Role of monocytes in mediating HIV-specific antibody-dependent cellular cytotoxicity

• DOI: 10.1016/j.jim.2012.07.006
• Publication Date: 2012-07-24T11:21:13Z
• Authors: M. Kramski; A. Schorcht; A.P.R. Johnston; G.F. Lichtfuss; S. Jegaskanda; R. De Rose; I. Stratov; A.D. Kelleher; M.A. French; R.J. Center; A. Jaworowski; S.J. Kent
• Keywords: 0303 health sciences; CD3 Complex; Antibody-Dependent Cell Cytotoxicity; Lipopolysaccharide Receptors; HIV; Succinimides; Flow Cytometry; Fluoresceins; Coculture Techniques; Monocytes; Cell Line; 3. Good health; Killer Cells, Natural; 03 medical and health sciences; Microscopy, Fluorescence; Phagocytosis; Leukocytes, Mononuclear; Humans; Organic Chemicals; Single-Cell Analysis; Cells, Cultured; Fluorescent Dyes

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.