Anti cd127 pe cy7

For flow cytometric analysis of surface markers, single-cell suspensions of lungs were incubated with a mixture containing anti-FcγRIII/II antibody (Biolegend) as well as mouse, rat, and hamster serum to block nonspecific binding. Cells were then incubated with optimal concentrations of the following specific antibodies against surface molecules: anti-CD90.2-APC-eFluor780, anti-CD127-PE-Cy7 (all from eBioscience), anti-CD44-FITC (Biolegend), anti-CD4-V500, anti-CD62L-APC, and anti-KLRG1-BV711 (all from BD Biosciences). For intracellular cytokine staining, 0.8 × 10⁶ cells were stimulated with plate-bound anti-CD3/anti-CD28 (each 5 µg/ml, BD Bioscience) or H1 (5 μg/ml) for 4.5 h in the presence of GolgiPlug™ (BD Biosciences). Cell were stained with optimal concentrations of anti-CD4-V500 (BD Biosciences), anti-CD44-FITC (Biolegend), and anti-CD90.2-APC-eFluor780 (eBioscience). Afterward cells were fixed and permeabilized with Cytofix/Cytoperm™ (BD Biosciences). Intracellularly accumulated cytokines were stained with anti-IFN-γ-PE (Biolegend) or anti-IFN-γ-V450 (BD Bioscience), respectively, anti-TNF-PE-Cy7 (Biolegend), anti-IL-17A-PerCP-Cy5.5 (eBioscience), and anti-IL-2-APC (BD Biosciences). Data were acquired on a FACSCanto™II (BD Bioscience) or on a LSRII (BD Bioscience) and analyzed with the FCS Express 5 Flow Cytometry software (DeNovo™ Software).

For HLA-0A2, HLA-B35, HLA-B27 and HLA-B07 positive participants identified using polymerase chain reaction, yellow fever specific CD8⁺ cells were identified using the tetramers described above. Twenty μL tetramer mix were added to 1–2 million cells per well in a 96-wells plate. After incubation for 30 minutes at 4°C, 30 μL of antibody mix including anti-CD3 V500 (BD Biosciences, (San Jose, CA, USA)), anti-CD8 BV785, anti-CD45RA BV650 from Biolegend (San Jose, CA, USA) anti-CD27 APC-eFluor 780 and anti-CD127 PE-Cy7 from eBioscience (San Diego, CA, USA) and Live/Dead fixable red cell stain kit (Invitrogen, Carlsbad, CA, USA) were added for 30 minutes. For intracellular staining, cells were fixated with the Fixation solution (eBioscience) for 20 minutes at room temperature and permeabilized with permeabilization solution (eBioscience). Cells were washed twice and a mix of intracellular antibodies comprising anti-Eomes PerCP-eFluor710 from BD Biosciences, anti-Ki67 BV711, anti-T-bet AF647 from Biolegend, anti-granzyme B AF700 from eBioscience, and anti-granzyme K PE from Immunotools (Friesoythe, Germany) was added for 30 minutes. Cells were then washed and re-suspended in 100 μl PBEA to be measured.