Research paperRole of monocytes in mediating HIV-specific antibody-dependent cellular cytotoxicity
Introduction
Virus-specific binding but non-neutralizing antibodies (Abs), in particular Abs that mediate antibody-dependent cellular cytotoxicity (ADCC) activity, have been of major interest in human immunodeficiency virus (HIV) research. Considerable evidence supports a role for ADCC activity in the control of HIV infection with a beneficial impact on disease progression (Ahmad et al., 2001, Huber and Trkola, 2007, Alter and Altfeld, 2009, Chung et al., 2011, Johansson et al., 2011). In the context of vaccination, ADCC-Abs correlate positively with protection in animal models of HIV infection (Gómez-Román et al., 2005, Hidajat et al., 2009, Xiao et al., 2010). Passive transfer experiments in macaques support a role for ADCC in assisting in the control of chimeric SIV-HIV infection (Hezareh et al., 2001, Hessell et al., 2007). The recent partially successful RV144 clinical HIV vaccination trial (Rerks-Ngarm et al., 2009) also suggested a potential role of non-neutralizing and ADCC Abs in protection (Haynes et al., 2012). Given the potential protective role for non-neutralizing antibodies robust assays are needed to measure HIV-specific ADCC responses.
ADCC responses to HIV-1 were first assayed using the 51Chromium release assay with envelope (Env) protein coated target cell lines (Baum et al., 1996, Cox, 1999, Cox et al., 1999, Battle-Miller et al., 2002, Nag et al., 2004, Yamada et al., 2004). More recently, several non-radioactive assays measuring ADCC responses have been developed (Sheehy et al., 2001, Pollara et al., 2011). The rapid and fluorometric antibody-dependent cellular cytotoxicity (RFADCC) assay developed by Robert-Guroff and colleagues (Gómez-Román et al., 2006) has been widely used in the context of HIV and SIV vaccine and immunology research (Gómez-Román et al., 2005, Chung et al., 2009, Vaine et al., 2010, Xiao et al., 2010, Patterson et al., 2011). The target cells used in the RFADCC assay are CEM.NKr-CCR5 cells, a CD4 + T cell line resistant to NK cell killing mediated by natural cytotoxicity receptors, that are coated with HIV-1 or SIV Env protein. To assess anti-HIV ADCC activity mediated by HIV-specific IgG present in HIV + patient sera the Env-coated CEM.NKr-CCR5 are double labelled with the cell membrane dye PKH26 and the cytoplasmic dye CFSE and co-cultured with PBMC from a healthy, HIV-uninfected subject in the presence of defined amounts of test serum. The killing by PBMC is defined by the loss of CFSE but the retention of PKH26 resulting in the emergence of a "killed" PKH26 + CFSE- population within the PKH26 + gate.
While using the RFADCC assay, we noted that the population of cells that has previously been accepted as “killed” target cells (PKH26 + CFSE-), based on their two-way CFSE-PKH26 + dot plot signature, appeared to emerge from the unlabelled PBMC population, rather than from the PKH26 + CFSE + CEM.NKr-CCR5 target cells. We investigated the PKH26 + CFSE- population further and found that antibody-dependent monocyte uptake of PKH26-stained target cell fragments is a more likely explanation of the biological events measured in this assay. An analysis of samples from 53 HIV + subjects confirmed that gating on PKH26 + monocytes was a more robust method to define ADCC in this assay. Further studies analysing the role of monocyte-mediated ADCC are warranted.
Section snippets
Plasma samples
Plasma was collected from HIV + subjects (n = 53) and HIV- subjects (n = 2). HIV + subjects were receiving antiretroviral therapy at the time of recruitment. HIV + subjects had a mean CD4 T cell count of 494 cells/μl (range 3–1360 cells/μl) and a mean plasma HIV RNA level of 7.1 × 104 copies/ml, (range 4 × 101–7.5 × 105 copies/ml) to reflect the spectrum of HIV disease states. All subjects provided written informed consent and the studies were approved by the relevant institutional ethics committees.
Purification of total IgG from plasma samples
Total IgG
Modified analysis and gating strategy using the RFADCC assay
The RFADCC assay has proved a very useful non-radioactive assay to measure ADCC, although in our hands the standard method of gating on PKH26 + targets cells and determining the proportion of CSFE-negative cells within this gate resulted in relatively high background levels of “killing” of non-antigen coated targets (Fig. S1) up to 20%. Although no information of background killing of uncoated target cells of previously published results is available, a mean background “killing” of 14.4% for
Discussion
ADCC is an immune response of emerging interest with a potential role in controlling HIV infection (Lambotte et al., 2009, Berger and Alter, 2011, Chung et al., 2011). ADCC activity is most commonly attributed to NK cells although monocytes and granulocytes also bear Fcγ receptors (FcγR), including FcγRI, FcγRIIa/b and FcγIII and have been shown to kill targets via ADCC (Barker and Reisfeld, 1993, Horner et al., 2007, Wu et al., 2008). Effector functions of monocytes/macrophages focus mainly on
Conclusion
Our studies clarify the understanding of the cellular events underlying IgG-mediated HIV-specific cellular effector function detected by the RFADCC assay. We demonstrate that this assay primarily reflects Ab-mediated monocyte function and not NK cell ADCC and therefore has to be treated with caution in regards to the interpretation for NK cell-mediated ADCC. Presumably, lysis is not mediated by complement activation because the effect is seen with purified IgG. Our amended gating on monocytes
Acknowledgements
This work was supported by Australian NHMRC award 510448 and NIH award R21AI081541. We are grateful to all the subjects who kindly provided blood samples and their carers. We also thank Damian Purcell for helpful discussions.
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