The IgG-degrading enzyme, Imlifidase, restores the therapeutic activity of FVIII in inhibitor-positive hemophilia A mice.

Neutralizing anti-factor VIII (FVIII) antibodies, known as FVIII inhibitors, represent a major drawback of replacement therapy in persons with congenital hemophilia A (PwHA), rendering further infusions of FVIII ineffective. FVIII inhibitors can also appear in non-hemophilic individuals causing acquired hemophilia A (AHA). The use of non-FVIII bypassing agents in cases of bleeds or surgery in inhibitor-positive patients is complicated by the lack of reliable biological monitoring and increased thrombotic risk. Imlifidase (IdeS) is an endopeptidase that degrades human immunoglobulin G (IgG); it was recently approved for hyperimmune patients undergoing renal transplants. Here we investigated the ability of IdeS to eliminate FVIII inhibitors in vitro and in a model of inhibitor-positive HA mice. IdeS cleaved anti-FVIII plasma IgG from PwHA and AHA patients, and hydrolyzed recombinant human anti-FVIII IgG independently from their subclass or specificity for the A2, A3, C1 or C2 domains of FVIII. In HA mice passively immunized with recombinant human anti-FVIII IgG, IdeS restored the hemostatic efficacy of FVIII, as evidenced by the correction of the bleeding tendency. Our results provide the proof of concept for the transient removal of FVIII inhibitors by IdeS, thereby opening a therapeutic window for efficient FVIII replacement therapy in inhibitor-positive patients.

Stimulation with FITC-labeled antigens confers B cells with regulatory properties.

B cells with regulatory properties (Bregs) were identified in human and in mice among different B-cell subsets. Their regulatory properties rely mainly on the production of anti-inflammatory cytokines, in particular IL10, IL-35 and TGFß, and were extensively studied in mouse models of autoimmune and inflammatory diseases. However, the exact nature of the stimulatory signals conferring regulatory properties to B cells is still not clear. We serendipitously observed that fluorescein isothiocyanate (FITC) binds to a significant proportion of naïve mouse B cells. Binding of FITC to the B-cell surface implicated at least in part the B-cell receptor. It triggered IL-10 production and allowed the endocytosis of FITC-coupled antigens followed by their presentation to CD4+ T cells. In particular, B cells incubated with FITC-OVA polarized OTII T cells towards a Tr1/Th2 phenotype in vitro. Further, the adoptive transfer of B cells incubated with FITC-labeled myelin oligodendrocyte glycoprotein peptide protected mice from experimental autoimmune encephalomyelitis, a T-cell-dependent autoimmune model. Together, the data show that FITC-stimulated B cells polarize immune responses towards Tr1/Th2 and acquire immuno-modulatory properties.

Correction of bleeding in experimental severe hemophilia A by systemic delivery of factor VIII-encoding mRNA.

The treatment or prevention of bleeding in patients with hemophilia A relies on replacement therapy with different factor VIII (FVIII)-containing products or on the use of by-passing agents, i.e., activated prothrombin complex concentrates or recombinant activated factor VII. Emerging approaches include the use of bispecific anti-factor IXa/factor X antibodies, anti-tissue factor pathway inhibitor antibodies, interfering RNA to antithrombin, and activated protein C-specific serpins or gene therapy. The latter strategies are, however, hampered by the short clinical experience and potential adverse effects including the absence of tight temporal and spatial control of coagulation and the risk of uncontrolled insertional mutagenesis. Systemic delivery of mRNA allows endogenous production of the corresponding encoded protein. Thus, injection of erythropoietin-encoding mRNA in a lipid nanoparticle formulation resulted in increased erythropoiesis in mice and macaques. Here, we demonstrate that a single injection of in vitro transcribed B domain-deleted FVIII-encoding mRNA to FVIII-deficient mice enables endogenous production of pro-coagulant FVIII. Circulating FVIII:C levels above 5% of normal levels were maintained for up to 72 h, with an estimated half-life of FVIII production of 17.9 h, and corrected the bleeding phenotype in a tail clipping assay. The endogenously produced FVIII did however exhibit low specific activity and induced a potent neutralizing IgG response upon repeated administration of the mRNA. Our results suggest that the administration of mRNA is a plausible strategy for the endogenous production of proteins characterized by poor translational efficacy. The use of alternative mRNA delivery systems and improved FVIII-encoding mRNA should foster the production of functional molecules and reduce their immunogenicity.

