Neonatal Fc Receptor For IgG Research Articles

Role of the Neonatal Fc Receptor for IgG in the Pathophysiology of Lupus

Role of the Neonatal Fc Receptor for IgG in the Pathophysiology of Lupus

Localization of neonatal Fc receptor for IgG in aggregated lymphoid nodules area in abomasum of Bactrian camels (Camelus bactrianus) of different ages.

BackgroundThe neonatal Fc receptor (FcRn) plays a crucial role in transporting IgG and associated antigens across polarized epithelial barriers in mucosal immunity. However, it was not clear that FcRn expression in aggregated lymphoid nodules area (ALNA) in abomasum, a unique and important mucosal immune structure discovered only in Bactrian camels. In the present study, 27 Alashan Bactrian camels were divided into the following five age groups: fetus (10–13 months of gestation), young (1–2 years), pubertal (3–5 years), middle-aged (6–16 years) and old (17–20 years). The FcRn expressions were observed and analyzed in detail with histology, immunohistochemistry, micro-image analysis and statistical methods.ResultsThe results showed that the FcRn was expressed in mucosal epithelial cells of ALNA from the fetus to the old group, although the expression level rapidly declined in old group; moreover, after the ALNA maturated, the FcRn expression level in the non-follicle-associated epithelium (non-FAE) was significantly higher than that in FAE (P < 0.05). In addition, the FcRn was also expressed in the vessel endothelium, smooth muscle tissue, and macrophages and dendritic cells (DCs) of secondary lymphoid follicles (sLFs).ConclusionsIt was demonstrated that FcRn was mainly expressed in non-FAE, the effector sites, although which was expressed in FAE, the inductive sites for mucosal immunity. And it was also expressed in DCs and macrophages in sLFs of all ages of Bactrian camels. The results provided a powerful evidence that IgG (including HCAb) could participate in mucosal immune response and tolerance in ALNA of Bactrian camels through FcRn transmembrane transport.

Single chain antibody fragments with pH dependent binding to FcRn enabled prolonged circulation of therapeutic peptide in vivo

The neonatal Fc receptor for IgG (FcRn) is considered critical for the regulation of endogenous IgG and serum albumin (SA) and their circulation half-life in vivo. Both IgG and SA can bind to FcRn tightly at acidic pH but not so much at neutral pH. Here we reported a few novel single chain antibody fragments (scFv) obtained based on screening of a phage library. FnAb-8 and FnAb-12 can bind to human FcRn with higher affinities than IgG at acidic pH but similar or lower affinities than IgG at pH7.4. Fusion proteins consisted of the therapeutic peptide, GLP-1 (Glucagon-like peptide-1) connected to the N-terminus of FnAb-8 and FnAb-12, named as G8 and G12, were shown to retain the pH-dependent binding capabilities to FcRn while also bound to the GLP-1 receptor. In vivo efficacy studies in diet induced diabetes mice confirmed the GLP-1 receptor (GLP-1R) agonist activities and sustained blood sugar lowering effect. In vivo pharmacokinetics (PK) studies were performed in nonhuman primates and FnAb-8 was found to have circulation half-life several folds longer than what have been reported for scFvs. G8 may be developed into long acting GLP-1R agonists with great potentials in clinical applications.

The intracellular localization and association of porcine Ia-associated invariant chain with the MHC class I-related porcine neonatal Fc receptor for IgG

The neonatal Fc receptor (FcRn) transports IgG from mother to young and is involved in antigen presentation. FcRn is structurally similar to MHC class I, but its intracellular trafficking pathway is much more analogous to that of MHC class II. Ia-associated invariant chain (Ii) molecules play an additional role in directing MHC class II trafficking within the endocytic compartments by physical association with MHC class II. This study addresses the question of whether pig Ii chain plays this important role in FcRn trafficking to the endoplasmic reticulum. Red or green fluorescent protein-fused Ii or FcRn was constructed, and the intracellular localization of pig Ii with FcRn was detected using confocal microscopy. Immunoprecipitation and western blotting were used to test for their association. The results indicate that pig Ii chain specifically interacts with both FcRn H chain alone and FcRn–β2m complex, and the CLIP in Ii was required for FcRn–Ii association. A truncated FcRn deletion in the cytoplasmic tail changed the intracellular localization of FcRn. However, the truncated FcRn can still combine Ii. This indicated that the cytoplasmic tail of FcRn fails to affect FcRn association with Ii. These results suggest that association of FcRn with Ii chain is relevant, and appreciation of this process is important to the understanding of how IgG is transported.

