FcRn: The Architect Behind the Immune and Nonimmune Functions of IgG and Albumin

FcRn: The architect behind the immune and non-immunefunctions of IgG and albumin

Michal Pyzik*,$, Timo Rath*,$,†, Wayne I. Lencer‡,¶,§, Kristi Baker*,$, and Richard S.Blumberg*,$,§

*Division of Gastroenterology, Hepatology and Endoscopy, Brigham and Women’s Hospital,Boston, MA 02115

$Department of Medicine, Harvard Medical School, Boston, MA 02115

†Division of Gastroenterology, Department of Medicine, Erlangen University Hospital, FriedrichAlexander University Erlangen-Nueremberg, 91054 Erlangen, Germany

‡Division of Gastroenterology, Hepatology and Nutrition, Boston Children’s Hospital Boston, MA02115

¶Department of Pediatrics, Harvard Medical School, Boston, MA 02115

§Harvard Digestive Diseases Center, Boston, MA 02115

AbstractThe neonatal Fc receptor (FcRn) belongs to the extensive and functionally divergent family ofMHC molecules. Contrary to classical MHC family members, FcRn possesses little diversity andis unable to present antigens. Instead, through its capacity to bind IgG and albumin with highaffinity at low pH, it regulates the serum half-lives of both of these proteins. In addition, FcRnplays important role in immunity at mucosal and systemic sites through both its ability to affectthe lifespan of IgG as well as its participation in innate and adaptive immune responses. Eventhough the details of its biology are still emerging, the property of FcRn to rescue albumin andIgG from early degradation represents an attractive approach to alter the plasma half-life ofpharmaceuticals. Here, we will review some of the most novel aspects of FcRn biology, bothimmune as well as non-immune, and provide some examples of FcRn-based therapies.

From Ehrlich passive immunity and Brambell Receptor to FcRnIt was more than one hundred and twenty years ago that Paul Ehrlich described the ability ofmaternal antibodies to cross to offspring and protect them from infections in early life (1).From the available studies, it was then recognized that the acquisition of this passiveimmunity varied depending on species. For instance, in humans and rabbits antibodytransfer occurred mainly before birth (antenatally) either transplacentally or via the yolk sac,respectively. In ruminants, horses and pigs, however, postnatal transmission took place,where antibodies were transferred in colostrum or milk, and were then absorbed trans-intestinally. In mice, rats, dogs and cats, antibody transfer occurred both before and after

Correspondence to: Richard S. Blumberg.

HHS Public AccessAuthor manuscriptJ Immunol. Author manuscript.

Published in final edited form as:J Immunol. 2015 May 15; 194(10): 4595–4603. doi:10.4049/jimmunol.1403014.

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birth, being more predominant in the neonatal period (2). Nonetheless, it was unknownwhether all types of globulins were transferred, how they were transported, and whetheridentical, equivalent or diverse transfer systems were operating in different species. In the1950s and 60s, by studying these phenomena, F. W. Rogers Brambell and colleaguesshowed that γ-globulins (consisting mostly of immunoglobulins, Ig) in particular wereselected for transmission while most other proteins from maternal circulation were not (3).Further, it was recognized that such transfer was completely dependent on the Fragmentcrystallisable (Fc) region of Ig and not on the Fragment antigen-binding (Fab) region (4).Brambell also investigated the fate of γ-globulins outside of pregnancy, reflecting on theirlong persistence in adult circulation (~20 versus ~five days for most of other plasmaproteins) and the characteristics of their elimination. He recognized that the long half-life ofIg required the Fc region and that very rapid elimination occurred upon high doseadministration, indicating that a saturable rescue process was involved (5, 6). Thus,Brambell postulated that a singular receptor may control both the transport of IgG duringearly life and the protection of IgG from catabolism in later life. Concurrently, Hermann E.Schultze and Joseph F. Heremans, observing fractional catabolic rates of different serumproteins, made similar predictions about the existence of an albumin specific protectionreceptor (7).

