A flow cytometry based assay that simultaneously measures cytotoxicity and monocyte mediated antibody dependent effector activity

Aljawharah Alrubayyi; Alexandra Schuetz; Kerri G Lal; Surat Jongrakthaitae; Kristopher M Paolino; Julie A Ake; Merlin L Robb; Mark S de Souza; Nelson L Michael; Dominic Paquin-Proulx; Michael A Eller

doi:10.1016/j.jim.2018.08.012

A flow cytometry based assay that simultaneously measures cytotoxicity and monocyte mediated antibody dependent effector activity

J Immunol Methods. 2018 Nov:462:74-82. doi: 10.1016/j.jim.2018.08.012. Epub 2018 Aug 24.

Affiliations

¹ U.S. Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, MD, USA; Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc., Bethesda, MD, USA.
² U.S. Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, MD, USA; Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc., Bethesda, MD, USA; Department of Retrovirology, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand.
³ Department of Retrovirology, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand.
⁴ Clinical Trials Center, Translational Medicine Branch, Walter Reed Army Institute of Research, Silver Spring, MD, USA.
⁵ U.S. Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, MD, USA.
⁶ SEARCH, Thai Red Cross AIDS Research Center, Bangkok, Thailand.
⁷ U.S. Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, MD, USA; Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc., Bethesda, MD, USA. Electronic address: dpaquin-proulx@hivresearch.org.

PMID: 30148978
DOI: 10.1016/j.jim.2018.08.012

Abstract

Antibody effector functions such as antibody dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis (ADCP) are considered important immunologic parameters following results from the RV144 clinical trial where a reduced risk of infection was associated with non-neutralizing antibody against the V1/V2 region of HIV envelope. The rapid and fluorometric ADCC (RFADCC) assay has been widely used to measure ADCC, however, the mechanism behind the activity measured remains unclear. Here, we demonstrate that monocytes acquire the PKH26 dye used in the RFADCC assay and that the commonly used RFADCC readout correlates with phagocytosis. The RFADCC assay was combined with an amine reactive dye staining to confirm target cell killing. Interestingly, the majority of RFADCC and amine indices were mutually exclusive. In fact, the amine reactive assay results correlated with results from another assays that directly measure NK cell antibody effector functions not associated with phagocytosis. Together, this combined assay offers the opportunity to discriminate monocytes and NK cell antibody effector functions simultaneously.

Keywords: ADCC; ADCP; Cytotoxicity; HIV antibodies; Monocytes; NK cells.

Publication types

Clinical Trial
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Antibody-Dependent Cell Cytotoxicity*
Female
Flow Cytometry / methods*
Humans
Killer Cells, Natural / immunology*
Male
Monocytes / immunology*
Organic Chemicals / chemistry
Phagocytosis*

Substances

Organic Chemicals
PKH 26