Immunoregulatory functions of immune complexes in vaccine and therapy

Abstract Clinical and experimental preparations of IgG/soluble antigen complexes, as well as those formed following antibody therapy in vivo, are multifaceted immune regulators. These immune complexes (ICs) have been tested in humans and animal models, mostly in forms of experimental or clinical vaccination, for at least a century. With intensified research on Fcγ receptor‐mediated immune modulation, as well as with immune complex‐directed antigen processing, presentation, and inflammatory responses, there are renewed interests of using ICs in vaccines and immunotherapies. Currently, IC‐based immune therapy has been broadly experimented in HBV and HIV viral infection control and antitumor treatments. However, mechanistic insights of IC‐based treatments are relatively recent subjects of study; strong efforts are needed to establish links to connect laboratory findings with clinical practices. This review covers the history, mechanisms, and in vivo outcomes of this safe and effective therapeutic tool, with a clear aim to bridge laboratory findings with evolving clinical applications.


Introduction
Immune complex, as a form of autoimmune disorder and a tool in clinical therapy, has been an important topic of modern immunology. Binding of antigen by antibody, an immunologically simple event, changes properties of the original ligand, resulting in modulated antigen processing, presentation, receptor signaling, and inflammatory responses. On a larger scheme, antibody immune therapy has become one of the successfully implemented biomedical technologies in modern health care. Immunoglobulin-based new drugs such as rituximab, ipilimumab, and alemtuzumab are among the highly effective in cancer treatments (Sondak et al, 2011;Scott et al, 2012). Intravenous immunoglobulin, IVIG, is also a choice treatment of autoimmunity, pediatric infections, and antibody deficiencies (Schwab & Nimmerjahn, 2013). In contrast, use of classically defined IC represents an old subcategory among the list of options. ICs in vaccine development and clinical interventions are becoming increasingly sophisticated, largely attributable to the newly gained understanding of Fcc receptors (FccR; Ravetch, 2010). However, IC-mediated functions are not solely controlled via those receptor engagements. For instance, its ability to transport antigen is also a consideration of anatomic location; its roles at different stages of immune induction and memory formation are modulated by additional spatiotemporal factors.
Over 100 years ago, IC vaccines were started as a practice of using antisera complexed to toxoids of Corynebacteria diphtheria or Streptococcus pyogenes to decrease the side effects in human vaccination (Copeman et al, 1922;Olitzki, 1935). One concern of this approach was that antigens coated with antibodies might have reduced exposure of surface sites, leading to limited antibody production. This concern was proven unnecessary by subsequent experiments (Olitzki, 1935). In 1950s, Terres et al reported that mice immunized with human or bovine serum albumin were sensitized faster if a specific rabbit antibody was also introduced to the recipients, an effect not seen with antigen alone or in combination with a control antiserum (Terres & Wolins, 1959a, 1961. The enhancement was optimal in a slight antigen excess (Terres & Wolins, 1959b, 1961Terres et al, 1960). After another three decades, using peptides from HIV gp120 to rejuvenate CD4 T cells in HIV carriers, Berzofsky et al reported that addition of antibodies recognizing these peptides substantially increased the proliferative index of CD4 + T cells in the patients (Berzofsky et al, 1988).
In the meantime, immune complexes were casted in a negative shadow, mainly because of their frequent presence in sites of autoimmunity and inflammation, such as lupus and membranous nephropathy. Collectively called type III hypersensitivity, IC deposition at rates faster than their clearance leads to robust activation of the complement system and Fc receptor signaling. As consequences, mononuclear cell degranulation, antibody-dependent cellular cytotoxicity (ADCC), and release of proinflammatory cytokines may result (Nydegger, 2007). Some efforts were made to characterize the pathogenic ICs. It was found that the IC-mediated attacks were mainly driven by inflammatory cytokines such as TNF-a (Warren et al, 1989). It was also found that ICs formed with low affinity antibodies tended to be present in high levels in the circulation and deposited more readily into subendothelia of renal capillary loops (Koyama et al, 1978). This was confirmed by mice genetically bred to produce only low and high-affinity antibodies, as ICs chronically formed in the former were deposited in glomeruli (Devey & Steward, 1980). The early work was not illuminated from mechanistic insights. A landmark turning point for IC's immune regulatory function was the discovery of FccRs. In an attempt to understand the molecular nature of antibodies adsorbed to splenocytes reported by Boyden & Sorkin (1960), Belkin and Benacerraf suggested in 1966 that this affinity was mainly afforded by the heavy chain of antibodies, implicating a structural presence on the surface of macrophages interacting with the Fc portion of IgG (Berken & Benacerraf, 1966). In the 1980s, all common forms of FccR were identified (Ravetch & Kinet, 1991), which subsequently developed into the current paradigm of Fc receptor biology (Nimmerjahn & Ravetch, 2007). Mouse activating FccR family consists of FccRI, RIII, and RIV, while human counterparts include FccRI, IIa, IIc, IIIa, and IIIb. Except for human FccRIIIb, these receptors transmit activating signals via an ITAM motif on the associated common c-chain or intrinsically present in their cytoplasmic domains. Both species have an additional inhibitory receptor, FccRIIb, that signals via a cytoplasmic ITIM domain. Meticulous work by Ravetch et al gradually delineated the subtype specificities and binding strengths to IgG subtypes. In addition, the presence of lectin-based type II FccRs (DC-SIGN and CD23) that are sensitive to the glycosylation state of Fc has been demonstrated in recent years (Ravetch, 2010;Bohm et al, 2014). Relevant to ICs, FccR signaling has implications in at least three aspects: (i) the activating and inhibiting receptors, their affinities, and the emerging lectin-based type II FccRs; (ii) the subclasses of antibodies binding to these receptors; and (iii) the highly diverse expression of these receptors on host cells and their levels of expression at different stages of immune activation. These considerations are becoming indispensable to modern applications of ICs (Nimmerjahn & Ravetch, 2005, 2008Guilliams et al, 2014). Under this backdrop, this review aims to provide a utilitarian look on ICs from a perspective of clinical application, to complement the current picture of antibody immune therapy.