Prevention of the anti-factor VIII memory B-cell response by inhibition of Bruton tyrosine kinase in experimental hemophilia A.

Hemophilia A is a rare hemorrhagic disorder caused by the lack of functional pro-coagulant factor VIII. Factor VIII replacement therapy in patients with severe hemophilia A results in the development of inhibitory anti-factor VIII IgG in up to 30% of cases. To date, immune tolerance induction, with daily injection of large amounts of factor VIII, is the only strategy to eradicate factor VIII inhibitors. This strategy is, however, efficient in only 60-80% of patients. We investigated whether blocking B-cell receptor signaling upon inhibition of Bruton tyrosine kinase prevents anti-factor VIII immune responses in a mouse model of severe hemophilia A. Factor VIII-naïve and factor VIII-sensitized factor VIII-deficient mice were fed with the selective inhibitor of Bruton tyrosine kinase, (R)-5-amino-1-(1-cyanopiperidin-3-yl)-3-(4-[2,4-difluorophenoxyl] phenyl)-1H pyrazole-4-carboxamide (PF-06250112), to inhibit B-cell receptor signaling prior to challenge with exogenous factor VIII. The consequences on the anti-factor VIII immune response were studied. Inhibition of Bruton tyrosine kinase during the primary anti-factor VIII immune response in factor VIII-naïve mice did not prevent the development of inhibitory anti-factor VIII IgG. In contrast, the anti-factor VIII memory B-cell response was consistently reduced upon treatment of factor VIII-sensitized mice with the Bruton tyrosine kinase inhibitor. The Bruton tyrosine kinase inhibitor reduced the differentiation of memory B cells ex vivo and in vivo following adoptive transfer to factor VIII-naïve animals. Taken together, our data identify inhibition of Bruton tyrosine kinase using PF-06250112 as a strategy to limit the reactivation of factor VIII-specific memory B cells upon re-challenge with therapeutic factor VIII.

Biochemical characterization and immunogenicity of Neureight, a recombinant full-length factor VIII produced by fed-batch process in disposable bioreactors.

Hemophilia A is a X-linked recessive bleeding disorder consecutive to the lack of circulating pro-coagulant factor VIII (FVIII). The most efficient strategy to treat or prevent bleeding in patients with hemophilia A relies on replacement therapy using exogenous FVIII. Commercially available recombinant FVIII are produced using an expensive perfusion technology in stainless steel fermenters. A fed-batch fermentation technology was recently developed to produce 'Neureight', a full-length recombinant human FVIII, in Chinese hamster ovary (CHO) cells. Here, we investigated the structural and functional integrity and lack of increased immunogenicity of Neureight, as compared to two commercially available full-length FVIII products, Helixate and Advate, produced in baby hamster kidney or CHO cells, respectively. Our results demonstrate the purity, stability and functional integrity of Neureight with a standard specific activity of 4235 ±â€¯556 IU/mg. The glycosylation and sulfation profiles of Neureight were similar to that of Advate, with the absence of the antigenic carbohydrate epitopes α-Gal and Neu5Gc, and with sulfation of Y1680, that is critical for FVIII binding to von Willebrand factor (VWF). The endocytosis of Neureight by human immature dendritic cells was inhibited by VWF, and its half-life in FVIII-deficient mice was similar to that of Advate, confirming unaltered binding to VWF. In vitro and in vivo assays indicated a similar immunogenicity for Neureight, Advate and Helixate. In conclusion, the production of full-length FVIII in a fed-batch fermentation mode generates a product that presents similar biochemical, functional and immunogenic properties as products developed using the classical perfusion technology.

