Sara van den Berg

148 Chapter 6 CMV infection affects expression of lifespan-associated markers in the CD4+ T-cell pool CD57 and KLRG-1 expression is often associated with proliferative capacity [32, 33] and CD95 is known as a death receptor. Therefore, we assessed if, apart from the changes in T-cell numbers and phenotype, CMV infection also affected other lifespan-associated markers. T-cell proliferation was assessed by measuring the expression of Ki-67, and apoptosis sensitivity by Bcl-2 ( Figure 2A ). Both CD4 + T EM and T EMRA cells expressed significantly lower levels of Ki-67 in CMV+ compared to CMV- individuals ( Figure 2A ), suggesting that a lower percentage of cells were undergoing cell division in CMV+ individuals at the moment of blood withdrawal. Expression of the anti-apoptotic protein Bcl-2 was significantly higher in CD4 + T EM cells of CMV+ individuals ( Figure 2A ). For CD8 + T-cells, no significant differences in expression of these markers were observed between CMV+ and CMV- individuals ( Figure 2A ). As a proxy for lifespan, we combined the information of both previous flow cytometry panels (CD28, KLRG-1, CD57, Ki-67 and Bcl-2) in a principal component (PC) analysis (explaining 73.3% of the variance with two PCs). We found that CD4 + T EM and T EMRA cells in CMV+ individuals clustered separately from CD4 + T EM and T EMRA in CMV- individuals ( Figure 2B ). This was also the case for CD8 + T EM cells albeit to a lesser extent, but not for CD8 + T EMRA cells ( Figure 2C ). Thus, the effect of CMV infection on the expression of senescence and lifespan-associated markers was most pronounced for CD4 + T EM and CD4 + T EMRA cells. HLA type Sex Age CMV-specific IgG level A1/ A36 A2 A3 A24 B7 B8 CMV-specific epitope Total CMV+ 69.14 781.58 2 3 2 2 2 1 E04 Male 68.8 185.9 + - - - - + A1-VTE, B8-ELR E08 Male 66.6 191.1 + + - + - - A2-NLV E34 Male 67.7 145.1 - + - - - - A2-NLV E39 Male 66.6 123.1 - - + - + - B7-TPR E59 Female 76 3262.7 - + + + + - B7-TPR Total CMV- 69.22 1.58 1 3 3 2 2 1 E35 Female 69.8 1.8 - + - - - - N/A E30 Male 66 1.4 + - + - - + N/A E23 Male 71.3 1.6 - - + + + - N/A E21 Male 69.7 1.5 - + - - + - N/A E10 Female 69.3 1.6 - + + + - - N/A Table 1. Selected individuals for heavy water labelling study CMV-specific CD8+ T-cells do not differ from bulk memory CD8+ T-cells in terms of lifespan-associated markers We next investigated if CMV-specific CD8 + T-cells differ in their expression of lifespan- associated markers compared to bulk memory CD8 + T-cells. CMV-specific CD8 + T-cells showed significantly lower expression of Ki-67 compared to bulk memory CD8 + T-cells of CMV+ individuals ( Supplementary Figure 2D ). Also within the memory T CM subset, CMV- specific CD8 + T-cells expressed significantly less Ki-67 compared to bulk T CM cells ( Figure