Mechanisms of immune regulatory functions of IC
Cross-presentation and cellular immunity For extracellular antigens, the default antigen presentation pathway takes in soluble cargos via endocytosis, processed in progressively acidic endolysosomal vesicles by pH-dependent chain activation of cathepsins (Amigorena & Savina, 2010), for loading onto the class II molecules. Solid particles engulfed via phagocytosis or soluble cargos endocytosed via receptor-mediated uptake can be routed into MHC class I cross-presentation via several mechanisms (Lizee et al, 2003;Burgdorf et al, 2007;Joffre et al, 2012). The latter activates CD8 + T cells, known as the cross-presentation, that becomes the mechanistic basis of cellular immunity against antigens originated outside antigen-presenting cells (APCs).
The involvement of ICs in cross-presentation was initially noted in an assay to study the in vivo tumor killing by antibodies. EG7 tumor was inhibited or eradicated with an antibody against CD4 Glossary Self-tolerance breaking While immune tolerance refers to the lack of immune response to an antigen as a result of central (thymic selections) or peripheral (lack of co-stimulation) tolerance education, an extrinsic trigger, such as the same antigen presented to the host in an immune complex, can initiate specific immune recognition against the tolerized antigen. The end results can be detrimental in cases of autoimmunity, or beneficial, as in the immune attack on tumor antigens.

Cross-presentation
The ability of antigen contained in endo/phagocytic vesicles to be presented by MHC class I molecules on the APC surface. This is in contrast with the conventional understanding that class I antigens originate from endogenous protein synthesis. This is an intensely studied and contested topic, as several leading hypotheses, including models of "ER-phagosome fusion", "endocytic/plasma membrane recycling", and "cytosolic translocation", are still being verified. Toxoids Richard Friedrich Johannes Pfeiffer classified bacterial toxins into endotoxins that were believed to be sequestered by the cell wall and exotoxins that were thought to be released into the surroundings. Those toxins, particularly the latter, can be thermally or chemically deactivated, that is, with formalin, to produce toxoids to be used as a form of deactivated vaccine. This is an old term with decreasing popularity in modern literature.

Fc and Fcc receptor (FccR)
Fc, fragment crystallizable region, simply refers to the constant region of antibodies of any type. FccRs are the cell surface receptors expressed mostly by cells of the immune system that have binding specificities to the Fc region of c-immunoglobulins. Biological retention Refers to any substance retained in a biological system. Used in the context of antibody induction or antibody-based treatments, the term usually refers to the deposition of IC in tissues that can either serve as a source for persistent antigenic stimulation, usually with desirable outcomes, or induce uncontrolled immune attacks, as often seen in autoimmunity. Plasmablast Less mature plasma cell precursors, which could still undergo cell division. Chemotaxis Generically refers to the movement of a biological entity in response to chemical stimulus in their environment. Immunologically, this term refers to the immune cell movement driven by their surface expression of chemotactic receptors to or away from a gradient of cognate chemokines. Erythroleukemia A form of acute myeloid leukemia that involves the cells that give rise to the erythrocytes. Antigenemia Persistence of antigen in circulating blood. Antigenemia is used as an indication for severity of infection or as a measurement for efficacy of immune intervention.
expressed by these cells. The antibody did not kill the tumor directly; the effect was entirely dependent on CD8 + T cells and FccR c-chain (Vasovic et al, 1997). A logical explanation today is that binding of CD4 by the antibody facilitated tumor antigen processing by DCs and drove antigen-specific CD8 + T-cell activation. As a vivid confirmation, in the RIP-mOVA mice whereby OVA is transcriptionally controlled by rat insulin promoter, infusion of OVA-specific CD8 + T cells (OT-I) was not efficient to trigger destruction of b-cells. Addition of an OVA-specific IgG antibody, however, led to severe autoimmune diabetes, suggesting that the presence of ICs can overcome the tolerance to "self" antigens. As expected, the breakdown of self-tolerance required the presence of FccR common c-chain (Harbers et al, 2007).