Neonatal Fc receptor for IgG (FcRn) expressed in the gastric epithelium regulates bacterial infection in mice.

Neonatal Fc receptors for immunoglobulin (Ig)G (FcRn) assume a central role in regulating host IgG levels and IgG transport across polarized epithelial barriers. We have attempted to elucidate the contribution of FcRn in controlling Helicobacter infection in the stomach. C57BL/6J wild-type or FcRn(-/-) mice were infected with Helicobacter heilmannii, and gastric lesions, bacterial load and the levels of antigen-specific IgG in serum and gastric juice were analyzed. The elevated levels of anti-H. heimannii IgG in gastric juice were observed exclusively in wild-type mice but not in FcRn(-/-) mice. In contrast, an increase in lymphoid follicles and bacterial loads along with deeper gastric epithelium invasion were noted in FcRn(-/-) mice. C57BL/6J wild-type or FcRn(-/-) mice were also infected with Helicobacter pylori SS1, and the results of the bacterial load in stomachs of these mice and the anti-H. pylori IgG levels in serum and gastric juice were similar to those from H. heilmannii infection. Our data suggest that FcRn can be functionally expressed in the stomach, which is involved in transcytosis of IgG, and prevent colonization by H. heilmannii and the associated pathological consequences of infection.

Intracellular neutralization of viral infection in polarized epithelial cells by neonatal Fc receptor (FcRn)-mediated IgG transport

IgG was traditionally thought to neutralize virions by blocking their attachment to or penetration into mucosal epithelial cells, a common site of exposure to viruses. However, we describe an intracellular neutralizing action for an influenza hemagglutinin-specific monoclonal antibody, Y8-10C2 (Y8), which has neutralizing activity only at an acidic pH. When Y8 was applied to the basolateral surface of Madin-Darby canine kidney cells expressing the rat neonatal Fc receptor for IgG (FcRn), it significantly reduced viral replication following apical exposure of the cell monolayer to influenza virus. Virus neutralization by Y8 mAb was dependent on FcRn expression and its transport of IgG. As both FcRn and Y8 mAb bind their partners only at acidic pH, the Y8 mAb is proposed to carry out its antiviral activity intracellularly. Furthermore, the virus, Y8 mAb, and FcRn colocalized within endosomes, possibly inhibiting the fusion of viral envelopes with endosomal membranes during primary uncoating, and preventing the accumulation of the neutralized viral nucleoprotein antigen in the nucleus. Prophylactic administration of Y8 mAb before viral challenge in WT mice, but not FcRn-KO mice, conferred protection from lethality, prevented weight loss, resulted in a significant reduction in pulmonary virus titers, and largely reduced virus-induced lung pathology. Thus, this study reveals an intracellular mechanism for viral neutralization in polarized epithelial cells that is dependent on FcRn-mediated transport of neutralizing IgG.

Neonatal Fc receptor for IgG (FcRn) regulates cross-presentation of IgG immune complexes by CD8−CD11b+dendritic cells

Cross-presentation of IgG-containing immune complexes (ICs) is an important means by which dendritic cells (DCs) activate CD8(+) T cells, yet it proceeds by an incompletely understood mechanism. We show that monocyte-derived CD8(-)CD11b(+) DCs require the neonatal Fc receptor for IgG (FcRn) to conduct cross-presentation of IgG ICs. Consequently, in the absence of FcRn, Fcγ receptor (FcγR)-mediated antigen uptake fails to initiate cross-presentation. FcRn is shown to regulate the intracellular sorting of IgG ICs to the proper destination for such cross-presentation to occur. We demonstrate that FcRn traps antigen and protects it from degradation within an acidic loading compartment in association with the rapid recruitment of key components of the phagosome-to-cytosol cross-presentation machinery. This unique mechanism thus enables cross-presentation to evolve from an atypically acidic loading compartment. FcRn-driven cross-presentation is further shown to control cross-priming of CD8(+) T-cell responses in vivo such that during chronic inflammation, FcRn deficiency results in inadequate induction of CD8(+) T cells. These studies thus demonstrate that cross-presentation in CD8(-)CD11b(+) DCs requires a two-step mechanism that involves FcγR-mediated internalization and FcRn-directed intracellular sorting of IgG ICs. Given the centrality of FcRn in controlling cross-presentation, these studies lay the foundation for a unique means to therapeutically manipulate CD8(+) T-cell responses.

