title: Rapid, flow cytometric assay for NK alloreactivity reveals exceptions to rules governing alloreactivity.
abstract: Alloreactive NK cells lyse target cells lacking self-HLA-C or the HLA-B-Bw4 epitope. Prior to haploidentical stem cell transplants, donor alloreactivity toward the patient is evaluated by natural killer (NK) cloning followed by testing of the clones in the (51)Cr-release assay. As only a few percent of NK clones are alloreactive, a large number of NK clones must be established and evaluated. This approach is laborious and time consuming, with a complete evaluation taking up to 6 weeks. We developed a flow cytometry-based cytotoxicity assay utilizing CD107a expression on 12-day polyclonally expanded NK cells and showed that NK alloreactivity mediated by inhibitory and activating KIR can be detected by measuring CD107a expression following incubation with targets lacking the appropriate class I epitope. The percentage of alloreactive NK cells varied greatly between individuals and was easily estimated by the CD107a assay. For each epitope (C1, C2, Bw4), donors were found who did not have alloreactivity, although alloreactivity was predicted by the current rules thought to govern alloreactivity. The data emphasize the importance of demonstrating alloreactivity in a functional assay.
authors.affiliation: School of Pathology and Laboratory Medicine, University of Western Australia, Nedlands, Australia.
authors.forename: Dianne
authors.initials: D
authors.lastname: De Santis
authors.forename: Bree A
authors.initials: BA
authors.lastname: Foley
authors.forename: Elisabeth
authors.initials: E
authors.lastname: John
authors.forename: David
authors.initials: D
authors.lastname: Senitzer
authors.forename: Frank T
authors.initials: FT
authors.lastname: Christiansen
authors.forename: Campbell S
authors.initials: CS
authors.lastname: Witt
mesh_terms: D018797:Cell Cycle Proteins / Q000378:metabolism; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D003601:Cytotoxicity Tests, Immunologic / Q000379:methods*; D003602:Cytotoxicity, Immunologic; D018985:Epitopes, B-Lymphocyte / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D005838:Genotype; D006087:Graft vs Host Reaction / Q000276:immunology*; D006680:HLA Antigens / Q000378:metabolism; D015395:Histocompatibility Antigens Class I / Q000378:metabolism; D006650:Histocompatibility Testing / Q000379:methods*; D006801:Humans; D007376:Interleukin-2 / Q000276:immunology; D007694:Killer Cells, Natural / Q000276:immunology*; D008024:Ligands; D008213:Lymphocyte Activation; D052118:Lysosomal-Associated Membrane Protein 1 / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D054340:Receptors, KIR / Q000235:genetics / Q000378:metabolism; D013997:Time Factors
chemical_list: D018797:Cell Cycle Proteins; D018985:Epitopes, B-Lymphocyte; C115270:G2A receptor; D006680:HLA Antigens; D015395:Histocompatibility Antigens Class I; D007376:Interleukin-2; D008024:Ligands; D052118:Lysosomal-Associated Membrane Protein 1; D043562:Receptors, G-Protein-Coupled; D054340:Receptors, KIR