An improved method to quantify human NK cell-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) per IgG FcR-positive NK cell without purification of NK cells

摘要

Abstract Natural killer (NK) lymphocyte ADCC supports anti-viral protection and monoclonal antibody (mAb) anti-tumor therapies. To predict in vivo ADCC therapeutic responses of different individuals, measurement of both ADCC cellular lytic capacity and their NK cellular receptor recognition of antibodies on ‘target’ cells are needed, using clinically available amounts of blood. Twenty ml of blood provides sufficient peripheral blood mononuclear cells (PBMCs) for the new assay for lytic capacity described here and for an antibody EC 50 assay for Fc-receptor recognition. For the lytic capacity assay, we employed flow cytometry to quantify the CD16A IgG Fc-receptor positive NK effector cells from PBMCs to avoid loss of NKs during isolation. Targets were 51 Cr-labeled Daudi B cells pretreated with excess obinutuzumab type 2 anti-CD20 mAb and washed; remaining free mAb was insufficient to convert B cells in the PBMCs into ‘targets’. We calculated: the percentage Daudis killed at a 1:1 ratio of CD16A-positive NK cells to Daudis (CX1:1); lytic slopes; and ADCC 50 lytic units. Among 27 donors, we detected wide ranges in CX1:1 (16–73% targets killed) and in lytic slopes. Slope variations prevented application of lytic units. We recommend CX1:1 to compare individuals' ADCC capacity. CX1:1 was similar for purified NK cells vs . PBMCs and independent of CD16A V & F genotypes and antibody EC 50 s. With high mAb bound onto targets and the high affinity of obinutuzumab Fc for CD16A, CX1:1 measurements discern ADCC lytic capacity rather than antibody recognition. This assay allows ADCC to be quantified without NK cell isolation and avoids distortion associated with lytic units. Graphical abstract Display Omitted Highlights ? NK cell ADCC lytic capacity differs from NK cell recognition of antibodies. ? PBMCs as ADCC effectors with type 2 αCD20 obinutuzmab pretreated Daudi targets. ? Flow cytometry with TruCount beads to quantify CD16pos NK cell effectors in PBMCs. ? ADCC lytic capacity calculated as CX1:1 [cytotoxicity at 1 ADCC effector: 1 target]. ? Individuals' 4h CX1:1 values (and lytic slopes) vary 5-fold among healthy donors.