Oxidation of factor VIII increases its immunogenicity in mice with severe hemophilia A.

The development of antibodies against therapeutic factor VIII (FVIII) represents the major complication of replacement therapy in patients with severe hemophilia A. Amongst the environmental risk factors that influence the anti-FVIII immune response, the presence of active bleeding or hemarthrosis has been evoked. Endothelium damage is typically associated with the release of oxidative compounds. Here, we addressed whether oxidation contributes to FVIII immunogenicity. The control with N-acetyl cysteine of the oxidative status in FVIII-deficient mice, a model of severe hemophilia A, reduced the immune response to exogenous FVIII. Ex vivo exposure of therapeutic FVIII to HOCl induced a mild oxidation of the molecule as evidenced by the loss of free amines and resulted in increased FVIII immunogenicity in vivo when compared to native FVIII. The increased immunogenicity of oxidized FVIII was not reverted by treatment of mice with N-acetyl cysteine, and did not implicate an increased maturation of professional antigen-presenting cells. Our data document that oxidation influences the immunogenicity of therapeutic FVIII.

Inhibitor Formation in Congenital Hemophilia A: an Immunological Perspective.

The immunogenicity of therapeutic factor VIII (FVIII) in patients with hemophilia A has been puzzling scientific and clinical communities for more than 3 decades. Indeed, the development of inhibitory antibodies to FVIII remains a major clinical challenge and is associated with enormous societal costs. Thus, the reasons for which a presumably innocuous, short-lived, intravenously administered glycoprotein triggers such a deleterious, long-lasting neutralizing immune response is an enigma. This review does not pretend to bring an answer to this challenging question. It will however summarize the latest findings regarding the molecular interactions at play in the recognition of FVIII by the immune cells, the validity of the proposed risk factors for FVIII alloimmunization, and the different solutions that allow induction of FVIII-specific tolerance in preclinical models of hemophilia A.

Complement C3 is a novel modulator of the anti-factor VIII immune response.

Development of neutralizing antibodies against therapeutic Factor VIII (FVIII) is the most serious complication of the treatment of hemophilia A. There is growing evidence to show the multifactorial origin of the anti-FVIII immune response, combining both genetic and environmental factors. While a role for the complement system on innate as well as adaptive immunity has been documented, the implication of complement activation on the onset of the anti-FVIII immune response is unknown. Here, using in vitro assays for FVIII endocytosis by human monocyte-derived dendritic cells and presentation to T cells, as well as in vivo complement depletion in FVIII-deficient mice, we show a novel role for complement C3 in enhancing the immune response against therapeutic FVIII. In vitro, complement C3 and its cleavage product C3b enhanced FVIII endocytosis by dendritic cells and presentation to a FVIII-specific CD4+ T-cell hybridoma. The C1 domain of FVIII had previously been shown to play an important role in FVIII endocytosis, and alanine substitutions of the K2092, F2093 and R2090 C1 residues drastically reduce FVIII uptake in vitro Interestingly, complement activation rescued the endocytosis of the FVIII C1 domain triple mutant. In a mouse model of severe hemophilia A, transient complement C3 depletion by humanized cobra venom factor, which does not generate anaphylatoxin C5a, significantly reduced the primary anti-FVIII immune response, but did not affect anti-FVIII recall immune responses. Taken together, our results suggest an important adjuvant role for the complement cascade in the initiation of the immune response to therapeutic FVIII.

The C1 and C2 domains of blood coagulation factor VIII mediate its endocytosis by dendritic cells.

