Anti cd69 bv421

HLA-DP-expressing Jurkat E6.1 cells were co-incubated with NKp44ζ⁺, NKp46ζ⁺ Jurkat reporter cells or untransduced Jurkats at an effector to target ratio of 1:10 for 5 h at 37 °C/5% CO₂. For peptide-pulsing experiments, HLA-DP-expressing Jurkat E6.1 cell lines were washed twice with serum-free medium and pulsed with 100 µM CLIP peptide (sequence: LPKPPKPVSKMRMATPLLMQALPM; GenScript) or an equivalent amount of DMSO for 18 h at 37 °C/5% CO₂. After peptide pulsing, JE6.1-DP cells were washed and co-incubated with Jurkat reporter cell lines. Subsequently, cells were stained with anti-CD3-BUV737 (BD Biosciences), anti-NKp44-PE (Biolegend) or anti-NKp46-PE (BD Biosciences), anti-CD69-BV421 (Biolegend), LiveDead NearIR (Life Technologies) and anti-HLA-ABC-APC (Biolegend), to discriminate between HLA-DP-expressing JE6.1 and Jurkat reporter cells, for 30 min at 4 °C. Cells were fixed with 4% PFA and analyzed on a BD LSR Fortessa. HLA-DP and CLIP surface expression of JE6.1-DP cells was assessed by staining CLIP-pulsed and DMSO-pulsed JE6.1-DP cells with anti-CD3-BUV737 (BD Biosciences), anti-HLA-DP-APC (Leinco Technologies), anti-CLIP-FITC (BD Biosciences) and LiveDead NearIR (Life Technologies) for 30 min at 4 °C. Cells were subsequently fixed and analyzed on a BD LSR Fortessa. (A detailed list of antibodies used is displayed in Supplementary Table 2.)