Sara van den Berg

96 Chapter 4 differentiated cells expressing KLRG-1 high CD57 medium were significantly increased in CMV+ individuals ( Figure 1B, Supplementary Figure 1A ). We next performed more detailed analysis on the expression of senescence-associated markers within the two age groups: young adults (20-52 years) and older adults (older than 60 years). CMV infection was associated with significantly increased expression of CD57 and KLRG-1 among older adults, but not in young adults ( Figure 1C,D ). Likewise, expression of PD-1 was only significantly reduced in CMV+ individuals (as compared to CMV-) in the older group, and not in the younger group ( Figure 1E ). In line with this, CMV infection was associated with increased frequencies of T EMRA cells in older individuals, but not in young individuals ( Figure 1F ). Together, this indicates that CMV infection establishes large changes in the CD8 + T-cell pool by inducing terminally differentiated and senescent T-cells mostly in older adults. Frequency of influenza virus-specific T-cells is decreased in CMV+ individuals, but only in older adults To investigate the hypothesis that CMV infection may negatively influence the immune response to other pathogens by outcompeting other antigen-specific T-cells, the frequency of influenza virus-specific T-cells in healthy individuals was determined using HLA-A2 tetramers containing the matrix protein-1 GILG-epitope. The frequency of influenza virus- specific T-cells was significantly lower in CMV+ compared to CMV- individuals (P=0.0005) ( Figure 1G ). Importantly, this lower percentage of influenza virus-specific T-cells was solely explained by a lower frequency in the older group, where some donors had no detectable HLA-A2 GILG-specific T-cells. Among the young adults no differences in the frequencies of influenza virus-specific T-cells between CMV- and CMV+ individuals were observed ( Figure 1H ). Thus, CMV infection results in lower frequencies of influenza virus-specific memory T-cells, but only in older adults. Characterization of the T-cell response during influenza virus infection in older adults To investigate the effect of CMV infection on the T-cell response during influenza virus infection in older adults, we characterized the T-cell response to influenza virus infection during the acute phase of infection, i.e. within 72 hours after start of fever, and 2 and 8 weeks later. The frequency of influenza virus-specific CD8 + T-cells was determined by tetramer staining for the HLA-A2 GILG-epitope. Influenza virus-specific CD8 + T-cell numbers were increased upon influenza virus infection at the 2 week time point compared to <72 hours after fever onset (median 0.03% to 0.15% of total CD8 + T-cells respectively), after which the response contracted (median of 0.08/total CD8 + T-cells) ( Figure 2A ). The increase in influenza virus-specific CD8 + T-cell numbers was mainly explained by the expansion of influenza virus-specific effector memory T-cells (T EM ) ( Figure 2B ), while influenza virus- specific T-cell numbers in the other subsets (i.e. naïve (T N ), central memory (T CM ) and T EMRA ) did not increase (data not shown). IFN γ responses after in vitro stimulation of PBMCs with peptide pools covering the influenza matrix-protein-1 revealed similar results ( Figure 2C ).