Next, in parallel to the demonstration of Fc dependent in utero rabbit Ab transfer, neonataltransmission of only γ-G globulin (IgG) across the intestinal mucosa in neonatal rats wasdefined at the functional and biochemical levels (8). In the 1970s, the proximal intestine wasidentified as the main site of transmission of passive immunity in neonatal rats (9).Subsequently, it was shown that this Fc receptor of the neonatal (FcRn) rat intestine orhuman intestine preferentially bound IgG under acidic conditions (8, 10). In the acidicenvironment typical for the duodenum and jejunum during early life, IgG was bound byFcRn on enterocyte surface, endocytosed and transcytosed, travelling from the lumen of theintestine, to the basolateral side where it was released at physiological pH (Figure 1B) (11,12). Following the purification of the heterodimeric receptor consisting of heavy (p51) andlight (p14) chains from rat enterocytes, the cloning the FcRn was achieved in 1989 (13, 14).During the same period, the initial observations on cellular and temporal expression of FcRnwere extended. Crystallization of the receptor and the identification of physical domains andamino acid residues responsible for Fc binding were accomplished rapidly thereafter (15–17). In the 1990s, the demonstration that animals deficient in the gene encoding the p14 lightchain did not express the p51 heavy chain, were unable to acquire IgG from maternal milk,and had low IgG circulating levels, undeniably demonstrated that the receptor responsiblefor IgG protection and fetal/neonate transfer were the same and only receptor; that is FcRn(18–20).

Albumin possesses an equally long half-life (~20 days) in circulation and the existence of analbumin protection receptor had also been hypothesized. Although several receptors able tobind albumin exist such as cubulin and megalin, none of them could account for the longalbumin persistence (21). As it was subsequently shown that FcRn binds albumin at acidicpH prolonging its half-life in the circulation, FcRn was recognized as the receptorresponsible for rescuing albumin from destruction (22). In support of this conclusion, theidentification of patients with defective FcRn function due to p14 light chain deficiency or

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the genetic deletion of the p51 heavy chain in animals clearly illustrated that this resulted inthe reduction of both albumin and IgG levels in the circulation (23, 24).

Recent years have brought forth several important advances in the study of FcRn biology.These include the recognition that FcRn expression continues after the neonatal period andis more widespread than initially suspected (25). Equally, in polarized epithelial cells FcRnis uniquely responsible for the bidirectional transcytosis of IgG, in contrast to the polymericIg receptor (pIgR) that transports polymeric IgA and IgM unidirectionally (26). This iscritically important to the immune responses at barrier surfaces that contain IgG. Further,FcRn is now recognized to exhibit significant expression throughout the hematopoieticsystem, notably in myeloid cells (27). Such targeted expression endows FcRn+ cells withparticular function: to distinguish between monomeric IgG and multimeric IgG-immunecomplexes (IC). The former recognition results in protection from degradation and themaintenance of serum IgG levels, while the latter initiates routing of the IgG-IC forbreakdown in antigen presentation compartments that leads to improved MHC class I and IIpresentation and the production of inflammatory cytokines (18, 28–30). The insights into thebiology of FcRn over the last 60 years are also now being converted into new therapeuticapproaches for several diseases. This review summarizes these recent advances and theirimplications.

General aspects of FcRn: From gene to proteinThe FCGRT (Fcgrt) gene is encoded on chromosome 19q13 in humans, and 7 in mice (31–33). Orthologues have been identified broadly in numerous mammalian and marsupialspecies (34, 35). The human FCGRT encoded receptor shares overall 66% homology withmouse FcRn while Fcgrt is highly conserved between mice and rats. The allelic diversity ofFCGRT/Fcgrt gene is low, with few polymorphisms identified (31–33). Of note, fivedifferent alleles of variable number of tandem repeats (VNTR1–5) in the 5′-flanking regionof hFCGRT have been described, with VNTR2 and VNTR3 being the most common (7.5%and 92% in Caucasians, and 3.2% and 96.8% in Japanese, respectively) (36, 37). Presence ofthe VNTR3 variant results in higher FCGRT transcriptional activity when compared with theVNTR2 allele, yet no significant effect on materno-fetal IgG transfer, or on the eliminationof therapeutic antibodies has been observed (38, 39). FCGRT encodes MHC class I-like α,heavy chain (p51) that non-covalently associates with light chain (p14), also known as β2-microglobulin (β2m), forming a heterodimer (13). The reported molecular mass of the αchain varies from 45 to 55 kDa depending on the species or cell type it is isolated from, andrelates mostly to variability in glycosylation with a single N-glycosylation site in humanFcRn compared with three in rodent FcRn (40). FcRn shares high sequence and structuralhomology with MHC class I molecules, however it is encoded outside of the HLA or H-2loci and is nonpolymorphic (31, 41). Thus FcRn is a type I glycoprotein consisting of α1,α2, α3 extracellular domains, a transmembrane region and cytoplasmic tail. The α1, α2domains form a narrow groove that is unable to present peptides but nonetheless inconjunction with the α3 domain and β2m form the ligand binding regions (13, 16).

