Tetramer Generation and Validation for CD8+ T Cell Analysis

pHLA class-I (pHLA-I) tetramers were generated by refolding each peptide with its restricted HLA α-heavy chain-BirA and β2-microglobulin^{53 (link),85 (link),86 (link)} before 8:1 conjugation with PE- or APC-streptavidin (BD, cat #554061 and #554067 respectively) to generate tetramers; B7/NP₃₀, B7/NS1₁₉₆, B8/NP₃₀, B8/NP₉₂, B8/NP₄₇₉, B35/HA₂₃₁ and B35/NS1₂₆₀. To test newly generated tetramers, 0.2–0.4 × 10⁶ frozen IBV pool-specific CD8⁺ T cells were thawed in RF10 supplemented with 2 µg/ml DNAse. Cells were incubated with anti-human FcR block (Miltenyi Biotec) for 15 min and stained with tetramer at room temperature for 1 h before surface staining on ice for 30 min with anti-CD3-BV510 (1:200, BioLegend #317332), anti-CD4-BV650 (1:200, BD Horizon #563875), Live/Dead NIR (1:800, Invitrogen #L34976) and anti-CD8-PerCP-Cy5.5 (1:50, BD Pharmingen #565310). Cells were then fixed with 1% paraformaldehyde (PFA) (Electron Microscopy Sciences) for 20 min on ice, acquired on an LSRII Fortessa and analyzed with FlowJo v10.8.1.