Daan Pieren

45 Novel features of ageing in murine T cells 1 2 3 4 5 6 0 20 40 60 80 1 2 3 4 5 6 7 8 9 0 20 40 60 1 2 3 4 5 6 7 0 20 40 60 80 1 2 3 4 5 6 7 8 9 10111213 0 10 20 30 40 1 2 3 4 5 6 7 8 9 10 0 20 40 60 80 1 2 3 4 5 6 7 8 9 0 20 40 60 Cell density viSNE Day 0 Treg Young Aged Day 0 Day 2 Day 4 Tc Young Aged Day 0 Day 2 Day 4 Th Young Aged Day 0 Day 2 Day 4 1 2 1 23 34 5 6 4 6 5 7 9 8 1 3 2 4 5 6 7 1 4 3 2 5 6 7 8 10 13 12 9 11 4 1 3 2 5 6 7 8 9 10 1 3 2 4 6 5 9 8 7 A Cluster size Heatmaps 0 3 6 Marker expression CD69 CD25 CD122 PD-1 1 2 3 4 5 6 7 8 9 Day 0 Day 2 Day 4 CD69 CD25 CD122 PD-1 1 2 3 4 5 6 7 8 9 10 Day 2 Day 4 Day 0 Cluster (number) Day 0 Day 2 Day 4 Day 0 Day 2 Day 4 % Cluster of total viSNE CD69 CD25 CD122 PD-1 Day 0 Day 2 Day 4 1 2 3 4 5 6 7 CD69 CD25 CD122 PD-1 1 2 3 4 5 6 7 8 9 101112 13 Day 0 Day 2 Day 4 Cluster (number) Day 0 Day 2 Day 4 Day 0 Day 2 Day 4 % Cluster of total viSNE Day 0 Day 2 Day 4 CD69 CD25 CD122 PD-1 1 2 3 4 5 6 CD69 CD25 CD122 PD-1 Day 0 Day 2 Day 4 1 2 3 4 5 6 7 8 9 Cluster (number) Day 0 Day 2 Day 4 Day 0 Day 2 Day 4 % Cluster of total viSNE B C Cell density viSNE Day 2 and 4 D Figure 6. Specific T-cell fingerprints show phenotypical alterations associated with ageing. Splenocytes of young (n=6, 2 months old) and aged mice (n=4, 28 months old) were analyzed using flow cytometry before and after stimulation for up to four days with an intermediate stimulatory strength. Cell density maps of dimensionally reduced single-cell data show clustering within either Th, Tc, or Treg cell populations of the pooled young and aged mice before stimulation (Day 0) ( A ), and cell density maps at day 2 and 4 after stimulation ( B ). Numbers within the gates correspond to the cluster numbers in the heatmaps ( C ) and bar graphs ( D ). Heatmaps depict the ArcSinh5-transformed median expression of the designated markers in Th, Tc, and Treg cell clusters at the indicated days of young and aged mice. Bar graphs indicate the cell proportions of the clusters identified by viSNE analysis within each T cell subset. Red arrows and bars indicate the non-activated (CD25 - CD69 - ) PD-1 + Th and Tc cell clusters in aged mice ( C,D ). Blue arrows and bars indicate the activated (CD25 + CD69 + ) PD-1 + Th and Tc cell clusters in aged mice ( C,D ). 2