FcRn expression is now recognized to be widespread, occurring throughout life. FcRn isexpressed by a wide variety of parenchymal cell types in many different species. These

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include vascular endothelium (including the central nervous system), most epithelial celltypes such as placental (syncytiotrophoblasts), epidermal (keratinocytes), intestinal(enterocytes), renal glomerular (podocytes), bronchial, mammary gland (ductal and acinar),retinal pigment epithelial cells, renal proximal tubular cells (PTC), hepatocytes,melanocytes, as well as cells of the choroid, ciliary body and iris in the eye (42, 43). FcRn isalso widely expressed by hematopoietic cells including monocytes, macrophages, dendriticcells (DC), neutrophils and B cells where, in contrast to polarized epithelial cells, it isdetected in significant quantities on the cell surface (27). In epithelial cells, which possessdistinct apical and basal membranes, FcRn is predominantly intracellular with a variabledistribution along the vesicular network and cell surface in a cell and species specificmanner (44). For instance, a greater quantity of FcRn has been observed in the apicalregions of syncytiotropoblasts and rodent enterocytes or primary renal PTC, whereas inmodel polarized cells such as Madin-Darby canine kidney (MDCK), human FcRn isexpressed predominantly basally including expression at the basolateral surface (45). Thisdifferential distribution has been attributed to the presence of dileucine and tryptophanmotifs, and several serine phosphorylation sites within the cytoplasmic tail, as well as theglycosylation status of the extracellular portion of the receptor (40, 45–47). The variablecellular localization of the receptor also affects the direction of IgG transport: in transfectedmodel systems and cell lines apical-to-basal transcytosis is greater with rodent FcRn incomparison to human FcRn, where basal-to-apical transport dominates (45, 47).

FcRn expression is also temporally and developmentally modulated. During fetal life, FcRnexpression in the syncytiotrophoblasts of the placenta is responsible for passive IgG transfer(Figure 1A). In rodents during the first weeks of life, the receptor is present at high levels inproximal intestinal epithelium coinciding with ingestion of mother’s milk (Figure 1B), andis rapidly down regulated after weaning (12, 48). In contrast, FcRn expression in humanenterocytes is lower than observed in neonatal rodents but does not decrease with age (26,49). Further, exposure of epithelial cells, human THP-1 (macrophage-like) cells, or humanPBMC to TNF-α, LPS or CpG induces a significant increase in FcRn expression in an NF-κB dependent manner, while IFN-γ priming results in FcRn down-regulation (50, 51). Theseresults suggest that FcRn expression can be modulated by the immunological context of thesurrounding milieu. Whether additional modes of spatiotemporal regulation of FcRnexpression, in humans or rodents, occur is presently unknown.

The ligands of FcRnFcRn binds IgG and albumin at acidic pH, which is characteristic of early and lateendosomes, the proximal intestine during neonatal life and, potentially, the extracellularmilieu associated with inflamed tissues (52). Albumin is the most abundant protein inmammalian plasma (~65% of the circulating proteins) and transports many endogenous andexogenous molecules, maintains colloidal osmotic pressure, and exerts antioxidant functions(53). Albumin is a 65–70 kDa protein consisting of three globular domains (DI, DII, andDIII). More than 60 variants of human albumin have been identified (53). Some, like theCasebrook variant, contains a single point mutation that decreases its affinity to FcRn (54).Likewise, IgGs represent the second most abundant proteins in serum, and are the mostfrequent antibody isotype found in the circulation. Of the four IgG subclasses in humans

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(IgG1, IgG2, IgG3 and IgG4), binding affinity to FcRn ranges from 20 nM (IgG1) to 80 nM(IgG4) (55). IgG3, with a longer hinge region relative to other IgG subclasses has the lowestpotential to bind FcRn and possesses a half-life of only nine days in circulation (56). Inmice, a similar range of affinities among the IgG subclasses (IgG1, IgG2a, IgG2b, IgG3) tobind FcRn has been demonstrated (57). Several polymorphisms and allotypes of the humanIgG heavy chain exist but whether these have differential binding to FcRn is unknown (58).Changes in the complementarity-determining region (CDR) of IgG have also been reportedto affect FcRn binding even though these are distant from the FcRn binding site (59, 60).Furthermore, variation in binding specificity is seen between FcRn orthologs. Whereas ratand mouse FcRn are more liberal in their ability to bind IgG from different species(including human, rabbit, mouse and bovine), human FcRn mainly interacts with human,rabbit and guinea pig IgG (61). Such disparity has also been illustrated for albumin binding,where rodent FcRn binds weakly to rhesus monkey or human albumin as compared withstrong binding to mouse and rat albumin, whereas human or rhesus monkey FcRn bindsmore strongly mouse and rat albumin than to the human or rhesus orthologue (62).