Cross-presentation of external antigens is enhanced by antigen association with surface receptors on DCs (Shakushiro et al, 2004;Burgdorf et al, 2007;Chatterjee et al, 2012). Independent studies arrived at a similar conclusion that the presence of ICs facilitated the antigen uptake by two to three logs (Amigorena & Bonnerot, 1999;Schuurhuis et al, 2002). However, the real enhancement in antigen presentation measured by the degree of T-cell activation was in the order of 4-5 logs (Regnault et al, 1999). c-chain ITAM domain-mutant NOTAM mice have normal surface expression of FccRs. This to some extent rescued the IC uptake; however, crosspresentation of OVA IC was lost (Boross et al, 2014). That report seems to indicate that the ITAM is not required for uptake and yet behaves in part to direct intracellular antigen trafficking. Interestingly, in DCs, Syk deficiency downstream of ITAM phosphorylation disrupted not only cross-presentation of IC antigen but antigen uptake as well (Sedlik et al, 2003). The difference is likely due to the fact that Syk is associated with a broader spectrum of inhibition on phagocyte signaling (Mocsai et al, 2010). Deep mechanistic understanding of how ICs facilitate cross-presentation is currently limited. In one report, IC-mediated enhanced cross-presentation required TAP proteins and was sensitive to proteasome inhibition, suggesting the involvement of cytosolic targeting of internalized antigens (Regnault et al, 1999). In some systems, IC-mediated antigen uptake retained the complex at early endosome, rather than permitting further maturation to more degradative lysosome (Chatterjee et al, 2012;Cohn et al, 2013). Although the molecular nature of this "shallow" targeting is unknown, the findings are in agreement with the contemporary understanding that reduced cargo digestion is the main advantage of DCs in antigen presentation over other APCs (Joffre et al, 2012;Kotsias et al, 2013;Hari et al, 2015).
CD8 + DC subsets in the mouse periphery are usually considered to be superior in cross-presentation, and a developmentally overlapping population of CD103 + DCs is known for similar functions. These cells are phenotypically mirrored by human CD141 + DCs (Platzer et al, 2014). However, how these cells are involved in ICmediated cross-presentation is not well understood (Joffre et al, 2012). Not in strict sense an standard IC, OVA linked to an antibody specific for a C-type lectin receptor DNGR-1 found on CD8 + conventional DCs elicited strong CD8 + T-cell activation and eliminated OVA-positive B16 tumor in vivo (Sancho et al, 2008). Yet, when delivered into the host, OVA conjugated to anti-DCIR-1 and anti-DEC205, expressed by CD8 À and CD8 + DCs respectively, suppressed OVA-expressing B16 tumor to a similar extent (Neubert et al, 2014). As IgG1 antibodies used in this study carried Fc mutation (N297A) that disrupts FccR binding, both preparations might function merely as a delivery vehicle. This paper therefore did not address the effect of FccR signaling in cross-presentation. In another report, Bevan's group suggested that FccR-mediated IC cross-presentation was only essential in CD8 À DCs, while IC uptake and processing by CD8 + DCs were not affected by the absence of FccRs (den Haan & Bevan, 2002).
DCs can rapidly engulf ICs and sustain the stimulation capacity for a long period after a short pulse (Bonifaz et al, 2004). In influenza virus-challenged mice, the absence of extracellular availability of ICs did not strongly affect the primary CTL response to flu NP antigen. However, the recall response was limited. Transfer of immune serum from control-infected mice restored the long-term CD8 + T activation to NP antigen (Leon et al, 2014). Crossing lMT (B-cell-deficient) mice to c-chain knockout mice, NP-specific CD8 response upon viral challenge was reduced. Transfer of immune serum restored both primary and secondary responses in lMT mice, yet failed to do so in the double-deficient recipients, suggesting the essential role of FccR signaling in cross-presentation. This observation was confirmed by a recent study that the ICs associated antigens were present in the endocytic vesicles from early endosome to lysosome one day after the uptake, with dissociation of antigen taking place along the route (Liu et al, 2016).