The development of inhibitory antibodies to therapeutic factor VIII is the major complication of replacement therapy in patients with hemophilia A. The first step in the initiation of the anti-factor VIII immune response is factor VIII interaction with receptor(s) on antigen-presenting cells, followed by endocytosis and presentation to naïve CD4+ T cells. Recent studies indicate a role for the C1 domain in factor VIII uptake. We investigated whether charged residues in the C2 domain participate in immunogenic factor VIII uptake. Co-incubation of factor VIII with BO2C11, a monoclonal C2-specific immunoglobulin G, reduced factor VIII endocytosis by dendritic cells and presentation to CD4+ T cells, and diminished factor VIII immunogenicity in factor VIII-deficient mice. The mutation of basic residues within the BO2C11 epitope of C2 replicated reduced in vitro immunogenic uptake, but failed to prevent factor VIII immunogenicity in mice. BO2C11 prevents factor VIII binding to von Willebrand factor, thus potentially biasing factor VIII immunogenicity by perturbing its half-life. Interestingly, a factor VIIIY1680C mutant, that does not bind von Willebrand factor, demonstrated unaltered endocytosis by dendritic cells as well as immunogenicity in factor VIII-deficient mice. Co-incubation of factor VIIIY1680C with BO2C11, however, resulted in decreased factor VIII immunogenicity in vivo In addition, a previously described triple C1 mutant showed decreased uptake in vitro, and reduced immunogenicity in vivo, but only in the absence of endogenous von Willebrand factor. Taken together, the results indicate that residues in the C1 and/or C2 domains of factor VIII are implicated in immunogenic factor VIII uptake, at least in vitro Conversely, in vivo, the binding to endogenous von Willebrand factor masks the reducing effect of mutations in the C domains on factor VIII immunogenicity.

The ADAMTS131239-1253 peptide is a dominant HLA-DR1-restricted CD4+ T-cell epitope.

Acquired thrombotic thrombocytopenic purpura is a rare and severe disease characterized by auto-antibodies directed against "A Disintegrin And Metalloproteinase with Thrombospondin type 1 repeats, 13th member" (ADAMTS13), a plasma protein involved in hemostasis. Involvement of CD4+ T cells in the pathogenesis of the disease is suggested by the IgG isotype of the antibodies. However, the nature of the CD4+ T-cell epitopes remains poorly characterized. Here, we determined the HLA-DR-restricted CD4+ T-cell epitopes of ADAMTS13. Candidate T-cell epitopes were predicted in silico and binding affinities were confirmed in competitive enzyme-linked immunosorbent assays. ADAMTS13-reactive CD4+ T-cell hybridomas were generated following immunization of HLA-DR1 transgenic mice (Sure-L1 strain) and used to screen the candidate epitopes. We identified the ADAMTS131239-1253 peptide as the single immunodominant HLA-DR1-restricted CD4+ T-cell epitope. This peptide is located in the CUB2 domain of ADAMTS13. It was processed by dendritic cells, stimulated CD4+ T cells from Sure-L1 mice and was recognized by CD4+ T cells from an HLA-DR1-positive patient with acute thrombotic thrombocytopenic purpura. Interestingly, the ADAMTS131239-1253 peptide demonstrated promiscuity towards HLA-DR11 and HLA-DR15. Our work paves the way towards the characterization of the ADAMTS13-specific CD4+ T-cell response in patients with thrombotic thrombocytopenic purpura using ADAMTS131239-1253-loaded HLA-DR tetramers.

Immunogenicity of long-lasting recombinant factor VIII products.

Replacement therapy for patients with hemophilia A using plasma-derived or recombinant factor VIII (FVIII) is complicated by the short half-life of the FVIII products and by the occurrence of neutralizing antibodies in a substantial number of patients. In the recent years, enormous efforts have been invested to develop new generations of coagulation factors with extended half-lives. Presumably, the use of long-lasting FVIII products should reduce the frequency of administration to the patients and drastically improve their quality of life. The question of their immunogenicity remains however unanswered as yet. The present review proposes a summary of the different strategies developed to enhance the half-life of FVIII, including fusion of FVIII to the Fc fragment of the human IgG1 or to human serum albumin, or attachment of polyethylene glycol. Based on the available literature, we hypothesize on the potential benefits or risks associated with each of the latter strategies in terms of immunogenicity of the newly derived hemostatic drugs.

