CE: Namrta; QAD/AIDS-D-18-00733; Total nos of Pages: 13; AIDS-D-18-00733 EDITORIAL REVIEW Antibody-dependent cellular cytotoxicity in HIV infection Donald N. Forthala,b and Andres Finzic,d Interactions between the Fc segment of IgG and its receptors (FcgRs) found on cells such as natural killer cells, monocytes, macrophages and neutrophils can potentially mediate antiviral effects in the setting of HIV and related infections. We review the potential role of Fc-FcR interactions in HIV, SIV and SHIV infections, with an emphasis on antibody- dependent cellular cytotoxicity (ADCC). Notably, these viruses employ various strate- gies, including CD4 down-regulation and BST-2/tetherin antagonism to limit the effect of ADCC. Although correlative data suggest that ADCC participates in both protection and control of established infection, there is little direct evidence in support of either role. Direct evidence does, however, implicate an FcgR-dependent function in aug- menting the beneficial in-vivo activity of neutralizing antibodies. Copyright ß 2018 Wolters Kluwer Health, Inc. All rights reserved. AIDS 2018, 32:000–000 Keywords: antibody-dependent cellular cytotoxicity, CD4, Fc receptor, HIV, natural killer cell, phagocytosis, simian immunodeficiency virus, simian/human immunodeficiency virus Introduction antibody-dependent enhancement, the interested reader is directed elsewhere [1,2]. In addition, detailed Much of the antiviral activity of antibody is mediated by treatments of FcR biology can be found in recent interactions between the Fc segment of immunoglobulin reviews [3,4]. and Fc receptors (FcRs) present on many different cell types. Such interactions could have a beneficial impact on ADCC occurs when antibody forms a bridge between a viral infection through, for example, antibody-dependent target cell bearing foreign antigens on its surface and an cellular cytotoxicity (ADCC), phagocytosis, or trogocy- effector cell, typically a natural killer cell expressing FcRs. tosis (wherein plasma membranes are sheared off of one The cross-linking of the FcRs initiates a cascade of signals cell by another cell). However, Fc–FcR interactions may leading to the release of lytic compounds from the also result in antibody-dependent enhancement of effector cell that ultimately result in the lysis of the target infection, a phenomenon best described with respect cell. Thus, as is the case with cytotoxic T-cell activity, to dengue virus infections. In this review, we will focus ADCC has the potential to remove cells producing virus. primarily on ADCC in the setting of HIV-1 and related ADCC is best described for IgG interacting with Fc lentiviral infections and also touch briefly on other receptors for IgG (FcgRs). However, IgA-FcaR and potentially beneficial functions. For a discussion of IgE-FceR interactions leading to ADCC also occur [5,6]. aDivision of Infectious Diseases, Department of Medicine, School of Medicine, bDepartment of Molecular Biology & Biochemistry, School of Biological Sciences, University of California, Irvine, USA, cDepartment of Microbiology, Infectious Diseases and Immunology, Universite de Montreal, Centre de Recherche du CHUM, and dDepartment of Microbiology and Immunology, McGill University, Montreal, Canada. Correspondence to Donald N. Forthal, UC Irvine School of Medicine, Irvine, California, 92697, USA. E-mail: [email protected] Received: 24 July 2018; accepted: 30 July 2018. DOI:10.1097/QAD.0000000000002011 ISSN 0269-9370 Copyright Q 2018 Wolters Kluwer Health, Inc. All rights reserved. 1 CE: Namrta; QAD/AIDS-D-18-00733; Total nos of Pages: 13; AIDS-D-18-00733 2 AIDS 2018, Vol 00 No 00 Of the IgG subclasses, IgG1 and IgG3 are best at engaging IFN-g or cell-surface expression of CD107a or by Fc receptors [3,7]. Fc glycosylation can also have a major measuring the loss of intracellular granzyme B [38,39]. impact on Fc–FcgR interactions [8]. Finally, polymor- Note that these latter assays do not directly measure phisms in the FcgRs themselves, particularly those in ADCC but more precisely measure activities that require FcgRIIIa and FcgRIIa, can influence the affinity for IgG natural killer effector cell responses similar to those and the ADCC activity in a subclass-specific manner [7,9]. occurring during ADCC. Antibody-dependent phagocytosis (ADP) occurs when It is important to consider that Env coating of CD4þ T antibody opsonizing a cell or a cell-free pathogen cross- cells is achieved through the interaction of gp120 with the links FcRs on phagocytic cells such as monocytes, CD4 receptor. Thus, these assays cannot detect activity macrophages, dendritic cells, or neutrophils. The mediated by antibodies that target the gp120 CD4- opsonized cell or pathogen is then internalized and, for binding site (CD4BS) [40–44]. Moreover, gp120-coated the most part, degraded in phagolysosomes. However, cells do not expose quaternary epitopes present in the FcR-mediated internalization of virus also represents a untriggered Env trimer, and therefore, are not useful to means of virus entry into susceptible target cells that could measure ADCC