title: Role of monocytes in mediating HIV-specific antibody-dependent cellular cytotoxicity.
abstract: Antibodies (Abs) that mediate antibody-dependent cellular cytotoxicity (ADCC) activity against HIV-1 are of major interest. A widely used method to measure ADCC Abs is the rapid and fluorometric antibody-dependent cellular cytotoxicity (RFADCC) assay. Antibody-dependent killing of a labelled target cell line by PBMC is assessed by loss of intracellular CFSE but retention of membrane dye PKH26 (CFSE-PKH26+). Cells of this phenotype are assumed to be derived from CFSE+PKH26+ target cells killed by NK cells. We assessed the effector cells that mediate ADCC in this assay. Backgating analysis and phenotyping of CFSE-PKH26+ revealed that the RFADCC assay's readout mainly represents CD3-CD14+ monocytes taking up the PKH26 dye. This was confirmed for 53 HIV+plasma-purified IgG samples when co-cultured with PBMC from three separate healthy donors. Emergence of the CFSE-PKH26+ monocyte population was observed upon co-culture of targets with purified monocytes but not with purified NK cells. Image flow cytometry and microscopy showed a monocyte-specific interaction with target cells without typical morphological changes associated with phagocytosis, suggesting a monocyte-mediated ADCC process. We conclude that the RFADCC assay primarily reflects Ab-mediated monocyte function. Further studies on the immunological importance of HIV-specific monocyte-mediated ADCC are warranted.
authors: Department of Microbiology and Immunology, University of Melbourne, Australia. mkramski@unimelb.edu.au
authors: M
authors: M
authors: Kramski
authors: A
authors: A
authors: Schorcht
authors: A P R
authors: AP
authors: Johnston
authors: G F
authors: GF
authors: Lichtfuss
authors: S
authors: S
authors: Jegaskanda
authors: R
authors: R
authors: De Rose
authors: I
authors: I
authors: Stratov
authors: A D
authors: AD
authors: Kelleher
authors: M A
authors: MA
authors: French
authors: R J
authors: RJ
authors: Center
authors: A
authors: A
authors: Jaworowski
authors: S J
authors: SJ
authors: Kent
mesh_terms: D000920:Antibody-Dependent Cell Cytotoxicity / Q000276:immunology*; D017252:CD3 Complex / Q000276:immunology / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D018920:Coculture Techniques; D005434:Flow Cytometry; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D006678:HIV / Q000276:immunology*; D006801:Humans; D007694:Killer Cells, Natural / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D018950:Lipopolysaccharide Receptors / Q000276:immunology / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009000:Monocytes / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D009930:Organic Chemicals / Q000737:chemistry; D010587:Phagocytosis / Q000276:immunology; D059010:Single-Cell Analysis / Q000379:methods; D013388:Succinimides / Q000737:chemistry
chemical_list: C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D017252:CD3 Complex; D005452:Fluoresceins; D005456:Fluorescent Dyes; D018950:Lipopolysaccharide Receptors; D009930:Organic Chemicals; C070080:PKH 26; D013388:Succinimides