MHC class I-related FcRn-mediated presentation of immune complexed antigen is associated with endosomal and phagosomal pH and antigen stability in macrophages and dendritic cells (100.56)

Abstract The Fc receptors for the immunoglobulin (Ig) G antibody (FcgammaRs) found on macrophages and dendritic cells (DCs) efficiently facilitate the presentation or cross-presentation of immune-complexed (IC)-antigens to T cells. We found that the neonatal Fc receptor for IgG (FcRn) in both macrophages and DCs failed to have a strong effect on the cross-presentation of IC ovalbumin (OVA) antigen to CD8+ T cells. Interestingly, endosomal FcRn enhanced the presentation of the monomeric OVA-IC to OT-II CD4+ T cells robustly, whereas FcRn in phagosomes exerted distinctive effects on antigen presentation between macrophages and DCs. The presentation of phagocytosed latex-OVA-ICs to CD4+ T cells was also considerably enhanced on wild-type versus FcRn-deficient macrophages, but was not affected in FcRn-deficient DCs. This functional discrepancy was associated with the dependence of IgG-FcRn binding in an acidic pH. Following phagocytosis, the phagosomal pH dropped rapidly to below 6.5 in macrophages but remained in the neutral range in DCs. This disparity in pH determined the rate of degradation of phagocytosed ICs. Thus, our findings reveal that FcRn expression has a different effect on antigen processing and presentation of ICs to CD4+ T cells in the endosomal versus phagosomal compartments of macrophages versus DCs.

Role of Fc Receptors as a Therapeutic Target

It has been forty years since the discovery of Fc Receptors and their function. Fc Receptors include the IgG receptors (FcgammaR), high-affinity IgE receptor (FcepsilonRI), IgA and IgA/IgM receptors, and neonatal Fc receptor for IgG (FcRn). In particular, the FcgammaRs have been well known to play an important role in many biologic processes including those associated with the response to infection and cancer as well as in the pathogenesis of immune-mediated diseases. Both positive and negative regulatory function has ascribed to Fc receptors and FcgammaRs in particular which serve to establish a threshold for immune cell activation. In other cases, Fc receptors such as FcRn possess a novel structure and function by playing a major role in the transport of IgG across polarized epithelial barriers at mucosal surfaces and in the regulation of IgG half-life. These diverse functions highlight the potential effectiveness of targeting Fc receptors for therapeutic purposes. This review summarizes new information available in the therapeutic applications of this biology.

Immune modulation by antibodies and antibody receptors

Mucosal immunoregulation involves the unique cross-talk between epithelial cells and various immune system components. Mucosal secretions contain significant quantities of immunoglobulin (Ig) G. The neonatal Fc receptor for IgG (FcRn) is important in regulating host IgG levels and in transporting IgG and its associated antigens across polarized epithelial barriers. Through its ability to secrete and absorb IgG, FcRn integrates its encounters with luminal antigens with the systemic immune compartments. The FcRn thereby provides essential host defense and immunoregulatory functions at mucosal surfaces. In this review, we examine recent data on the use of FcRn and anti-CD3 for immune modulation. Recent preclinical studies have revealed the significant efficacy of using FcRn or orally administered antibodies as a means of immune modulation. For example, the oral administration of anti-CD3 was recently shown to be effective in the prevention and treatment of several animal models of immune-mediated disorders. In addition, the use of FcRn or orally administered antibodies were recently described in two patents as potential methods for inducing systemic tolerance and for treating immune-mediated disorders. The FcRn patent generally discussed the methods and products for initiating an immune response against an antigen. In particular, this patent described the trans-epithelial delivery of antigens to provoke tolerance and immunity. The anti-CD3 patent emphasized the advantages of an oral route of administration compared to a parenteral one. These advantages included easier chronic use and a reduction in the side effects that are associated with the parenteral use of anti-CD3.