High factor VIII concentrations interfere with glycoprotein VI-mediated platelet activation in vitro.

BACKGROUND: The recruitment of activated factor VIII (FVIII) at the surface of activated platelets is a key step toward the burst of thrombin and fibrin generation during thrombus formation at the site of vascular injury. It involves binding to phosphatidylserine and, possibly, to fibrin-bound αIIbß3. Seminal work had shown the binding of FVIII to resting platelets, yet without a clear understanding of a putative physiological relevance. OBJECTIVES: To characterize the effects of FVIII-platelet interaction and its potential modulation of platelet function. METHODS: FVIII was incubated with washed platelets. The effects on platelet activation (spontaneously or triggered by collagen and thrombin) were studied by flow cytometry and light transmission aggregometry. We explored the involvement of downstream pathways by studying phosphorylation profiles (Western blot). The FVIII-glycoprotein (GP) VI interaction was investigated by ELISA, confocal microscopy, and proximity ligation assay. RESULTS: FVIII bound to the surface of resting and activated platelets in a dose-dependent manner. FVIII at supraphysiological concentrations did not induce platelet activation but rather specifically inhibited collagen-induced platelet aggregation and altered glycoprotein VI (GPVI)-dependent phosphorylation. FVIII, freed of its chaperone protein von Willebrand factor (VWF), interacted in close proximity with GPVI at the platelet surface. CONCLUSION: We showed that VWF-free FVIII binding to, or close to, GPVI modulates platelet activation in vitro. This may represent an uncharacterized negative feedback loop to control overt platelet activation. Whether locally activated FVIII concentrations achieved during platelet accumulation and thrombus formation at the site of vascular injury in vivo are compatible with such a function remains to be determined.

Imlifidase, a new option to optimize the management of patients with hemophilia A on emicizumab.

BACKGROUND: Emicizumab is a bispecific, chimeric, humanized immunoglobulin G (IgG)4 that mimics the procoagulant activity of factor (F) VIII (FVIII). Its long half-life and subcutaneous route of administration have been life-changing in treating patients with hemophilia A (HA) with or without FVIII inhibitors. However, emicizumab only partially mimics FVIII activity; it prevents but does not treat acute bleeds. Emergency management is particularly complicated in patients with FVIII inhibitors receiving emicizumab prophylaxis in whom exogenous FVIII is inefficient. We have shown recently that Imlifidase (IdeS), a bacterial IgG-degrading enzyme, efficiently eliminates human anti-FVIII IgG in a mouse model of severe HA with inhibitors and opens a therapeutic window for the administration of exogenous FVIII. OBJECTIVES: To investigate the impact of IdeS treatment in inhibitor-positive HA mice injected with emicizumab. METHODS: IdeS was injected to HA mice reconstituted with human neutralizing anti-FVIII IgG and treated with emicizumab. RESULTS: IdeS hydrolyzed emicizumab in vitro and in vivo, albeit, at slower rates than another recombinant human monoclonal IgG4. While F(ab')2 fragments were rapidly cleared from the circulation, thus leading to a rapid loss of emicizumab procoagulant activity, low amounts of single-cleaved intermediate IgG persisted for several days. Moreover, the IdeS-mediated elimination of the neutralizing anti-FVIII IgG and restoration of the hemostatic efficacy of exogenous FVIII were not impaired by the presence of emicizumab and polyclonal human IgG in inhibitor-positive HA mice. CONCLUSION: Our results suggest that IdeS could be administered to inhibitor-positive patients with HA receiving emicizumab prophylaxis to improve and ease the management of breakthrough bleeds or programmed major surgeries.

Transplacental delivery of therapeutic proteins by engineered immunoglobulin G: a step toward perinatal replacement therapy.

