RESUMO
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.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos/imunologia , HIV/imunologia , Células Matadoras Naturais/imunologia , Monócitos/imunologia , Complexo CD3/imunologia , Complexo CD3/metabolismo , Linhagem Celular , Células Cultivadas , Técnicas de Cocultura , Citometria de Fluxo , Fluoresceínas/química , Corantes Fluorescentes/química , Humanos , Células Matadoras Naturais/química , Células Matadoras Naturais/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Receptores de Lipopolissacarídeos/imunologia , Receptores de Lipopolissacarídeos/metabolismo , Microscopia de Fluorescência , Monócitos/citologia , Monócitos/metabolismo , Compostos Orgânicos/química , Fagocitose/imunologia , Análise de Célula Única/métodos , Succinimidas/químicaRESUMO
We developed a real-time PCR to quantify 16S rRNA gene levels in plasma from HIV-infected patients as a marker of microbial translocation. The assay uses shrimp nuclease (SNuc) to eliminate DNA contamination, giving high sensitivity and low variability. The 16S rRNA gene levels measured in plasma from HIV patients correlated significantly with lipopolysaccharide levels.