Helper T cells and antibody responses
FccR-mediated IC signaling likely supports a simultaneous activation of both CD8 + and CD4 + T cells. For instance, prostate-specific antigen-containing ICs, when targeted via FccR, led to a combined activation of both CD4 + and CD8 + T cells, while mannose receptor targeting was more associated with CD4 + T-cell response alone (Berlyn et al, 2001). Regarding intracellular antigen trafficking, Syk signaling is an essential event for efficient MHC class II epitope peptide loading, as in the case of cross-presentation. A mutation in FccR that disrupts Syk association failed to deliver internalized antigen in ICs to lysosome (Bonnerot et al, 1998). Syk deficiency resulted in reduced MHC class II/DM molecule association with internalized antigens (Le Roux et al, 2007). As evidence of IC-facilitated class II antigen presentation, a viral glycoprotein rabies G as a free antigen entered Rab5 + early endosome with a kinetics similar to that of antibody (ARG1)-bound protein. Thereafter, the IC became localized to Rab9 + late endo/lysosome but at no time was co-localized with Rab11 (slow recycling endosome) and Rab4 (rapid recycling endosome) markers, while the free antigen was more associated with Rab11 + vesicles (St Pierre et al, 2011). Therefore, ICs may facilitate antigen delivery to vesicles in late endocytic maturation steps for MHC class II presentation. This notion inevitably disagrees with the "shallow" targeting reported in cross-presentation as discussed previously. This discrepancy possibly resulted from different antibodies used in experiments, which could have preferences in FccR subtype association, leading to distinct routes of trafficking. However, considering ICs in general enhance both class I and class II antigen presentation, one can postulate that binding of antibodies in a uncharacterized manner intercepts the robust proteolytic "grinding" in the endolysosomal system, saving antigens at multiple stages for eventual surface presentation.
One main mechanism for enhanced antigen presentation of ICs to B cells is longer biological retention. This is most evident in the trapping of ICs by follicular dendritic cells (FDCs) (Szakal et al, 1988a,b;Kosco et al, 1989). It was reported that great quantities of ICs attached to the long membrane processes of FDCs (Szakal et al, 1988b). FDCs have high FccRIIb expression at protein level (Qin et al, 2000) and reports suggested that ICs were internalized by FccRIIb (Bergtold et al, 2005), consistent with the observation that FDCs and B cells only express this Fc receptor. The ICs internalized by FccRIIb were retained in the cytoplasm and antigens within the complex were kept in their native form and were made available to B cells via surface recycling (Bergtold et al, 2005). The exact order of baton exchange has been reported. ICs were initially trapped by lymph node subcapsular macrophages; B cells then acted as an intermediator fetching and sending the complexes to FDCs (Phan et al, 2007). FDCs received complement-coated ICs from nonspecific B cells, and following endocytosis sorted, the complexes to a non-degradative compartment for extended resurfacing (Heesters et al, 2013). Furthermore, FDC-associated ICs played a role for BCR hypermutagenesis to generate high-affinity antibodies (Wu et al, 2008).
Type II FccRs are also implicated in B-cell activation. IgG is glycosylated at asparagine 297 with a biantennary core glycan that serves as the base for further addition of sugar moieties, including fucose, galactose, bisecting N-acetylglucosamine, or sialic acid, etc. This complex glycosylation event controls IgG conformational state and determines the preferential binding to either type I or type II FccRs. For instance, sialylation reduces the binding of Fc to all type I while increasing the binding to type II receptors (Pincetic et al, 2014). Treatment to remove this moiety suppressed ankle swelling in a model of serum transfer arthritis and prolonged survival of a mouse strain from an autoantibody-induced lupus-like disease (Albert et al, 2008). Targeting OVA to DCs via DEC-205 without concomitant activation via CD40 induced IgG1 antibody response and but a suppressed DTH (delayed type hypersensitivity) response. When the antiserum from those mice was transferred to new hosts that were challenged with the antigen in the full complement of anti-CD40, an extensive reduction of antigen-specific IgG1, IgG2b, and IgG2c was detected, accompanied by a reduced DTH response as well. These results suggested that antibodies produced in the original hosts without DC activation signal were immunosuppressive. Indeed, the antibody produced without anti-CD40 had a N297 sialylation rate higher than those with the treatment, due to higher levels of sialyltransferase in the plasma cells (Oefner et al, 2012). As this reduction in antibody production and DTH response was independent of FccRIIb, the involvement of type II receptors was implicated (Oefner et al, 2012). Type II FccR engagement is an important consideration in vaccination. In a trivalent inactivated influenza vaccine (TIV)-immunized individuals, mass-spec analysis did not show a significant glycosylation pattern change in the recipients. Interestingly, the subtle differences in glycosylation in the existing antibody pool were found to be a predictor of responders and nonresponders to the vaccine (Wang et al, 2015a). In addition, it was recently reported that in human volunteers, TIV led to a peak production of HA-specific sialylated IgG from plasmablast one week after the immunization, followed by a drop in abundance by week three. This elevation was resulted from glycosyltransferase expression in these early responding B cells. Importantly, this sialylated IgG, via CD23, triggered FccRIIb expression, which is a known threshold setter for BCR signaling (Wang et al, 2015b) resulting in altered antibody-binding affinity. The pattern of Fc glycosylation is associated with protection efficacy of a given antibody.
