- Poster presentation
- Open Access
Critical role for monocytes in mediating HIV-specific antibody-dependent cellular cytotoxicity
© Kramski et al; licensee BioMed Central Ltd. 2012
- Published: 13 September 2012
- Healthy Donor Blood
- Live Cell Imaging
- Monocyte Function
- ADCC Activity
- Image Stream
Antibodies (Abs) that mediate antibody-dependent cellular cytotoxicity (ADCC) activity against HIV-1 are of major interest. Considerable evidence supports a role for ADCC activity in the control of HIV-1 infection and in the context of vaccination. One method widely used to assess the role of ADCC is the rapid and fluorometric antibody-dependent cellular cytotoxicity (RFADCC) assay. In the RFADCC assay specific killing of target cells by PBMC is assessed by loss of intracellular CFSE but retention of membrane dye PKH26 (CFSE-PKH26+), which is assumed to be derived from CFSE+PKH26+ target cells killed by NK cells. We have revisited this assay to assess the role of effector cells in mediating ADCC.
Multi-color flow cytometry was used to analyse gp140-pulsed, CFSE and PKH26 double labeled CEM.NKr-CCR5 cells incubated with HIV+ plasma or purified IgG samples (n=57) and co-cultured with PBMC, purified NK cells, or monocytes prepared from healthy donor blood. Effector/target cell interaction was visualized using image stream flow cytometry and live cell imaging.
Backgating analysis and phenotyping of CFSE-PKH26+ cells identified CD3-CD14+ monocytes as the major effector cell type. This was confirmed for all 57 HIV+ plasma samples tested. Emergence of the CFSE-PKH26+ cell population was observed following co-culture with purified monocytes but not purified NK cells. No significant IFNγ production or CD107a degranulation was detected in NK cells in this assay. Image flow cytometry and microscopy confirmed a monocyte-specific interaction with target cells. Monocytes acquire PKH26+ cell membrane presumably derived from killed target cells without typical morphological changes associated with phagocytosis, suggesting monocyte-mediated ADCC.
Our studies advance the understanding of the cellular events underlying HIV-specific ADCC. The RFADCC assay primarily reflects Ab-mediated monocyte function and has to be treated with caution in regard to NK cell-mediated ADCC. Further studies on the biological importance of HIV-specific monocyte-mediated ADCC are warranted.
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.