activity mediated by antibodies recog- potentially result in enhanced infection [10,11]. Using nizing such quaternary epitopes (e.g. PG9, PG16, etc.) green fluorescent protein (GFP)-expressing HIV-1, it has [45–48]. These considerations and others have led to been shown that virions are poor targets for monocyte- several approaches to measure ADCC against infected mediated ADP by Env-specific IgG, likely because of the target cells. Such target cells can be made by infecting paucity of Env spikes on the virion surface and because of CD4þ T cells, mostly CEM-NKr cells or primary CD4þ the inability of IgG antibodies to aggregate virus [12]. T cells, with infectious HIV-1 molecular clones (IMCs). However, cells decorated with virus on their surface, a However, an overlooked problem of this approach is the state imitating target cells in the process of being infected, intrinsic ability of gp120 to be shed from the surface of may undergo ADP or trogocytosis [12–15]. Whether or infected cells. Shed gp120 binds to uninfected, bystander not phagocytosis or trogocytosis of such cells, or of T cells through a CD4 interaction, thus providing another infected cells, contributes to virus clearance is unknown. susceptible target cell population in addition to the infected cells (Fig. 1). This natural phenomenon greatly Apart from functions that result in target cell death, complicates ADCC measurements. Although infected triggering of FcRs by opsonizing antibodies can result in cells express trimeric Env at their surface, uninfected the secretion of various chemokines and cytokines with bystander cells are coated with monomeric gp120. It is, direct and indirect antiviral and inflammation-modulat- therefore, paramount to discern ADCC responses against ing effects [16,17]. Finally, the sum total of FcR-triggered infected versus uninfected cells, as anti-Env Abs have effects on virus replication can be measured using the different specificities [40]. Indeed, whereas some antibody-dependent cell-mediated virus inhibition preferentially recognize monomeric gp120, others only (ADCVI) assay, in which viral yield is measured after bind the untriggered ‘closed’ trimer (discussed below). incubating infected lymphocytes with effector cells and antibody [16,18]. Though natural killer cell activation and GranToxiLux assays fail to differentiate responses against infected versus uninfected cells, other approaches do. Infected cells can easily be identified by performing intracellular p24 Measuring antibody-dependent cellular staining; ADCC activity is then determined by calculat- cytotoxicity: concepts and controversies ing the loss of the percentage of infected (p24þ) cells by flow cytometry [36,40,49–55]. Similar flow cytometry Env-coated versus infected target cells approaches use modified IMC coding for reporter genes One of the earliest and most widely used techniques to such as GFP [52,53,56–60]. Another method to measure measure anti-HIV ADCC activity relies on the coating of ADCC activity against the infected cell population is the CD4þ T cells – either cell lines such as CEM-NKr or use of IMCs coding for the luciferase reporter gene primary CD4þ T cells – with recombinant Env proteins (LucR). This approach is amenable to high-throughput gp120 or gp140 or with inactivated viral particles [19– assays and has been widely used in the field [37,61–74]. 32]. Killing of Env-coated target cells is then measured by However, great caution should be exerted in selecting the release of 51Chromium (chromium release assay) these reporter IMCs in order to ensure that insertion of [26,33,34] or by calculating the emergence of prelabeled the reporter gene does not affect expression of viral coated cells that lose a viability dye [rapid fluorometric proteins, particularly Nef, that play a critical role in ADCC (RFADCC) assay] [25,32,35]. ADCC activity has protecting infected cells from ADCC responses (detailed also been evaluated by quantifying the cleavage of a below; Fig. 1) [75]. Finally, another luciferase-based fluorogenic peptide substrate that generates a fluorescent method uses a T-cell line encoding a Tat-driven luciferase signal when cleaved by granzyme B (ADCC GranTox- gene, which allows luciferase to be expressed only upon iLux assay) [27,28,36,37], by detecting intracellular productive HIV-1 or simian immunodeficiency virus CE: Namrta; QAD/AIDS-D-18-00733; Total nos of Pages: 13; AIDS-D-18-00733 Antibody-dependent cellular cytotoxicity in HIV Forthal and Finzi 3 (a) Protection Non-neutralizing CD4+-induced Abs + BST2 CD4 Closed Env Vpu Nef HIV-1-infected CD4+ T cell gp120 shedding Deletion of Nef and/or Vpu ADCC (c) ADCC (b) NK cell NK cell CD16/FcγRIIIa CD16/FcγRIIIa CD4+-bound CD4+-bound Env Env HIV-1-infected CD4+ T cell Uninfected bystander CD4+ T cell Fig. 1. HIV-1-infected cells are protected from antibody-dependent cellular cytotoxicity responses. To avoid exposing vulnerable CD4-induced epitopes, HIV-1 controls the level of cell surface CD4 through the action of Nef and Vpu and limits Env accumulation through Vpu-mediated BST-2/tetherin downmodulation (a).