BACKGROUND: Transplacental delivery of maternal immunoglobulin G (IgG) provides humoral protection during the first months of life until the newborn's immune system reaches maturity. The maternofetal interface has been exploited therapeutically to replace missing enzymes in the fetus, as shown in experimental mucopolysaccharidoses, or to shape adaptive immune repertoires during fetal development and induce tolerance to self-antigens or immunogenic therapeutic molecules. OBJECTIVES: To investigate whether proteins that are administered to pregnant mice or endogenously present in their circulation may be delivered through the placenta. METHODS: We engineered monovalent immunoglobulin G (FabFc) specific for different domains of human factor VIII (FVIII), a therapeutically relevant model antigen. FabFc was injected with exogenous FVIII into pregnant severe hemophilia A mice or pregnant mice expressing human FVIII following AAV8-mediated gene therapy. FabFc and FVIII were detected in the pregnant mice and/or fetuses by enzyme-linked immunosorbent assay and immunohistochemistry. RESULTS: Administration of FabFc to pregnant mice allowed the maternofetal delivery of FVIII in a FcRn-dependent manner. FVIII antigen levels achieved in the fetuses represented 10% of normal plasma levels in the human. We identified antigen/FabFc complex stability, antigen size, and shielding of promiscuous protein patches as key parameters to foster optimal antigen delivery. CONCLUSION: Our results pave the way toward the development of novel strategies for the in utero delivery of endogenous maternal proteins to replace genetically deficient fetal proteins or to educate the immune system and favor active immune tolerance upon protein encounter later in life.

Thermodynamic analysis of the interaction of factor VIII with von Willebrand factor.

Factor VIII (FVIII) is a glycoprotein that plays an important role in the intrinsic pathway of coagulation. In circulation, FVIII is protected upon binding to von Willebrand factor (VWF), a chaperone molecule that regulates its half-life, distribution, and activity. Despite the biological significance of this interaction, its molecular mechanisms are not fully characterized. We determined the equilibrium and activation thermodynamics of the interaction between FVIII and VWF. The equilibrium affinity determined by surface plasmon resonance was temperature-dependent with a value of 0.8 nM at 35 °C. The FVIII-VWF interaction was characterized by very fast association (8.56 × 10(6) M(-1) s(-1)) and fast dissociation (6.89 × 10(-3) s(-1)) rates. Both the equilibrium association and association rate constants, but not the dissociation rate constant, were dependent on temperature. Binding of FVIII to VWF was characterized by favorable changes in the equilibrium and activation entropy (TΔS° = 89.4 kJ/mol, and -TΔS(++) = -8.9 kJ/mol) and unfavorable changes in the equilibrium and activation enthalpy (ΔH° = 39.1 kJ/mol, and ΔH(++) = 44.1 kJ/mol), yielding a negative change in the equilibrium Gibbs energy. Binding of FVIII to VWF in solid-phase assays demonstrated a high sensitivity to acidic pH and a sensitivity to ionic strength. Our data indicate that the interaction between FVIII and VWF is mediated mainly by electrostatic forces, and that it is not accompanied by entropic constraints, suggesting the absence of conformational adaptation but the presence of rigid "pre-optimized" binding surfaces.

Induction of heme oxygenase-1 in factor VIII-deficient mice reduces the immune response to therapeutic factor VIII.

Replacement therapy with exogenous factor VIII (FVIII) to treat hemorrhages induces anti-FVIII inhibitory immunoglobulin G in up to 30% of patients with hemophilia A. Chronic inflammation associated with recurrent bleedings is a proposed risk factor for FVIII inhibitor development. Heme oxygenase-1 (HO-1) is a stress-inducible enzyme with potent anti-inflammatory activity. Here, we demonstrate that induction of HO-1 before FVIII administration drastically reduces the onset of the anti-FVIII humoral immune response. The protective effect was specific for HO-1 because it was reproduced on administration of the end products of HO-1 activity, carbon monoxide, and bilirubin, and prevented by the pharmacologic inhibition of HO-1 using tin mesoporphyrin IX. HO-1 induction was associated with decreased major histocompatibility complex class II expression by splenic antigen-presenting cells and reduced T-cell proliferation. Triggering the endogenous anti-inflammatory machinery before FVIII administration may represent a novel therapeutic option for preventing the development of FVIII inhibitors in hemophilia A patients.