Structural studies have shown that FcRn binds to IgG with 1:1 or 2:1 stoichiometry undernon-equilibrium or equilibrium conditions, respectively (63, 64). In contrast, one FcRnreceptor binds to one albumin molecule (65). FcRn interacts with each of its two ligandsthrough contacts associated with opposite surfaces, such that FcRn can bind IgG andalbumin simultaneously without either competition or cooperation occurring between eachother (65). Biochemical and crystallographic data indicate that upon binding at pH 6.0,neither FcRn nor IgG undergo major conformational changes. Instead, it is the protonationof histidine residues (H310, H435, H436) in the CH2-CH3 hinge region of IgG1 whichenable binding (66). This allows for the formation of salt bridges at the FcRn-Fc interface,specifically the acidic residues on the C-terminal portion of the α2 domain (E117, E132 andD137) in FcRn, and the first residue of β2m (55, 66, 67). Notably, the sites within IgG thatbind to FcRn competitively overlap with IgG Fc binding to Staphylococcal protein A (68).In contrast, FcRn binding to albumin is mostly hydrophobic in nature and thought to bestabilized by a pH dependent hydrogen bonding network internal to each protein. Thisinteraction involves two tryptophan (W53 and W59) residues of FcRn and three histidine(H464, H510 and H535) residues within albumin third domain (DIII), which arefundamental for pH-dependent FcRn binding, as well as some contribution of the firstdomain (DI) (54, 69, 70). Contrary to FcRn-IgG interactions, no pH-dependentintermolecular salt bridges exist in hydrophobic FcRn-albumin binding. Available datasuggest that FcRn possesses higher affinity for IgG than for albumin (54, 65). These uniqueinteraction properties of FcRn and its ligands form the basis for a wide variety ofphysiologically important FcRn-driven functions.

FcRn functions: Not just bi-directional transporterFcRn-mediated recycling and the protection of monomeric IgG and albumin fromdegradation

FcRn plays a major role in the maintenance of serum IgG levels. Early studies with FcRnsuggested that the major cell type involved in this process was vascular endothelium (71).Yet, chimerism of WT mice with Fcgrt−/− bone marrow Tie-2-Cre driven, deletion of Fcgrt,

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have revealed that cells of the hematopoietic origin participate in the IgG salvage pathway(28, 72, 73). Given that only small amounts of FcRn receptor expression has been detectedon the surface of endothelial cells, IgG uptake is believed to occur mostly via non-specific,fluid-phase pinocytosis rather than by receptor-mediated endocytosis (18). Once inside thecell, IgG binding to FcRn is thought to occur as the early endosome becomes increasinglyacidic and permissive for pH-dependent interaction of FcRn with IgG. FcRn bound IgG isthen sorted into common recycling endosomes which recycle IgG away from lysosomes andback to the cell surface via rapid and slow release routes where IgG is extruded into theextracellular milieu due to the neutral pH in that locale (Figure 1C) (74).

Less is known about the FcRn dependent salvage of albumin, which is partly extrapolatedfrom insights derived from IgG-FcRn interactions. Based upon this, an equivalent pathwayfor FcRn receptor-mediated salvage of albumin is thought to exist (22). However, theefficiency of these two processes is vastly different and it is estimated that for every sixFcRn-recycled albumin molecules in humans, only one IgG molecule is rescued, while theratio in mice is even lower at 30:1 (75). Nonetheless, this level of recycling is sufficient forsustaining high IgG plasma concentrations with a relatively minor contribution provided byIgG production. In contrast, high serum albumin levels depend more on a high rate ofhepatic synthesis rather than on FcRn-dependent salvage (75). Thus the manner in whichFcRn maintains IgG and albumin in the circulation may share some but not all mechanisticfeatures. Furthermore, it remains unknown whether cells of hematopeitic and parenchymalorigin participate equivalently in IgG and albumin retention.