Palivizumab, a humanized mAb against respiratory syncytial virus (RSV) derived from animal cell culture, has a heterogeneous glycosylation pattern. The same antibody derived from plant cells (Nicotiana benthamiana) that had their xylosyl-and fucosyltransferase activities deleted produced G0-dominated glycans, palivizumab-N. This form of glycosylation showed enhanced FccRIII binding and better protection against RSV challenge (Hiatt et al, 2014).

Regulation of antigen-presenting cells
ICs can directly activate APCs via activating FccRs. Upon IC binding to these receptors, the ITAM/Syk interaction is the primary signaling axis in phagocyte activation, leading to the full spectrum of signaling events involving PI3Ks, PLCs, MAPKs, and NF-jB (Mocsai et al, 2010). Accordingly, ICs can trigger DC expression of CD40, CD86, IL-6, IL-13, and unexpectedly IL-2 (Matsubara et al, 2006;Perreau et al, 2008;Boross et al, 2014). In addition, ICs themselves were sufficient to trigger CD11c + DC aggregation and entrance into lymphatic vessels for migration to LNs in a CCR7/ MMP-9-dependent manner (Clatworthy et al, 2014). While in general the activation of APCs is linked to the FccR signaling, it has been recently suggested that once the ICs are inside the cell, cytosolic antibody receptor TRIM21 can bind to the Fc portion and activate a K63-specific Ubc13-mediated ubiquitination event. This event can independently activate NFkb, AP-1, and IRF pathways, adding another level of complexity to IC signaling (McEwan et al, 2013).
Immune-suppressive effects of ICs are equally noted. The small immune complexes formed in IVIG can inhibit macrophage responses to IFN-c, an effect mainly mediated by IgG Fc binding to FccRIII, and surprisingly independent of FccRIIb (Park-Min et al, 2007). In complement C5a-induced inflammatory recruitment, IgG1 ICs suppressed the neutrophil and macrophage chemotaxis. This inhibition was a result of an unexpected collaboration between ITIM tyrosine phosphorylation in FccRIIb itself and a ITAM phosphorylation of Syk downstream of dectin-1 (Karsten et al, 2012). In contrast to the conventional understanding that ICs formed in chronic infections cause inflammatory response by FccR cross-linking, it was recently found that in mice with clone 13 LCMV (long term) infection, the presence of ICs resisted CD4 + T-and B-cell depletion by anti-CD4 and anti-CD20 (rituximab) antibodies and blocked the activation of DCs by agonistic anti-CD40. The effect can be explained by competitive inhibition in the long-term presence of excessive ICs (Wieland et al, 2015). A similar report in the same issue of immunity confirmed the finding and demonstrated additional defects in cross-presentation. It was shown in the latter that the suppression was dependent on CD4 + T-supported antibody production (Yamada et al, 2015). This conclusion somewhat echoes the observation that in influenza infection, peak of antibody production formed ICs that blocked antibody-dependent phagocytosis (Astry & Jakab, 1984), both pointing to IC's negative impact on immune activation.
To summarize the discussion on the immune regulatory functions of IC engagement of FccRs, Fig 1 categorizes

IC vaccines in persistent viral infections
In HIV infection, experimental work on ICs started by using antibodies targeting the viral epitopes, as an approach to form ICs in vivo. ICs formed by antibodies recognizing HIV CD4 binding sites (CD4bs) and V3 region of gp120 have been promising, eliciting strong neutralizing antibodies against the virus in mice (Visciano et al, 2008;Hioe et al, 2009). One of V3 region-specific antibodies mAb 654-D showed high affinity to large variants of gp120 subtypes. The binding of this antibody significantly increased the accessibility of V3 region by other mAbs, suggesting that the attachment of 645-D opened up the V3 region that is masked by other structures, a common detection-averting strategy of HIV (Kwong et al, 2002;Pan et al, 2015). This IC elicited anti-V3 neutralizing antibody in BALB/c mice (Hioe et al, 2009). IC formed with mAb A32, binding to gp120 outside the CD4 binding site, induced neutralizing antibodies against a panel of HIV strains, although the enhancement over gp120 alone was not pronounced.
