Cd3 sk7 af700

HLA-A2 positive healthy individuals were selected based on availability from young and old healthy individuals for subsequent IAV-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 dextramer for epitope GILG of the M1 protein of IAV (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) (53 (link)) with for every donor 10.000 CD8⁺ T cells. Donors with less than 10.000 CD8⁺ T cells were excluded from t-SNE analysis (n=3).

Of all HLA-A2 positive individuals undergoing IAV infection (n=36), both IAV-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 dextramer for the GILG epitope of the M1 protein of IAV (A*0201/GILGFVFTL-APC, Immudex) (± 8 mln PBMCs) (detected in n=17) and the HLA-A2 dextramer 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), 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.