The FcRn-dependent diversion of IgG and albumin away from lysosomal degradation is thebasis for designing new or modified drugs with enhanced FcRn binding capacity and, thus,prolonged serum half-lives and potentially improved pharmacokinetics. A large number ofmodifications within the CH2-CH3 domains of IgG spanning the FcRn contact surface (suchas L253, H410 and H435 residues) have been described to significantly enhance pHdependent binding of IgG to FcRn (reviewed in (76)). Although they have not yet enteredclinical use, a number of preclinical studies in non-human primates have shown increasedefficacy of such engineered therapeutics in anti-cancer and anti-infection therapeuticapproaches (77–79). Alternatively, pharmacologic FcRn inhibition using peptide mimetics,anti-FcRn monoclonal antibodies or antibodies modified to possess Fc-dependent and lowpH-independent binding (so-called Abdegs) have been shown to decrease circulating levelsof pathogenic IgG and confer protection in models of myasthenia gravis (MG), systemiclupus erythematosus (SLE), rheumatoid arthritis (RA) and experimental autoimmuneencephalomyelitis (EAE)(77). Similarly, engineering IgGs to lack the ability to bind toFcRn, (such as the mutation of the I253, H410 and H435 residues (IgGIHH)), enablesaccelerated catabolism of IgG-radioimmuno conjugates during molecular imaging,endowing them with fewer toxic side effects than conventional chemotherapeutic drugs (80).Similar approaches aiming to develop long-lived albumin-based therapies demonstrate theenormous potential derived from understanding of mechanisms associated with FcRn-mediated recycling.

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Transcytosis of IgG and its implications for drug delivery and vaccine development

In polarized cells such as the epithelium, FcRn is able to transport IgG bi-directionally viatranscytosis which allows for the transfer of IgG from mother to young (Figure 1A, B), andthe delivery of IgG-complexed environmental antigens and microbial products into the hostresulting in the induction of tolerance or immunity as discussed further below (81, 82).Similar to the recycling pathway, IgG is likely internalized by fluid phase pinocytosis intopolarized epithelial cells. Still, the trafficking of FcRn is highly dynamic, and while most ofthe receptor occupies endosomal compartments at any single time, there is always someFcRn located at the cell surface with a flux of FcRn moving through the plasma membraneover time. Thus, receptor-mediated endocytosis is also thought to occur in light of earlyobservations wherein FcRn expression was detected on the apical enterocyte surfacemembrane and in the context of an acidic microenvironment in the neonate proximalintestine (74, 83, 84). That said, there is a paucity of data in support of the existence of anacidic pH environment in the neonate intestine which is further complicated by the fact thatthe uptake of IgG by neonatal rodent enterocytes has been observed to take place in theabsence of FcRn raising the possibility that the initial internalization of IgG in the neonatalenterocyte might be FcRn and pH independent (85). “Whether fluid phase and receptormediated pathways of IgG uptake operate concurrently and whether they escort IgG to thesame intracellular compartments still needs to be assessed.

Subsequent to internalization, IgG is firstly diverted into early sorting endosomes and thenthe common recycling endosome (86). The acidification of endosomes allows for FcRn tobind IgG in these compartments thereby enabling active FcRn-mediated transport of IgGacross the cell and its subsequent release on the opposite side, once exposed to the neutralextracellular pH (Figure 1B). The intracellular mechanisms that determine whether apical orbasal recycling versus apical-to-basal or basal-to-apical transcytosis occurs have onlyrecently begun to come to light. Apical to basolateral transport, for example, depends onmotifs present in the cytoplasmic tail, as well as on the ability of FcRn to bind calmodulin inpolarized epithelial model systems (46, 87, 88).

FcRn dependent transcytosis of IgG and likely albumin also occurs in vivo. In rodents,forced FcRn expression restricted to intestinal epithelial cells results in IgG secretion intothe gut lumen (82). In addition, Fc-dependent absorption of IgG has been described in thelungs and intestines of humanized mice that only express human FcRn (FCGRT/B2M Tg/Fcgrt−/− mice) (Figure 2A) (89). Similarly, Fc-fusion proteins, but not Fc-fusion proteinsdisabled in FcRn binding, can be absorbed in the lungs of mice and non-human primates(90, 91). Further, consistent with the near absence of serum proteins in the urine, albumin isre-absorbed in the renal PTC through an active process involving FcRn, suggestive oftranscytosis (92). In this pathway, albumin in the renal ultrafiltrate is internalized by bindingto the megalin-cubulin complex via receptor-mediated endocytosis on the apical surface ofPTCs. Subsequently, this complex is trafficked to late endosomes where, at acidic pH,albumin is captured by FcRn and directed back into the circulation (86, 93, 94). Theinvolvement of FcRn in albumin re-absorbtion by PTC requires additional substantiation andconsideration as FcRn-deficient mice, for instance, may not display significant albuminuria(95). This may be due to low albumin levels in the circulation of these animals

