3 CD69 CD25 CD122 PD-1 CTLA-4 GARP 1 2 2 1 FoxP3 CD69 CD25 CD122 PD-1 CTLA-4 GARP Memory CD4 - cells (CD3 + CD4 - CD44 Hi ) 2 1 3 FoxP3 CD69 CD25 CD122 PD-1 CTLA-4 GARP 1 2 3 2 1 3 FoxP3 CD69 CD25 CD122 PD-1 CTLA-4 GARP 1 2 3 Cluster size A B C G D E F Cluster (number) Cluster (number) Cluster (number) Cluster (number) 0 2 4 Marker expression 0 20 40 60 80 100 % CD44 Lo and CD44 Hi of CD4+ and CD4- CD3+ T cells CD4+ CD4+ CD4- CD4- *** *** *** *** Naive Naive Memory Memory Young Aged 1 2 3 0 20 40 60 80 100 %Clusterof totalviSNE 1 2 3 4 0 20 40 60 %Clusterof totalviSNE 1 2 3 0 20 40 60 80 %Clusterof totalviSNE 1 2 0 20 40 60 80 100 %Clusterof totalviSNE Y A Y A 0 20 40 60 %Treg of naiveCD4+ ** Y A 0 20 40 60 %Tregof memCD4+ ns Y A 0 20 40 60 80 100 % Tc reg of naive CD4- ** Y A 0 20 40 60 80 100 %Tc regof memCD4- ns Figure 5. Aging impacts the heterogeneity found within naive and memory T cells and en- riches the naive compartment with cells expressing a Treg phenotype. Naive (CD44 Lo ) and memory (CD44 Hi ) cells were identified in the CD4 + and CD4 - T-cell populations by expression of CD44 ( A ). Frequencies of naive and memory cells in the CD4 + and CD4 - T cell populations were determined in young (n=6, 2 months old) and aged (n=4, 28 months old) mice ( B ). Cell density maps of dimensionality reduced single-cell data show clustering within either naive or memory populations of CD4 + ( C,D ) and CD4 - T cells ( E,F ) from pooled samples of young or aged mice. Numbers in the clusters on the density maps correspond to the cluster numbers in the heatmaps shown to the right of each cell density plot ( C-F ). Bar graphs below the density and heatmaps indicate the proportions of each cluster defined within the total viSNE ( C-F ). Heatmaps depict the ArcSinh5-transformed median expression of the designated markers of the respective clusters. The frequency of Treg cells of CD4 + naive and memory cells (FoxP3 + of CD3 + CD4 + CD44 Lo/Hi ) and Tc reg cells of CD4 - naive and memory cells (PD-1 + CD122 + of CD3 + CD4 - CD44 Lo/Hi ) was analyzed by flow cytometry in young (Y) and aged (A) mice ( G ). Mean ± SD; ** p < 0.01, *** p < 0.001 for difference between young and aged mice using Mann-Whitney test. 2