Daan Pieren

136 Chapter 5 0 10 20 30 40 0 20 40 60 80 100 % KIR + RA + T cells in CD8 + T cells % CD8 + T-cell proliferation (relative to control) 0 20 40 60 80 100 0 20 40 60 80 100 Age (years) % CD8 + T-cell proliferation (relative to control) A B C I E CD8 (BUV-395) 38.9 48.4 25.6 CD69 (BV786) KIR - NKG2A + KIR + NKG2A - KIR - NKG2A - H F CellTrace (BV421) KIR - NKG2A - KIR - NKG2A + KIR + NKG2A - r = -0.618 p = <0.001 r = -0.655 p <0.001 G NKG2A (APC) 21.6 73.5 4.3 5.5 89.1 5.3 + aCD3 Unstimulated KIR (PE) D 0 20 40 60 80 100 % CD69 + cells of subset - + * aCD3 - + - + KIR - NKG2A - KIR + NKG2A - KIR - NKG2A + * ns - + - + - + 0 20 40 60 80 100 % Subset in CD8 + T cells ns **** **** aCD3 KIR - NKG2A - KIR + NKG2A - KIR - NKG2A + KIR - NKG2A - KIR + NKG2A - KIR - NKG2A + 0 20 40 60 80 100 % Subset proliferation (relative to control) * *** * T N T CM T EM T EMRA KIR + RA + NKG2A + RA + 0 20 40 60 80 % CD107a + cells of subset (relative to control) *** *** *** ** ** T N T CM T EM T EMRA KIR + RA + NKG2A + RA + 0 20 40 60 80 100 % IFNy + cells of subset (relative to control) * ns ns T N T CM T EM T EMRA KIR + RA + NKG2A + RA + 0 20 40 60 80 % IL-2 + cells of subset (relative to control) * ns ns ****** T N T CM T EM T EMRA KIR + RA + NKG2A + RA + 0 20 40 60 80 % TNFa + cells of subset (relative to control) ** ns ns Figure 4. KIR + RA + T cells are responsive to stimulation but have low proliferative capacity. Accumulation of intracellular cytokines was measured after exposure of total PBMCs of healthy individuals (n=10) to PMA/Ionomycin for six hours. Frequency of ( A ) CD107a + , ( B ) IL-2 + , ( C ) IFN- γ + , and ( D ) TNF- α + cells within the indicated CD8 + T-cell subsets (relative to medium control as calculated by % positive cells cultured with PMA/ionomycin minus % positive cells in medium control). ( E ) PBMCs of healthy individuals (n=15) were cultured for three days with (+) or without (-) stimulatory anti-CD3, after which the frequency of CD69 + cells was determined in CD8 + T cells that were KIR - NKG2A - , KIR + NKG2A - , or KIR - NKG2A + as shown in the bar graph and representative flow cytometry plots. CellTrace-labeled PBMCs of healthy individuals (n=50) were cultured with stimulatory anti-CD3 for three days to detect proliferation of CD8 + T cells. Relationship between the frequency of proliferated CD8 + T cells (relative to medium control) and ( F ) age, and ( G ) ex vivo frequency of KIR + RA + cells within the total CD8 + T-cell population.