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(hypoalbuminemia) which results in reduced amounts of albumin in the glomerularultrafiltrate. As such, the loss of albumin in the urine of FcRn-deficient mice should beinvestigated in animals with normal circulating albumin levels. FcRn-mediated IgG handlingin the kidneys differs from albumin. In order to prevent potential accumulation of proteincomplexes that could obstruct glomerular filtration, IgG is removed from the glomerularbasement membrane in an FcRn-mediated process that allows for the movement of IgGacross the podocyte and its excretion into the glomerular capsular space and thus away fromthe circulation (96). As IgG is not subsequently re-absorbed by the PTC, it is lost in theurine (93). The latter process may be the basis for immune protection within the urinaryexcretory system.

The transcytosis of IgG or IgG-IC by FcRn at mucosal barriers such as the lung, intestines orgenitourinary tract can have important consequences for the host immune response. Inaddition to conferring passive immunity, mouse dams tolerized to antigens are able to confertolerance to their offspring during the period of suckling by transfer of specific IgG or IgG-IC (97, 98). Intriguingly, IgG-IC that formed in the context of antibody excess have beenfound to be immunosuppressive in contrast to IC formation in setting of antigen excesswherein they are immunogenic (99). More recently, using the prototype antigen OVA in anallergic airway disease model, airborne antigen exposure of lactating mice resulted indecreased airway hyper-reactivity only in breastfed offspring (100). It was subsequentlyshown that the milk from OVA sensitized mothers contained TGF-β as well as IgG-IC,which was transferred to the newborn via FcRn and that both are required for the inductionof tolerance (101). Moreover, due the production of IgG with anti-IgE specificities, FcRncan mediate the transfer of IgE from the lumen into the circulation in form of IgG anti-IgE-IC, which may also play a role in pathways of early life tolerance (102).

In vivo studies with FcRn humanized mice have demonstrated that circulating IgG can bedelivered into the intestinal lumen where they bind orally administered antigens and formIgG-IC which are then transported back into the lamina propria in an FcRn-dependentmanner (Figure 2A) (82). Since the IgG-IC were shown to subsequently be taken up byCD11c+ DCs that induced antigen specific CD4+ T cell responses, the FcRn mediatedtranscytosis of IgG and IgG-IC across mucosal barriers in adult life confers a sensingcapacity on IgG which scavenges luminal antigens and permits their efficient delivery to theimmune system. Indeed, the presence of anti-pathogen specific IgG reduced disease severityonly in FcRn competent mice upon challenge with pathogens such as Helicobacter pylori,Citrobacter rodentium or Chlamydia muridarum (89, 103, 104).

In the context of viral infection, the protective role of FcRn can be variable. Analysis ofherpes simplex virus 2 (HSV-2) infection has shown that HSV-2-specific antibodies aretranscytosed from the systemic circulation into the genital lumen in wild type (WT) mice,but not in FcRn-deficient mice, and that this confers protection against intra-vaginal viralchallenge (105, 106). Similar protective FcRn-mediated effects were noticeable afteradministration of anti-influenza hemagglutinin-specific monoclonal antibody upon influenzainfection (107). In contrast, during cytomegalovirus (CMV) infection, infectious virionswere able to disseminate to the placenta via their associations with poorly, but not strongly,neutralizing anti-CMV antibodies when transported by FcRn (108). FcRn transcytosis of

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IgG-virion complexes across mucosal genital surfaces has also been illustrated for HIVinfections (109, 110). In a study designed to test the ability of neutralizing and non-neutralizing antibodies to protect macaques from vaginal simian HIV (SHIV) challenge, thestrongly neutralizing IgG provided sterilizing immunity while non-neutralizing IgG did not(111). Indeed, passive administration of a highly neutralizing human anti-HIV antibody(VRCO1) engineered to enhance pH dependent binding to FcRn protected from the mucosaltransmission of SHIV virus infection (78). Thus, depending on antibody neutralizingcapacities, site and tissue pH, FcRn may be responsible for shuttling infectious agents whicheither facilitate or prevent infection.

