Daan Pieren

148 Chapter 5 SUPPLEMENTAL FIGURES 0 20 40 60 80 100 0 20 40 60 80 % T N within total CD8 + 0 20 40 60 80 100 0 20 40 60 80 % T CM within total CD8 + 0 20 40 60 80 100 0 10 20 30 40 50 % T EM within total CD8 + 0 20 40 60 80 100 0 20 40 60 % T EMRA within total CD8 + r = -0.525 p < 0.001 r = 0.001 ns r = 0.461 p < 0.001 r = 0.461 p < 0.001 T NAIVE cells T CM cells T EM cells T EMRA cells B Age (years) A Lymphocytes Single cells Live CD3 + T cells CD8 + T cells CD45RA separation KIR and NKG2A subsets T NAIVE and T EMRA T EM and T CM CD45RA CD27 CD8 KIR NKG2A CD4 SSC-A FSC-A FSC-A FSC-H Live/Dead CD3 T EM T CM CD27 CD3 CD27 CD3 NKG2A + RA + T NAIVE T EMRA KIR + RA + Supplementary Figure 1. Gating strategy of CD8 + T-cell subsets and their proportion with age ( A ) Gating strategy towards the six CD8 + T-cell subsets in healthy individuals (n=50, 20-82 years of age) using flow cytometry. CD8 + T cells were identified based on gating of total lymphocytes, single cells, and live CD3 + T cells. CD8 + T cells were then separated into CD45RA + and CD45RA - cells. Within the CD45RA + T-cell population, KIR + RA + T cells (CD3 + CD8 + CD45RA + KIR + NKG2A - , blue gate) and NKG2A + RA + T cells (CD3 + CD8 + CD45RA + KIR - NKG2A + , orange gate) were identified. Remaining CD45RA + KIR - NKG2A - cells were divided into naive T cells (T NAIVE : CD3 + CD8 + CD45RA + KIR - NKG2A - CD27 + ) and terminally differentiated effector memory cells re-expressing RA (T EMRA : CD3 + CD8 + CD45RA + KIR - NKG2A - CD27 - ). CD45RA - CD8 + T cells were separated into central memory cells (T CM : CD3 + CD8 + CD45RA - CD27 + ) and effector memory cells (T EM : CD3 + CD8 + CD45RA - CD27 - ). ( B ) Relationship between the frequency of T NAIVE , T CM , T EM , or T EMRA cells within the total CD8 + T-cell population and age. Correlations ( r values) were assessed by Spearman test.