Sara van den Berg

100 Chapter 4 with age and is associated with decreased protection against influenza [41, 48], we assessed this ratio. No difference was observed ( Figure 4B ) in the IFN γ :IL-10 ratio between CMV+ and CMV- individuals at any of the time points, despite that both IFN γ and IL-10 levels were elevated at the earliest timepoint ( Supplementary Figure 4 ) . Although increased levels of the cytokines IL-6, CRP, IFN γ and IL-10 were observed in the acute phase of influenza virus infection, no association between these cytokine levels and the height of the influenza virus- specific T-cell response was observed at time point 1 ( Figure 4C ) or 2 weeks later (data not shown). Together, this suggests that CMV infection in older adults does not affect cytokine levels in serum thereby influencing the influenza virus specific T-cell response. Figure 3. No reduced influenza-specific T-cell response in number of T-cells. Influenza virus-specific IFN γ T-cell response in CMV- individuals and CMV+ individuals differentiated on the CMV-specific T-cell response. Influenza-specific IFN γ T-cell responses are depicted in blue open circles in scatter plot for CMV- individuals and in red solid circles in correlation of CMV-specific IFN γ T-cell responses for CMV+ individuals . CMV-specific T-cell responses were measured by ELISPOT for IFN γ spots to overlapping pp65, IE-1 and UL55 peptide pools. Differences were tested using Mann-Whitney to compare CMV+ and CMV- individuals. Correlation between CMV-specific T-cell responses and IFN γ response to influenza was tested by Spearman correlation. T-cell IFN γ responses are presented per 1x10 6 PBMCs. Severity of symptoms of influenza virus infection is not increased by CMV Since early T-cell responses during influenza virus infection play an important role in limiting disease severity [24, 25], we also investigated whether the height of the influenza virus- specific T-cell response was associated with severity of symptoms of influenza virus infection. In influenza virus-infected older adults, the number and duration of seven symptoms was assessed (fever (≥37.8 °C), cough, sore throat, runny nose, headache, pain while breathing and muscle pain). The severity of symptoms of influenza virus infection was positively associated with the height of the influenza virus-specific T-cell response at timepoint 3 (8 weeks after start of fever) ( Figure 5A ), but not at time point 1 (<72 hours after start of fever) or 2 (2 weeks after start of fever). The positive association between the height of the T-cell response against influenza at week 8 after start of fever with severity of symptoms was mainly based on the number of symptoms, and not on duration of symptoms ( Supplementary Figure 5 ) and not associated with CMV-specific antibody levels(data not shown). Thus, a T-cell response 8 weeks after infection, and not an early T-cell response, is linked to the severity of symptoms of the influenza virus infection in this cohort.