FcRn dependent regulation of IgG-IC by haematopoietic cells and the roleplayed in innate and adaptive immunity

Both mouse and human hematopoietic cells have been demonstrated to express FcRn (27).Notably, these include all subsets of DC, macrophages and monocytes, as well asneutrophils and B cells (27, 72, 73). Most FcRn carrying hematopoietic cells are thusproficient in antigen presentation or phagocytosis. Indeed, in addition to being a major siteof monomeric IgG protection from degradation, FcRn expression by DC has been shown toplay an important role in the ability of an antigen carried by IgG to be processed anddisplayed by MHC class I molecules via cross-presentation and MHC class II molecules forpresentation to CD8+ and CD4+ T cells, respectively (29, 30). This is consistent with theimportance of FcRn in facilitating the degradation of IgG-IC in vivo (28, 73, 112). Inneutrophils such functions of FcRn have been linked to the uptake of IgG-opsonized bacteriaand their delivery into phagolysosomes (113). The size of the IC also determines the degreeto which IgG and antigen are diverted to lysosomes as large IgG-IC are more likely toexhibit this fate in comparison to small IgG-IC (114). In the latter case, small IgG-IC’s arehandled similarly to monomeric IgG and are protected from degradation (28). In epithelialcells, IgG-IC are also diverted to lysosomes as shown by the behavior of neutralizing anti-influenza monoclonal antibodies (107), making such observations a consequence of FcRninteractions with multimeric IgG rather than that of the cell type per se. These findingsclearly indicate a fundamental functional difference in how FcRn traffics monomeric IgGand multimeric IgG in form of IgG-IC.

The expression of classical Fcγ receptors (FcγR) on the cell surface enables APCs to captureand internalize IgG-ICs (115). In doing so, APC primed with IgG-IC effectively activateCD4+ and CD8+ T cells (116–119). It has recently been demonstrated, that FcγR and FcRncooperate in the processes of MHC class II presentation and MHC class I cross-presentation.Both of these receptors possess distinct pH dependent binding to IgG: FcγR at neutral pHand the FcRn at acidic pH. This is consistent with the concept of IgG-IC “hand-off” withinacidified endosomes where FcγR enables IgG-IC internalization initially, and FcRn shapesthe subsequent fate of the IgG-IC (Figure 2B). Consistent with this, DCs exposed to IgG-IC,but not to the FcRn non-binding IgGIHH-IC, induce greater CD4+ T cell proliferation both invitro and in vivo when compared to FcRn-deficient DCs (28, 29). Therefore, FcRncooperates with FcγR in antigen presentation and cross-presentation initiated by the uptakeof IgG captured antigens.

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Although FcRn is expressed by all types of APC, an analysis of the subsets of APC involvedin MHC class I cross-presentation have shown that FcRn enables IgG-dependent cross-presentation most strongly in the CD8−CD11b+ DC subset. The CD8−CD11b+ DC, incontrast to the CD8+ DC, possess acidic endolysosomal compartments conducive to IgG-FcRn binding (29). In a model of chronic colitis with high levels of anti-bacterial IgGantibodies, inflammatory CD8−CD11b+ DCs expanded and, in WT but not Fcgrt−/− mice,enabled efficient IgG-dependent activation of CD8+ T cells (29). Similar mechanismspresumably account for the improved expansion of antigen-specific CD8+ T cells aftervaccination with haptenated Ag (120). Therefore, this suggests that FcRn couples IgG-ICresponses with the efficient induction and activation of CD4+ and CD8+ T cells dependingupon the DC subset involved.

This is physiologically relevant in steady state settings given that conditional deletion ofFcRn within the CD11c+ cell fraction results in decreased quantities of CD8+ T cells atmucosal sites (30). Further, deficient CD8+ T cell cytokine production and an inability toblock the growth of induced or spontaneous colorectal or lung cancers was also observed(30). This defect in CD8+ T cell numbers was dependent on FcRn expressing CD8−CD11b+

DC fraction, as adoptive transfer of WT DC conferred protection to Fcgrt−/− recipients.Interestingly, these cellular defects of Fcgrt−/− mice were not accompanied by decreasedIgG levels within the intestine despite the quasi absence of IgG in the serum (30). Thissupports the notion that a major function of FcRn in tissues is the maintenance of cellularimmunity through the processing of antigen-antibody complexes.

Studies of multimeric, IgG-IC interactions with FcRn have further revealed that upon cross-linking, FcRn is capable of orchestrating a signaling cascade that is associated with secretionof cytokines skewed towards T helper 1 (TH1) and T cytotoxicity 1 (TC1) responses,particularly IL-12 (Figure 2B). In humans, immunohistochemical staining demonstrated thatFcRn+CD11c+ DCs were present in situ within colorectal cancers and adjacent colon inclose juxtaposition to CD8+ T cells. Most importantly, colorectal cancer patients with higherfrequency of FcRn+CD11c+ DCs at or near cancer sites had significantly longer survivaltimes than did those with fewer FcRn+CD11c+ cells (30). These observations support thenotion that FcRn+ APC regulate CD8+ T cells and their function in anti-tumor andpresumably other similar types of immunity. It may be surmised that a major function ofFcRn in tissues is in its direct effects on innate and adaptive immunity through regulation ofcytokine tone and antigen presentation that supports TH1 and Tc1 responses.

