Daan Pieren

57 Novel features of ageing in murine T cells Memory Tc cells (CD3 + CD4 - CD44 Hi ) Naive Tc cells (CD3 + CD4 - CD44 Lo ) Viable CD3 + cells Naive CD4 + cells Memory CD4 + cells Naive Treg cells (CD3 + CD4 + CD44 Lo FoxP3 + ) Naive Th cells (CD3 + CD4 + CD44 Lo FoxP3 - ) Memory Treg cells (CD3 + CD4 + CD44 Hi FoxP3 + ) Memory Th cells (CD3 + CD4 + CD44 hi FoxP3 - ) FoxP3 FoxP3 CD4 CD4 CD44 CD4 A B C Supplementary Figure 4 Supplementary Figure 4. Gating strategy towards naive and memory Th, Tc, and Treg cell subsets for viSNE analyses. For our viSNE analysis we gated on naive and memory T cell subsets based on CD44 expression, here shown for one young mouse. Viable CD3 + T cells were gated on ( A ) naive Tc cells (CD3 + CD4 - CD44 Lo ), memory Tc cells (CD3 + CD4 - CD44 Hi ), naive CD4 + T cells (CD3 + CD4 + CD44 Lo ), and memory CD4 + T cells (CD3 + CD4 + CD44 Hi ). On these four subsets, we performed the viSNE analyses shown in Figure 5. Based on the viSNE analyses, we further divided ( B ) naive CD4 + T cells (CD3 + CD4 + CD44 Lo ) and ( c ) memory CD4 + T cells (CD3 + CD4 + CD44 Hi ) into naive Treg cells (CD3 + CD4 + CD44 Lo FoxP3 + ), memory Treg cells (CD3 + CD4 + CD44 Hi FoxP3 + ), naive Th cells (CD3 + CD4 + CD44 Lo FoxP3 - ), and memory Th cells (CD3 + CD4 + CD44 Hi FoxP3 - ). PD-1 CD122 Naïve Tc reg cells (CD3 + CD4 - CD44 Lo ) Memory Tc reg cells (CD3 + CD4 + CD44 Hi ) Young (2 months) Aged (28 months) 3.5 60.0 10.5 15.9 Supplementary Figure 5 Supplementary Figure 5. Gating strategy of regulatory Tc cells. Plots show gating of naive and memory Tc reg cells by expression of CD122 and PD-1 in a young (2 months old) and an aged (28 months old) mouse that represent their age group. Based on simultaneous expression of CD122 and PD-1 to identify Tc reg as previously reported, naive Tc reg cells were defined as CD3 + CD4 - CD44 Lo CD122 + PD-1 + and memory Tc reg cells were defined as CD3 + CD4 - CD44 Hi CD122 + PD-1 + . 2