The MHC Class II-Associated Invariant Chain Interacts with the Neonatal Fcγ Receptor and Modulates Its Trafficking to Endosomal/Lysosomal Compartments

The neonatal Fc receptor for IgG (FcRn) transfers maternal IgG to the offspring and protects IgG from degradation. The FcRn resides in an acidic intracellular compartment, allowing it to bind IgG. In this study, we found the association of FcRn and invariant chain (Ii). The interaction was initiated within the endoplasmic reticulum by Ii binding to either the FcRn H chain alone or FcRn H chain-beta(2)-microglobulin complex and appeared to be maintained throughout the endocytic pathway. The CLIP in Ii was not required for FcRn-Ii association. The interaction was also detected in IFN-gamma-treated THP-1, epithelial and endothelial cells, and immature mouse DCs. A truncated FcRn without the cytoplasmic tail was unable to traffic to early endosomes; however, its location in early endosomes was restored by Ii expression. FcRn was also detected in the late endosome/lysosome only in the presence of Ii or on exposure to IFN-gamma. In immature human or mouse DCs, FcRn was barely detected in the late endosome/lysosome in the absence of Ii. Furthermore, the cytoplasmic tail of Ii conferred tailless FcRn to route to both the early endosome and late endosome/lysosome in a hybrid molecule. Because the FcRn is expressed in macrophages and DCs or epithelial and endothelial cells where Ii is induced under inflammation and infection, these results reveal the complexity of FcRn trafficking in which Ii is capable of expanding the boundary of FcRn trafficking. Taken together, the intracellular trafficking of FcRn is regulated by its intrinsic sorting information and/or an interaction with Ii chain.

Dependence of antibody-mediated presentation of antigen on FcRn

The neonatal Fc receptor for IgG (FcRn) is a distant member of the MHC class I protein family. It binds IgG and albumin in a pH-dependent manner and protects these from catabolism by diverting them from a degradative fate in lysosomes. In addition, FcRn-mediated IgG transport across epithelial barriers is responsible for the transmission of IgG from mother to infant and can also enhance IgG-mediated antigen uptake across mucosal epithelia. We now show a previously undescribed role for FcRn in mediating the presentation of antigens by dendritic cells when antigens are present as a complex with antibody by uniquely directing multimeric immune complexes, but not monomeric IgG, to lysosomes.

Activation of the JAK/STAT-1 Signaling Pathway by IFN-γ Can Down-Regulate Functional Expression of the MHC Class I-Related Neonatal Fc Receptor for IgG

Expression of many MHC genes is enhanced at the transcriptional or posttranscriptional level following exposure to the cytokine IFN-gamma. However, in this study we found that IFN-gamma down-regulated the constitutive expression of the neonatal Fc receptor (FcRn), an MHC class I-related molecule that functions to transport maternal IgG and protect IgG and albumin from degradation. Epithelial cell, macrophage-like THP-1 cell, and freshly isolated human PBMC exposure to IFN-gamma resulted in a significant decrease of FcRn expression as assessed by real-time RT-PCR and Western blotting. The down-regulation of FcRn was not caused by apoptosis or the instability of FcRn mRNA. Chromatin immunoprecipitation and gel mobility shift assays showed that STAT-1 bound to an IFN-gamma activation site in the human FcRn promoter region. Luciferase expression from an FcRn promoter-luciferase reporter gene construct was not altered in JAK1- and STAT-1-deficient cells following exposure to IFN-gamma, whereas expression of JAK1 or STAT-1 protein restored the IFN-gamma inhibitory effect on luciferase activity. The repressive effect of IFN-gamma on the FcRn promoter was selectively reversed or blocked by mutations of the core nucleotides in the IFN-gamma activation site sequence and by overexpression of the STAT-1 inhibitor PIAS1 or the dominant negative phospho-STAT-1 mutations at Tyr-701 and/or Ser-727 residues. Furthermore, STAT-1 might down-regulate FcRn transcription through sequestering the transcriptional coactivator CREB binding protein/p300. Functionally, IFN-gamma stimulation dampened bidirectional transport of IgG across a polarized Calu-3 lung epithelial monolayer. Taken together, our results indicate that the JAK/STAT-1 signaling pathway was necessary and sufficient to mediate the down-regulation of FcRn gene expression by IFN-gamma.

