Daan Pieren

58 Chapter 2 Memory CD3 + CD4 + cells (CD44 Hi ) Naive CD3 + CD4 + cells (CD44 Lo ) Memory CD3 + Tc cells (CD4 - CD44 Hi ) Naive CD3 + Tc cells (CD4 - CD44 Lo ) MemoryTh (FoxP3 - ) MemoryTreg (FoxP3 + ) Naïve Th (FoxP3 - ) Naive Treg (FoxP3 + ) % PD-1 + % CD25 + % CD122 + % GARP + A B Y A 0 2 4 6 8 10 % CD25 + of naive Th ns Y A 0 20 40 60 80 100 % PD-1 + of naive Th ** Y A 0 10 20 30 40 % CD122 + of naive Th ** Y A 0 2 4 6 8 10 % GARP + of naive Th ** Y A 0 20 40 60 80 100 % Naive Th ** Y A 0 20 40 60 % Naive Treg ** Y A 0 20 40 60 % Memory Treg ns Y A 0 20 40 60 80 100 % PD-1 + of naive Treg ** Y A 0 20 40 60 80 100 % CD25 + of naive Treg ** Y A 0 5 10 15 % CD122 + of naive Treg * Y A 0 20 40 60 % GARP + of naive Treg ** Y A 0 20 40 60 80 100 % PD-1 + of mem Th ** Y A 0 10 20 30 40 % CD122 + of mem Th * Y A 0 2 4 6 8 10 % CD25 + of mem Th ns Y A 0 2 4 6 8 10 % GARP + of naive Tc ** Y A 0 2 4 6 8 10 % GARP + of mem Tc ns Y A 0 2 4 6 8 10 % CD25 + of naive Tc ** Y A 0 2 4 6 8 10 % CD25 + of mem Tc ns Y A 0 20 40 60 80 100 % CD122 + of naive Tc ** Y A 0 20 40 60 80 100 % CD122 + of mem Tc ns Y A 0 20 40 60 80 100 % PD-1 + of naive Tc ** Y A 0 20 40 60 80 100 % PD-1 + of memory Tc ns Y A 0 5 10 15 % CD122 + of mem Treg * Y A 0 20 40 60 % GARP + of mem Treg ** Y A 0 20 40 60 80 100 % CD25 + of mem Treg ns Y A 0 20 40 60 80 100 % PD-1 + of mem Treg ** Y A 0 2 4 6 8 10 % GARP + of mem Th ns Y A 0 20 40 60 80 100 % Memory Th ns Supplementary Figure 6. Frequencies of PD-1 + , CD25 + , CD122 + , and GARP + naive and memory T cell subsets. Graphs show the frequencies of PD-1 + , CD25 + , CD122 + , and GARP + T-cell subsets used to verify results generated by viSNE. ( A ) Upper (boxed) naive and memory T-cell subsets are those that have been analyzed by viSNE. Based on FoxP3 expression, naive CD3 + CD4 + cells were subdivided into naive Th cells (CD44 Lo Foxp3 - ) and naive Treg cells (CD44 Lo Foxp3 + ), and memory CD3 + CD4 + cells were subdivided into memory Th cells (CD44 Hi Foxp3 - ) and memory Treg cells (CD44 Hi Foxp3 + ). ( B ) Frequencies of PD-1 + , CD25 + , CD122 + , and GARP + cells were determined in the naive and memory subsets of Th, Treg, and Tc cells in spleens from young (n=6, 2 months old) and aged (n=4, 28 months old) mice. Mean ± SD; * p < 0.05, ** p < 0.01, ns = not statistically significant for difference between young and aged mice using Mann- Whitney test.