In the presence of cholera toxin and LPS as adjuvants, the breadth of the resulting antisera in neutralizing diverse HIV isolates was widened (Liao et al, 2004). Recently, broadly neutralizing antibodies (bNAb) against HIV-1 have seen great success. bNAb-producing B cells identified in HIV ICs preformed as vaccine preparations or as a consequence of antibody binding to endogenous antigens can signal via activating receptors to induce ADCC in macrophages and NK cells, leading to lysis of tumor or infected cells. The same signal via FccRs on dendritic cells results in enhanced antigen uptake and upregulated antigen presentation, both to MHC class II-restricted CD4 + T cells and to CD8 + T cells via cross-presentation. ICs retained in lymphoid tissues via deposition or FccRIIbmediated endocytosis extend the antigen availability for B-cell activation, resulting in increased antibody response and immune memory induction. Different glycosylation patterns on Fc regulate the preferential binding to type I or type II FccRs, controlling the state of inflammation. Sialylation of Fc with preferential binding to type II FccR in particular helps to set the threshold for the production of high-affinity antibodies.  2014). Beyond direct eradication of HIV, in a mouse model to approximate human mother to child transmission, FrCasE virusinfected neonatal mice develop erythroleukemia once reaching adulthood. Neonatal delivery of a specific mAb 667 boosted antibody protection and T-cell response that resulted in survival of treated mice in comparison with the total mortality in untreated controls. This protection was mediated by ADCC and formation of virus/antibody ICs (Michaud et al, 2010). Viral hepatitis B is another persistent infection treated with ICs as therapeutic vaccine. In a HBV vaccination model, ducklings were infected with duck hepatitis B virus (DHBV). They were treated with IC (DHBsAg complexed to rabbit anti-DHBs) linked to Staphylococcus aureas bacteria as a solid matrix complex. About 70% and 50% of treated ducks were cleared of viremia and antigenemia after three injections (Wen et al, 1994). The efficacy of IC-based vaccines in HBV infections was studied in mice in 1980s. ICs formed with nonspecific IgG or IgM antibodies showed minimal enhancement, as did the Fab fraction of those specific antibodies . HBsAg ICs stimulated T cell's avidity, which in turn induced antibody production in vivo (Celis et al, 1987). IC-mediated effects were followed by a seroconversion to HBsAb positivity in HBsAgtransgenic mice, indicating self-antigen tolerance breaking (Zheng et al, 2001). Subsequently, DCs induced from chronic hepatitis B patient PBMCs with GM-CSF/IL4 were stimulated by either HBsAg, antibody, or IC in vitro. Expression of functional markers including MHC class II, CD80/CD86, and CD40 was found increased only with the IC stimulation. Furthermore, when T cells from the patients were incubated with the stimulated DCs, higher levels of IFN-c and IL-2 were observed in the IC-stimulated group (Yao et al, 2007). Using a lentivirus vector coding HBsAg either alone or fused to Fc domain of mouse IgG2a, the fusion version induced much higher CD8 + T-cell activation in recipient mice, as well as elevated CD4 + T-cell response and antibody production (Hong et al, 2011). To simplify the vaccination protocol, Meng et al produced HBsAg-Fc (from IgG1) fusion protein and used it to immunize HBsAg-transgenic mice. This resulted in a predominant production of Th1 cytokines and reduced serum HBsAg level (Meng et al, 2012). Encouraged by these experimental findings, human HBsAg IC was adsorbed to alum and used to immunize 14 HBV chronic hepatitis B patients in a pilot study. The recipients showed substantial reduction in serum HBV DNA and HBeAg was converted to negative (Wen et al, 1995). In a phase 1 clinical trial, healthy subjects receiving various amounts of HBsAg IC preparations developed IgG1 and IgG3 antibodies against the immunogen with a simultaneous increase in serum IFN-c and IL-2 (Xu et al, 2005). From this outcome, one of the doses was used to immunize chronic hepatitis B patients. Five of ten patients responded and showed ≥ 2 logs decrease in serum HBV DNA, with a loss or marked reduction of HBeAg and an appearance of anti-HBeAg; and two of these patients developed anti-HBsAg antibodies (Xu et al, 2005). In a phase 2 clinical trial, HBeAg-positive chronic hepatitis B patients were immunized with six injections of the IC. At the end of post-trial follow-up, HBeAg seroconversion rate was 21.8% (17/78) and 9% (7/78) in the IC and placebo groups, respectively (P < 0.05; Xu et al, 2008).