ConclusionsThe name of FcRn largely originates from the initial historical observations focusing on theneonatal IgG transmission in rodents (10). With the rediscovery and confirmation ofBrambell’s, Hereman’s and Schultze’s original hypotheses that FcRn regulates systemic IgGand albumin homeostasis and documentation that FcRn is broadly expressed throughout life,this neonate nomenclature, although archaic, still applies.

FcRn’s functions have recently expanded from passive immunity and IgG protection to anactive IgG and antigen trafficking receptor. The strategic presence at mucosal barrier sites

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takes advantage of FcRn’s ability to bi-directionally transport IgG and to deliver antigens tothe mucosal immune system. Then again FcRn expression in APC and specific transfer ofmultimeric IgG-IC to antigen processing vesicles allows for efficient MHC class I and IIpresentation. The ensuing effect is more potent elicitation of CD4+ and CD8+ T celldependent responses and enhanced protection against both bacterial and viral pathogens.This further suggests that in tissues FcRn directed degradation of IgG-IC might be its majorfunction. FcRn ability to bi-directionally move IgG, to exploit IgG’s abundance in particularat mucosal sites and its specificity towards diverse soluble antigens, endows this receptor toconvey IgG-IC for efficient T cell priming within APCs reuniting ideally the humoral andcellular arms of immunity.

AcknowledgmentsThis work was supported by NIH grants DK044319, DK051362, DK053056, DK088199 and the Harvard DigestiveDiseases Center (HDDC) DK034854.

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Figure 1.A) Transport of IgG across the placenta in humans or fetal yolk sac of rabbits. In theendoderm of the fetal yolk sac IgG is internalized by fluid-phase endocytosis and encountersFcRn in early endosome; B) Transport of IgG across the intestine in rodents or ruminants. Innew-born rats IgG is taken up by fluid-phase endocytosis or through binding to FcRn at theapical cell surface of enterocytes. This enables active FcRn-mediated transport of IgG acrossthe cell and its subsequent release on the opposite, extracellular side at neutral pH. In suchcircumstances, FcRn transcytosis exhibits a dominant vector of transport from apical-to-basolateral directions which likely reflects concentration and pH gradients as well as cell-dependent factors; C) The catabolism of IgG (and potentially albumin) by FcRn inendothelial cells or hemaotpoietic cells (not shown here). Serum IgG is internalized by fluid-phase endocytosis and binds to FcRn in an acidic endosomal compartment. FcRn thenrecycles IgG back into neutral pH milieu of the circulation, thus extending its serum half-life. IgG not bound to FcRn due to levels that exceed FcRn capacity or other serum proteinsare destined for lysosomal degradation.

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Figure 2.A) In the adult human gut, both enterocytes and antigen-presenting cells (APCs) in thelamina propria express FcRn. Enterocytes transcytose IgG into the gut lumen where it bindsto antigens. The IgG–immune complexes (IC, IgG-IC) are then delivered to dendritic cells(DC) in the lamina propria. Antigen-loaded DCs then migrate to the draining lymph nodes toprime T-cell responses. B) The IgG-IC can bind to FcγR on the surface of DC at neutral pH,initiating receptor-mediated endocytosis. This delivers the IgG-IC into the endolysosomalcompartments. As these vesicles mature they become more acidic. Acidification allows IgG-IC to dissociate from FcγR and favours binding to FcRn. Such a “hand-off” enables efficienttrafficking of the IgG-IC and the delivery of antigen into antigen processing pathways thatpromote the loading onto MHC class I and MHC class II molecules. Ligation of FcRn byIgG-IC also induces the production of IL-12 by the DC. The peptide loaded MHC moleculesderived from IgG-IC, are then able to prime CD8+ and CD4+ T cells. While the secretedIL-12 acts upon the primed CD4+ T cells to induce TH1 polarization, upon CD8+ T cells itpromotes activation, cytotoxicity and a TC1 phenotype. For simplification, although a

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monomeric IgG immune complex is shown in panels A and B, multimeric IgG-IC are thetypes responsible for antigen processing and induction of IL-12 secretion.

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FcRn: The Architect Behind the Immune and Nonimmune Functions of IgG and Albumin

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