Comparing high-throughput methods to measure NK cell-mediated antibody dependent cellular cytotoxicity during HIV-infection

HIV-specific binding antibody responses, including those mediating antibody-dependent cellular cytotoxicity (ADCC), provided the best functional correlate of lower risk of infection in the RV144 HIV-1 vaccine clinical trial. The aim of this study was to compare two high-throughput flow cytometry based methods to measure HIV-specific ADCC responses, the GranToxilux and PanToxilux assays. Plasma from nine HIV-1 seropositive individuals was screened for binding antibody titres against HIV-1 subtype C gp120 by ELISA and western blot. Plasma from six HIV-negative individuals was included as controls. Both ADCC assays used subtype C gp120-coated CEM.NKRCCR5 cells as targets. The PanToxilux assay (which measured both granzyme B and caspase activity) measured higher levels of direct natural killer (NK) cell killing of K562 tumour cells than the GranToxilux assay (granzyme B alone; p<0.05). In ADCC assays in which NK cell killing was directed against gp120-coated CEM.NKRCCR5 cells in an antibody-dependent manner, plasma from HIV-positive individuals yielded significantly higher levels of ADCC activity than the HIV-negative controls. In contrast to direct killing, the GranToxilux assay measured similar levels of ADCC killing as the PanToxilux assay but had significantly lower background cytotoxicity against target cells coated with HIV negative serum. In conclusion, the PanToxilux assay was more sensitive for detecting direct NK cell killing of K562 cells than the GranToxilux assay, although the GranToxilux assay performed better at detecting HIV-specific ADCC activity, because of lower background cytotoxicity from HIV-negative serum. This is the first study to compare GranToxilux and PanToxilux ability to detect ADCC during HIV infection.