IC in other microbial infections
In addition to the vaccine trials against HIV and HBV, smaller scale efforts to control other infections have been reported. CTL response against influenza virus infection is reduced in aging populations. While young BALB/c mounted strong cytolytic response with no discernible differences in intensity to flu NP antigen following inoculation of live, killed H1N1 virus, or a preparation of the virus plus a NP-specific antibody, only the IC significantly restored the same response in the aged mice. This enhanced killing was associated with increased IFN-c production in both CD4 + and CD8 + T cells . Due to its rapid spread and high rate of mutagenesis, the emphasis of influenza infection control has been to generate bNAbs against conserved regions of hemagglutinin (HA) (Okuno et al, 1993(Okuno et al, , 1994Ekiert et al, 2011). One version of the antibody, CT149, targeting the stem region of HAs of both type I and type II isolates blocked the membrane fusion. In addition, the binding by this antibody induced FccR-dependent ADCC, suggesting the involvement of ICs (Wu et al, 2015). For Ebola infection, several B-cell clones of highly potent GP-specific neutralizing antibodies from a disease survivor have been tested. These antibodies also mediated ADCC via FccR as mutants in the Fc that interfered with the receptor binding lost the cytotoxicity (Corti et al, 2016). Francisella tularensis (FT) is the causative pathogen of tularemia. DCs coated with monoclonal antibody-treated FT delivered intranasally protected hosts from subsequent challenges. This effect was not affected by the absence of FccRIIb, suggesting a preferential engagement of activating FccRs (Pham et al, 2014). In malaria infection, it was found that the FccR binding was critical for the uptake of IC (McClintock et al, 2005). In one study, the mortality of Plasmodium berghei infection that is often lethal was prevented if the antiserum from immune mice was transferred into infected mice. This protection was eliminated in the absence of FccR c-chain (Pleass, 2009). In addition, Phoolcharoen et al transiently expressed Ebola virus GP1 glycoprotein fused to an antibody heavy chain from a humanized GP1-specific antibody in Nicotiana benthamiana (tobacco plant). The purified fusion protein induced high titers of Ebolaspecific IgG antibodies, similar to those induced by GP1 virus-like particles, suggesting a scheme of vastly increased production capacity for IC preparations (Phoolcharoen et al, 2011). Urgent productions of ICs to protect populations under threat from rapidly emerging viral infections such as Middle East respiratory syndrome (MERS) are also being tested . Although ICs are in general considered to be conducive to antiviral immunity, it should be noted that in some cases the presence of virus-specific antibodies can increase infectivity, a phenomenon called antibody-dependent enhancement (ADE) of viral infection (Tirado & Yoon, 2003). In the STEP trials, HIV gag, pol, and nef gene cassettes were inserted into the adeno vector, however individuals with preexisting immunity against the virus showed greater risk of HIV infection following the immunization (Buchbinder et al, 2008). The same adverse effect was seen for additional viruses, such as dengue (Goncalvez et al, 2007), measles (Iankov et al, 2006), and over a dozen others (Suhrbier & La Linn, 2003). FccR-facilitated virus entry and a skewed cytokine profile may be the main culprits. These are important considerations in assessing ICs' immune regulatory functions.

IC in tumor immunity
Cancer immunotherapy has seen tremendous strides with technological developments in the immune checkpoint blockage. The basic concept is to reduce negative regulators of adaptive immunity, mainly targeting CTLA-4 and PD-1/PD-L1. Another modality of antibody-based cancer therapy is to target surface factors critical to tumor cell growth, such as EGFR and VEGF, of which optimized FccR ligation is an important consideration (Nimmerjahn & Ravetch, 2012). Concurrent to these progresses, IC-based intervention has remained an alternative due to its low side effects. Antibody binding to tumor surface antigens triggers ADCC and FccR engagement is an important activation signal for tumor lysis (Moalli et al, 2010). A large number of experiments have been carried out using B16 or its variants as the tumor model. Here, ICs have been shown to mediate tumor suppression via prophylactic, therapeutic and memoryinducing effects (Rafiq et al, 2002). In mice challenged with B16 tumor-expressing OVA antigen (MO4), injection of DCs pulsed with OVA-containing IC, but not with OVA or antibody alone, inhibited the tumor growth. Furthermore, treatment with DCs loaded with ICs cured 40% of mice bearing established tumors. Protection mediated by DC/IC immunization was long-standing and induced memory responses. In recipients that had been vaccinated with OVA   (Rafiq et al, 2002). In addition, specific antibody targeting HER2/neu antigen expressed by a whole-cell vaccine (3T3-neu/GM) was shown to enhance antigen-specific CD8 + T-cell activation and memory development and protected the hosts from the tumor challenge (Wolpoe et al, 2003;Kim et al, 2008)). Loading antibody against myeloma cell surface syndecan-1, a heparan sulfate proteoglycan expressed on cancer cells, induced HLA A2.1-mediated antigen presentation to MHC allele-matched donors, resulting in cytotoxic T-cell activation (Dhodapkar et al, 