Sara van den Berg

93 4 CMV on influenza infection GMBH) according to manufacturer’s instructions. Participants with a CMV antibody level of ≥12U/ml or higher were considered CMV+, those with a level of ≤8U/ml were considered CMV-, and those with a level between 8 and 12U/ml were considered inconclusive and hence excluded for further analysis. For older healthy adults and influenza virus A-infected individuals, CMV-specific antibody levels were measured in serum by an in-house-developed multiplex immunoassay [45]. Individuals with a CMV-specific antibody level of ≤4 arbitrary units/ml were considered to be CMV- and individuals with an antibody level > 7.5 RU/ml were considered CMV+, and those with a level between 4 and 7.5 arbitrary units/ml were considered inconclusive and hence excluded from further analysis. To reduce inter-assay variation, all samples from the same individual were measured on the same plate. Antigen-specific T-cells by flow cytometry Healthy individuals HLA-A2 positive healthy individuals were selected based on availability from young and old healthy individuals for subsequent influenza virus-specific T-cell analysis, by staining PBMCs for expression of HLA-A2 with the HLA-A2(BB7.2)-V450 antibody (BD Bioscience). Of the HLA-A2 positive individuals, ±4 million PBMC’s were stained using the HLA-class I tetramer for epitope GILG of the M1 protein of influenza virus (A*0201/GILGFVFTL-APC, Immudex) for 20 minutes at room temperature. Surface staining was performed for 30 minutes at 4 °C with the following antibodies: Fixable Viability Staining-780 (BD bioscience), CD3 (SK7)-AF700(BD bioscience), CD8(RPA-T8)-BrilliantViolet510, CD45RO(UCHL1)-BrilliantViolet711, CD27(O323)- BrilliantViolet786, CCR7(150503)-BrilliantUV395 (BD bioscience), KLRG-1(13F12F2)-PE-Cy7 (eBioscience), PD-1(EH12.2H7)-PerCP Cy5.5, CD95(DX2)-BrilliantViolet421 (BD Biosciences), CD127(A019D5)-BrilliantViolet650, CD57(HCD57)-PE and CXCR3(G025H7)-PE-Dazzle. All antibodies were purchased from Biolegend, unless stated otherwise. Acquisition was performed on a LSRFortessaX20 and data analysis was performed using FlowJo (Treestar). tSNE-analyses were performed using Cytobank ( www.cytobank.org ) [46]with for every donor 10.000 CD8 + T-cells. Donors with less than 10.000 CD8 + T-cells were exlcuded from t-SNE analysis (n=3). Influenza virus A-infected individuals Of all HLA-A2 positive individuals undergoing influenza virus infection (n=17), both influenza virus-specific T-cells and CMV-specific T-cells were assessed within the first 72 hours 2 and 8 weeks after fever onset. PBMCs were stained using the HLA-A2 tetramer for the GILG epitope of the M1 protein of influenza virus (A*0201/GILGFVFTL-APC, Immudex) (±8 mln PBMCs) and the HLA-A2 tetramer for the NLV epitope of the pp65protein of CMV (A*0201/NLVPMVATV)-APC (Immudex) (1 mln PBMCs) for 20 minutes at room temperature. Extracellular staining was performed for 30 minutes at 4 °C with the following antibodies: Fixable Viability Staining-780 (BD bioscience), CD3 (SK7)-AF700(BD bioscience), CD8(RPA- T8)-BrilliantViolet510 (Biolegend), CD45RO(UCHL1)-BrilliantViolet711 (Biolegend), CD27(O323)-BrilliantViolet786 (Biolegend), CCR7(150503)-BrilliantUV395(BD bioscience), KLRG-1(13F12F2)-PE-Cy7 (eBioscience), PD-1(EH12.2H7)-PerCP Cy5.5 (Biolegend),