Daan Pieren

152 Chapter 5 0 20 40 60 0 20 40 60 80 100 % T EMRA in total CD8 + Relative Proliferation % Total CD8 + 0 1 2 3 4 0 20 40 60 80 100 % NKG2A + RA + in total CD8 + Relative Proliferation % Total CD8 + T N T CM T EM T EMRA KIR + RA + NKG2A + RA + 0 10 20 30 % Polyfunctional (IFNy + TNFa + IL-2 + ) cells in subset ns ns Sorted CD8 + T-cell subsets r = -0.075 p = 0.61 r = -0.43 p = 0.002 r = -0.29 p = 0.04 r = -0.02 p = 0.87 r = 0.63 p < 0.001 A B CD69 (BV786) 74.2 KIR - NKG2A + KIR + NKG2A - KIR - NKG2A - CD8 (BUV395) 2.86 70.9 0 20 40 60 80 0 20 40 60 80 100 % T NAIVE in total CD8 + Relative Proliferation % Total CD8 + 0 20 40 60 80 0 20 40 60 80 100 % T CM in total CD8 + Relative Proliferation % Total CD8 + 0 10 20 30 40 50 0 20 40 60 80 100 % T EM in total CD8 + Relative Proliferation % Total CD8 + C Supplementary Figure 5. Analysis of the KIR + RA + T-cell response. Accumulation of intracellular cytokines was measured after exposure of total PBMCs of healthy individuals (n=10) to PMA/Ionomycin for six hours. ( A ) Frequency of polyfunctional (IFN- γ + IL-2 + TNF- α + ) cells within the indicated CD8 + T-cell subsets (relative to medium control). ( B ) Frequency of CD69 + cells in sorted CD8 + KIR - NKG2A - T cells, CD8 + CD45RA + KIR + NKG2A + T cells, and CD8 + CD45RA + KIR - NKG2A + T cells after culturing with anti-CD3/anti-CD28 beads (1:12 bead-to-cell ratio) for three days (representing findings for n=4). ( C ) Relationships between the frequency of proliferated CD8 + T cells (relative to medium control) and ex vivo frequency of T NAIVE , T CM , T EM , T EMRA , or NKG2A + RA + cells within the total CD8 + T-cell population of healthy individuals (20-82 years of age, n=50). Correlations ( r values) were assessed by Spearman test. Statistical significance of data presented in the bar graph (means ± s.d.) was determined using row-matched one-way ANOVA (with Geisser-Greenhouse correction and Dunnett’s post-test). (ns=not significant).