2002). Here, antisyndecan-1 antibody served as a facilitator of overall antigen presentation by DCs. The ensuing cytotoxicity elicited from human HLA-A2.1-restricted CTLs was directed toward other tumor antigens, such as MAGE-3 and NY-Eso-1 (Dhodapkar et al, 2002). FccRs play an important role in IC-mediated tumor immunity. In the syndecan-1 experiment, it was found the antibody coating did not alter the level of uptake of tumor cells yet blocking of FccRs significantly reduced antigen-specific T-cell proliferation (Dhodapkar et al, 2002), suggesting that the regulatory role of FccRs is mostly at steps beyond opsonization, primarily at DC maturation. Preferential induction of tumor immunity can be achieved by finetuning the signaling intensity ratios of activating vs inhibitory FccRs. This concept was proposed by Ravetch et al (Nimmerjahn & Ravetch, 2005). For instance, a tumor-specific antibody (TA99) was found to have mIgG2a Fc that binds to the activating FccRs with much higher intensity than the inhibitory FccRIIb. This preferential binding, particularly to FccRIV, endowed a strong antitumor effect (Nimmerjahn & Ravetch, 2005). The signaling of FccRs in ICmediated immune therapy can lead to a vaccinal effect, especially under properly selected FccR engagement. In a recent paper, mice challenged with CD20-positive EL4 tumor survived in the presence of mIgG2a CD20-specific antibody. This protection was lost in the absence of total or all activating FccRs. As expected, those primed mice developed an immune response that rejected a subsequent challenge of neo-antigen CD20-positive EL4 tumor (DiLillo & Ravetch, 2015). When the lymphocytes from primed mice or from mice with a DC-specific deletion of FccRIV were transferred into new hosts, the former transfusion showed a better tumor resistance than the latter in the naïve mice, suggesting the role of this receptor in mediating the vaccine effect (DiLillo & Ravetch, 2015). When the concept was tested in a humanized mouse system with the Fc region of mouse CD20-specific mAb replaced with the human counterpart and a total replacement of mFccRs with hFccRs in the host, it was found that mutations in the Fc that allowed better binding to hFccRIIIa and IIa, as compared to IIIa alone protected the host to the best extent (DiLillo & Ravetch, 2015). Overall, as in the case of antipersistent viral infections, the selective pairing of IgG FccR subtypes in vivo may represent the most promising direction in IC-mediated cancer therapy. Lastly, intracellular tumor antigens that are often shielded from immune recognition can become stimulatory in the presence of ICs. NY-seo-1 is a tumor antigen considered to be strictly intracellular. However, in combination with 5-fluorouracil, which likely stresses tumor cells for ensuing releases of intracellular contents, injection of mAb against NY-seo-1 resulted in significant  Wolins (1959a, 1961) tumor growth inhibition. This specific combination caused antigen epitope spreading, whereby other intracellular tumor antigens also induced T-cell activation (Noguchi et al, 2012).

Future prospective
The demand to employ IC technology in disease prevention and treatment makes it an urgent task to study their effects in both basic and clinical settings ( Table 1). As one of the oldest technologies of immune therapy, the track records of safety and simplicity of ICs are valuable on this day of enormous breakthroughs at other directions. The downside of IC-based interventions is also clear: relative low clinical efficacy and often unpredictable clinical and experimental results. However, with growing understanding of FccRs and their preferential bindings to IgG subtypes, and modern biomedical engineering to steer the desired pairing, the future outcomes will likely change. In hindsight, if these understandings were built into vaccine designs at the time, several large-scale clinical trials might have seen different and likely more favorable effects. In the case of HIV prophylaxis, IC formation that results in the undesired cytokine profile and the existing antibodies targeting the adeno vector are two factors likely addressable via precision targeting of activating FccRs. In IC-based anti-HBV vaccination and immune therapy, first-hand monitoring of responder cells and their FccR expression should be built into the protocols of next-stage clinical trials. At this time of personalized medicine, IC preparations tailored to individual FccR polymorphism could be within reach as well. Addressing FccR binding is only a mandatory starting point. Additional efforts will be needed to understand the precise antigen trafficking as a consequence of antibody binding, as well as to map inflammatory pathways and tissue targeting. As the desired immune protections vary from pathogen to pathogen, and entry portals are also vastly different, specific IC preparations can be made to induce CD4/CD8 and antibody responses for optimal protection. For instance, for antitumor and antiviral immunity that relies on cytolytic T-cell activation, an IC vaccine designed to target the "shallow" endocytic compartment will be highly ideal. Admittedly, IC-mediated immune regulations have not been as intensely investigated as other classes of immune adjuvants. However, mechanistic research will be most crucial for future breakthroughs in IC-based human disease intervention and is the strongest assurance to maintain the continuing development of this old biomedical technology.