KIR-Fc Binding and Jurkat Reporter Assay

KIR-Fc staining was performed as described [12 (link),13 (link),24 (link)]. Measurement of KIR-Fc binding was assessed as percentage of PE-positive cells (SUP1). KIR Jurkat reporter cell assays were performed as described [16 (link)]. Briefly, peptide pulsed 221-TAPko-HLA-C∗03 : 04 cells were coincubated with Jurkat Reporter cells expressing KIR2DL3, with the intracellular tail exchanged through CD3ξ chain resulting in activation of the cell after ligand binding in a ratio of 1 : 10. After 5 h of incubation at 37°C/5%, CO₂ cells were stained with anti-CD69-BV421 (Biolegend, Inc. San Diego, California, USA), anti-CD3-PerCP-Cy5.5 (Biolegend) and anti-KIR2DL3-APC (Miltenyi Biotec). Washed and fixed cells were analysed using flow cytometry (BD LSRFortessa: BD Biosciences Franklin Lakes, New Jersey, USA). Gates were set to only include CD3⁺/KIR2DL3⁺ cells. Activation of reporter cells was quantified as median MFI of CD69 expression normalized to background expression.