Sara van den Berg

94 Chapter 4 CD95(DX2)-BrilliantViolet421 (BD Biosciences), CD127(A019D5)-BrilliantViolet650 (Biolegend), CD57(HCD57)-PE (Biolegend) and CXCR3(G025H7)-PE-Dazzle (Biolegend), CD38(HIT2)-PE-Dazzle (BD bioscience) and HLA-DR(TU39)-BrilliantUV737 (BD Bioscience). Analysis was performed on an LSRFortessaX20. Influenza virus-specific and CMV-specific IFNy T-cell response by ELISpot Virus-specific T-cell responses were quantified using the IFNy enzyme-linked immunospot (ELISPOT) assay at all three time points for those of which enough cells were available (respectively n=66, n=61, n=58 out of n=72). Briefly, 400.000 PBMCs were stimulated with a 15-mer peptide-pool with 11 amino acids overlap, covering the total influenza M1 protein (1 µg/ml) (JPT) and incubated for 16-20 hours at 37 °C on 96-well membrane-bottomed plates coated with anti-IFN γ mAbs. If indicated, IFN γ responses were corrected for the percentage of T-cells in lymphocytes based on flow cytometry data. For CMV responses, 100,000 PBMCs were stimulated with a 15-mer peptide-pool with 11 amino acids overlap, covering either the UL55 (1 μ g/ml) (JPT), the IE-1 (1 μ g/ml) (JPT), or the pp65 (1 μ g/ml) (JPT) CMV protein. The sum of the response to these three CMV peptide pools is presented in this study. Cytokine and chemokine levels in serum Cytokines and chemokine levels in serum were assessed for all influenza virus-infected individuals within 72 hours, and 2 and 8 weeks after fever onset. Levels were measured by bead-based multiplex LEGENDplex TM (BioLegend) according to the manufacturer’s instructions. The pro-inflammatory cytokines IL-6, IFNy, IL-10 and CRP and chemokines CXCL-10, CXCL-9 and CXCL-11 were analyzed for this study. Reactions were performed in duplicate. Analysis was performed on a Canto II. Data were analyzed via Legendplex V8.0 software (Biolegend). All data were transformed into averages of the logarithms of two measurements, and each data point was corrected by subtraction of the within-run averages to correct for batch effects. Severity of symptoms assessment Symptom assessment was performed by the participants, by filling in a diary during influenza virus infection. Presence and duration (start date and end date) of the following symptoms were collected: fever (≥37.8 °C), cough, sore throat, runny nose, headache, pain while breathing and muscle pain. Symptoms were noted in hindsight maximum 10 days before onset of fever, as symptoms before then could not have been related to the influenza virus infection. Z-values, e.g. the number of standard deviations by which the value of the score of an individual is above or below the mean value were calculated for each symptom. Overall severity of symptoms during influenza virus infection was assessed as the average of the Z-values of the seven symptoms. Statistical analysis Differences between groups (for example CMV- compared to CMV+) were assessed using Mann-Whitney U test, and comparisons within the same individuals with the Wilcoxon