Fc-mediated antibody effector functions serve as a mechanism whereby antibodies can provide therapeutic benefit. In HIV, genetic evidence has been split as to the role of antibody engagement of innate immunity in protection from or after infection. High-affinity polymorphisms of FcγR3a, while protective in monoclonal antibody therapy of cancer, have been associated with HIV progression (43
). Similarly, the allele of FcγR2a with improved recognition of IgG2 subclass antibodies has been found to be a risk factor in neonatal HIV transmission (22
), whereas it has been associated with protection from progression in adults (21
). Yet passive-transfer studies of both neutralizing monoclonal and vaccine-induced nonneutralizing antibodies have implicated antibody effector functions in protection (9
). More recently, there has been speculation that the modest degree of protection observed in the RV144 trial may have been due to antibody effector mechanisms (46
Here we show that antibodies from controllers exhibit enhanced humoral phagocytic potential and that this potentiation is related to the natural induction of antibodies with a propensity to bind the activating FcγR2a over its inhibitory FcγR2b counterpart. Given their high degree of sequence similarity, we were surprised to observe differential binding of antibodies to these receptors. Specificity to the activating or inhibitory receptor can be mediated by amino acid point mutations in the antibody Fc domain (47
), and differences in binding to FcγR2a and FcγR2b among IgG subclasses have also been noted (1
), supporting the possibility that these Fc receptors display some unique specificity for their antibody ligands. Receptor blocking experiments likewise demonstrated distinct mechanistic differences in FcγR usage from each subject group. These data strongly suggest that the immune system is able to tune the antibody effector profile to naturally produce antibodies with specific effector functions. While strong glycan-based modulations of interactions with FcγR3a have been described, glycan modifications that differentially modulate FcγR2a and FcγR2b binding have yet to be described. Careful dissection of the antibody profiles among subjects may provide new opportunities to define the specific glycan profiles that modulate binding to FcγR2a relative to FcγR2b, potentially providing critical information to drive the production of antibodies potentiated for this particular effector function.
Perhaps surprisingly, the phagocytic activity of antibodies from controllers was most similar to that of untreated subjects, in whom circulating virus, inflammation, and immune activation are high. Interestingly, while controllers durably maintain viral replication to undetectable levels in plasma, recent reports suggest that these individuals have elevated blood levels of microbial products, indicating that they may have residual viral replication within their gut (48
). Residual replication may induce low-grade inflammation driving hypergammaglobulinemia, the production of antibodies with an inflammatory glycan, and the skewed interactions with FcγR2a and FcγR2b associated with potentiated capacity to induce phagocytosis observed in this study. While the subclass skewing in controllers was more subtle than in other HIV-positive subject classes, the presence of (i) high antibody titers, (ii) decreased plasma IgG2 levels and a trend toward elevated IgG1, and (iii) increased binding of plasma IgG to the complement protein MBP are marks of inflammation and stimulation even in the absence of detectable viral replication in this patient population.
While this study focused on antibody-driven phagocytosis, striking differences in viral suppression mediated by the same antibody samples were observed depending on the effector cell type utilized. Indeed, complex antibody functions do not necessarily correlate with titer or among different effector cell populations (49
). The observation that antibody titer does not predict effector function indicates that there are qualitative antibody features that can disparately affect function in different cellular assays; the “active” fraction of antibody may be only a component of the total antibody measured, and this fraction may vary among subjects. In this study, NK cell ADCVI activity differed significantly from monocyte ADCVI activity. Despite the fact that IgG1 and IgG3 antibodies are implicated as being important to both NK ADCC and phagocytosis, there are a number of differences between these effector cell types that may account for their differential engagement by antibody.
First, NK cells express only FcγR3a (51
), and this receptor is sensitive to Fc domain fucosylation (52
). Phagocytes can express FcγR3a but typically do so at lower levels, relying primarily on FcγR2a, which may also bind to a broader array of subclasses, but is insensitive to Fc domain fucosylation, for activity (18
). Thus, differences in IgG subclass distribution and glycosylation among or even within subclasses (particularly IgG1 and IgG3) within the broader pool of HIV-specific antibodies present in each patient population may lead to altered recruitment of innate immune cell subsets expressing different FcγRs, resulting in the differential antiviral clearance observed in this study. Consistent with the striking differences observed between effector cell types here, in a previous study IgG fucosylation was found to have opposing impacts on polymorphonuclear leukocyte (PMN) and mononuclear cell ADCC activity even in the context of a monoclonal antibody (50
). Future in-depth analysis of glycosylation of HIV-specific antibodies may provide key insights into these divergent functional profiles.
Larger questions pertain to defining the humoral mechanism that may afford the greatest level of protection in HIV, and evidence as to the possible importance of phagocytosis in contributing to slower HIV disease progression has been accumulating (21
). Moreover, often in the in vivo
data in which ADCC or ADCVI has been implicated in protection, it has been difficult to separate whether distinct FcγR-based mechanisms, such as ADCC or phagocytosis or a combination of these and other effector activities, are involved in the protection observed. Studies aimed at defining the role of NK cells within the gut have demonstrated that these cells are found at relatively low frequencies, and a neutralizing antibody with potentiated NK effector function did not provide improved protection, suggesting that other Fc receptor-bearing innate immune cells may play a more central role in antiviral containment at this site (27
). In contrast, the gut and other mucosal membranes are abundantly lined with phagocytes. Thus, it is plausible that the activity difference observed in this in vitro
study may have an impact in vivo
. Furthermore, phagocytosis may be important not only in the rapid removal of inflammatory immune complexes or infectious particles but also in driving and regulating the adaptive immune response via phagocytic antigen-presenting cells (56
In this study, we have shown that antibodies from controllers and untreated chronic progressors exhibit increased phagocytic activity relative to antibodies from treated progressors. Beyond differences in phagocytic uptake, antibodies from controllers exhibited differential interactions with the activating FcγR2a and the inhibitory FcγR2b compared to chronic progressors, exhibiting a preference for the FcγR2a yet greater inhibition of phagocytosis driven by FcγR2b. Because the route of phagocytosis and receptors involved have been shown to alter downstream processing and cross-presentation of pathogens (58
), patterns of receptor usage may dramatically impact the rate, downstream signaling, and outcome of immune complex clearance via this mechanism. Further elucidation of the role of these receptors in different cellular subsets will be critical to understanding their impact on HIV acquisition and progression. Continued research to define the properties that may provide specificity for FcγR2a over FcγR2b will be important for the design of potential monoclonal therapeutics for passive transfer as well as vaccines that can specifically induce these types of humoral immune responses. In HIV, however, defining specific features of the antibody Fc domain and effector mechanisms that may provide robust protection against or after infection remains a critical goal.