NF-κB Signaling Regulates Functional Expression of the MHC Class I-Related Neonatal Fc Receptor for IgG via Intronic Binding Sequences

The neonatal Fc receptor for IgG (FcRn) functions to transport maternal IgG to a fetus or newborn and to protect IgG from degradation. Although FcRn is expressed in a variety of tissues and cell types, the extent to which FcRn expression is regulated by immunological and inflammatory events remains unknown. Stimulation of intestinal epithelial cell lines, macrophage-like THP-1, and freshly isolated human monocytes with the cytokine TNF-alpha rapidly up-regulated FcRn gene expression. In addition, the TLR ligands LPS and CpG oligodeoxynucleotide enhanced the level of FcRn expression in THP-1 and monocytes. Treatment of TNF-stimulated THP-1 cells with the NF-kappaB-specific inhibitor or overexpression of a dominant negative mutant inhibitory NF-kappaB (IkappaBalpha; S32A/S36A) resulted in down-regulation of FcRn expression. By using chromatin immunoprecipitation we identified three NF-kappaB binding sequences within introns 2 and 4 of the human FcRn gene. An EMSA confirmed the p50/p50 and/or p65/p50 complex (s) bound to intron 2- or 4-derived oligonucleotides containing putative NF-kappaB binding sequences, respectively. The intronic NF-kappaB sequences in combination with the promoter or alone regulated the expression of a luciferase reporter gene in response to TNF-alpha stimulation or overexpression of NF-kappaB p65 and p50. DNA looping interactions potentially occurred after the stimulation between intronic NF-kappaB sequences and the FcRn promoter as shown by a chromosome conformation capture assay. Finally, TNF-alpha stimulations enhanced IgG transport across an intestinal Caco-2 epithelial monolayer. Together, these data provide the first evidence that NF-kappaB signaling via intronic sequences regulates FcRn expression and function.

FcRn: the neonatal Fc receptor comes of age

The neonatal Fc receptor for IgG (FcRn) has been well characterized in the transfer of passive humoral immunity from a mother to her fetus. In addition, throughout life, FcRn protects IgG from degradation, thereby explaining the long half-life of this class of antibody in the serum. In recent years, it has become clear that FcRn is expressed in various sites in adults, where its potential function is now beginning to emerge. In addition, recent studies have examined the interaction between FcRn and the Fc portion of IgG with the aim of either improving the serum half-life of therapeutic monoclonal antibodies or reducing the half-life of pathogenic antibodies. This Review summarizes these two areas of FcRn biology.

Identification and characterization of a novel splicing variant of the MHC class I-related neonatal Fc receptor (FcRn) for IgG (93.9)

Abstract The neonatal Fc receptor for IgG (FcRn), a MHC class I-related molecule, functions to transport maternal IgG into the fetus or newborn and protects IgG from catabolism. The mRNAs of several MHC class I-related molecules have multiple splicing variants. By cloning porcine FcRn cDNA from the intestinal epithelial cell line IPEC, two cDNAs were found: a long form of 1071 bp (pFcRn-L) and a short form of 795 bp (pFcRn-S). These mRNAs were also detected in porcine kidney epithelial cell line LLC-PK1, peripheral blood mononuclear cells, as well as a variety of porcine tissues by RT-PCR and Northern blot. Sequence analysis showed that pFcRn-L cDNA encodes the full-length pFcRn, whereas pFcRn-S cDNA represents a splicing variant of pFcRn mRNA, coding for a truncated pFcRn with deletion of 92 amino acids matching to the alpha2 domain of pFcRn-L. Recombinant pFcRn-L bound IgG at pH 6.0, but not at pH7.5. However, recombinant pFcRn-S bound IgG at both pH 5.0–6.0 and pH 7.5. Furthermore, the intracellular trafficking patterns of pFcRn-L and pFcRn-S exhibited remarkable differences. The pFcRn-L was expressed on the cell surface and mainly localized in the early endosomal compartment, whereas pFcRn-S was primarily found in late endosome/lysosome. In contrast to pFcRn-L, pFcRn-S trafficking to the lysosomal compartment is independent of porcine beta2m expression as assessed in a beta2m-deficient cell line FO-1. Therefore, the pattern of FcRn gene expression which is characterized by the complexity of RNA splicing further implies the functional complication of FcRn.

