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.
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