The Neonate Fc Receptor (FcRn) For IgG Is Important For The Development of Rotavirus-Specific IgG Responses And Clearance of Rotavirus From The Intestines Of Mice (53.5)

Abstract Rotavirus infection and virus-like particle vaccines induce intestinal rotavirus-specific IgG in mice that appears to play a role in protection. The mechanism by which rotavirus-specific IgG reaches the intestinal lumen is unknown. The neonatal Fc receptor for IgG (FcRn) is the only known transporter of IgG in the intestines in neonatal and adult mice. Additionally, FcRn is implicated in antigen presentation in the intestines of adult mice. We tested whether FcRn is important in protecting the intestine against rotavirus infection and in transport of rotavirus-specific IgG into the intestinal lumen. Naïve FcRn deficient (FcRn−/−) and wild type BALB/c controls were orally inoculated with 103ID50 ECWT rotavirus. Fecal samples were collected 0–12 days post inoculation (dpi) and tested for rotavirus antigen and rotavirus-specific IgG by ELISA. Virus clearance from the intestine was significantly delayed by 3 days in FcRn−/− compared to BALB/c mice. Rotavirus-specific intestinal IgG titers were also significantly lower in FcRn−/− versus BALB/c mice. These data indicate that FcRn plays a role in clearance of a primary rotavirus infection from the intestine and intestinal transport of rotavirus-specific IgG. Studies are underway to elucidate the mechanism(s) by which FcRn contributes to clearance of intestinal rotavirus infection. VA Merit Review Grant, NIH RO1 AI24998

IgG transport across mucosal barriers by neonatal Fc receptor for IgG and mucosal immunity

Mucosal secretions of the human gastrointestinal, respiratory, and genital tracts contain significant quantities of IgG. The neonatal Fc receptor for IgG (FcRn) plays a major role in regulating host IgG levels and transporting IgG and associated antigens across polarized epithelial barriers. The FcRn can then recycle the IgG/antigen complex back across the intestinal barrier into the lamina propria for processing by dendritic cells and presentation to CD4(+) T cells in regional organized lymphoid structures. FcRn, through its ability to secrete and absorb IgG, thus integrates luminal antigen encounters with systemic immune compartments and, as such, provides essential host defense and immunoregulatory functions at the mucosal surfaces.

Neonatal Fc receptor for IgG regulates mucosal immune responses to luminal bacteria

The neonatal Fc receptor for IgG (FcRn) plays a major role in regulating host IgG levels and transporting IgG and associated antigens across polarized epithelial barriers. Selective expression of FcRn in the epithelium is shown here to be associated with secretion of IgG into the lumen that allows for defense against an epithelium-associated pathogen (Citrobacter rodentium). This pathway of host resistance to a bacterial pathogen as mediated by FcRn involves retrieval of bacterial antigens from the lumen and initiation of adaptive immune responses in regional lymphoid structures. Epithelial-associated FcRn, through its ability to secrete and absorb IgG, may thus integrate luminal antigen encounters with systemic immune compartments and as such provide essential host defense and immunoregulatory functions at the mucosal surfaces.

Calnexin and ERp57 Facilitate the Assembly of the Neonatal Fc Receptor for IgG with β2-Microglobulin in the Endoplasmic Reticulum

The neonatal FcR (FcRn) consists of an MHC class I-like H chain in noncovalent association with beta(2)-microglobulin (beta(2)m). The proper folding of FcRn in the endoplasmic reticulum is essential for FcRn function. Using a low stringency immunoprecipitation of human FcRn, we observed the coprecipitation of an 88-kDa band. Mass spectrometry analysis revealed that this band was identical with calnexin (CNX). This association was verified by Western blotting the CNX or FcRn immunoprecipitates with either an anti-FcRn or anti-CNX Ab. In the beta(2)m-null FO-1 cell transfected with FcRn H chain alone or both FcRn H chain and beta(2)m, CNX bound to the FcRn H chain before the FcRn H chain association with beta(2)m. However, calreticulin only bound to the FcRn H chain-beta(2)m complex. Furthermore, the thiol oxidoreductase ERp57 was detected in FcRn-CNX complexes, suggesting its role in disulfide bond formation of the FcRn H chain. Removal of the N-linked glycosylation site from the FcRn H chain resulted in a decreased association of the FcRn H chain for beta(2)m. However, the absence of CNX did not significantly affect FcRn assembly as defined by the ability of FcRn to bind IgG and exit to the cell surface. This suggests that other chaperones compensate for the function of CNX in FcRn assembly. In addition, we found that tapasin and TAP were not involved in FcRn assembly, as shown by coimmunoprecipitation in THP-1 cells and IgG-binding assays in 721.220 (tapasin-deficient) and 721.174 (TAP-deficient) cells transfected with FcRn. These findings show the importance of chaperones in FcRn assembly.