[Origin and nature of the neutralizing immune response against therapeutic factor VIII]. / Origine et nature de la réponse immunitaire neutralisante contre le facteur VIII thérapeutique.

The use of therapeutic proteins induces in some patients the appearance of neutralizing antibodies. This is the case of pro-coagulant factor VIII (FVIII) used in patients with hemophilia A. Several parameters related to the protein itself, to the type of pathology or to the patients, condition the immunogenicity of a therapeutic protein. Understanding these parameters would help to anticipate or prevent the development of neutralizing antibodies. In the case of FVIII, we propose that the development of neutralizing antibodies does not result from an unpredicted immune response but rather from the inability of the patient's organism to develop an anti-inflammatory or regulatory response.

TITLE: Origine et nature de la réponse immunitaire neutralisante contre le facteur VIII thérapeutique. ABSTRACT: L'utilisation de protéines thérapeutiques se heurte, chez certains patients, à l'apparition d'anticorps neutralisants. C'est le cas, par exemple, du facteur VIII pro-coagulant qui est utilisé pour traiter les patients atteints d'hémophilie A. Plusieurs paramètres, liés à la protéine elle-même, au type de pathologie ou aux patients, conditionnent l'immunogénicité d'une protéine thérapeutique. Les comprendre permettrait d'anticiper ou de prévenir la survenue d'anticorps neutralisants. Nous proposons dans cette revue de montrer que, dans le cas du facteur VIII, la survenue de ces anticorps neutralisants ne résulte pas d'une réponse immunitaire inopinée, mais plutôt de l'incapacité de l'organisme des patients à développer une réponse anti-inflammatoire ou régulatrice.

Therapeutic normal IgG intravenous immunoglobulin activates Wnt-ß-catenin pathway in dendritic cells.

Therapeutic normal IgG intravenous immunoglobulin (IVIG) is a well-established first-line immunotherapy for many autoimmune and inflammatory diseases. Though several mechanisms have been proposed for the anti-inflammatory actions of IVIG, associated signaling pathways are not well studied. As ß-catenin, the central component of the canonical Wnt pathway, plays an important role in imparting tolerogenic properties to dendritic cells (DCs) and in reducing inflammation, we explored whether IVIG induces the ß-catenin pathway to exert anti-inflammatory effects. We show that IVIG in an IgG-sialylation independent manner activates ß-catenin in human DCs along with upregulation of Wnt5a secretion. Mechanistically, ß-catenin activation by IVIG requires intact IgG and LRP5/6 co-receptors, but FcγRIIA and Syk are not implicated. Despite induction of ß-catenin, this pathway is dispensable for anti-inflammatory actions of IVIG in vitro and for mediating the protection against experimental autoimmune encephalomyelitis in vivo in mice, and reciprocal regulation of effector Th17/Th1 and regulatory T cells.

Relevance of the Materno-Fetal Interface for the Induction of Antigen-Specific Immune Tolerance.

In humans, maternal IgGs are transferred to the fetus from the second trimester of pregnancy onwards. The transplacental delivery of maternal IgG is mediated by its binding to the neonatal Fc receptor (FcRn) after endocytosis by the syncytiotrophoblast. IgGs present in the maternal milk are also transferred to the newborn through the digestive epithelium upon binding to the FcRn. Importantly, the binding of IgGs to the FcRn is also responsible for the recycling of circulating IgGs that confers them with a long half-life. Maternally delivered IgG provides passive immunity to the newborn, for instance by conferring protective anti-flu or anti-pertussis toxin IgGs. It may, however, lead to the development of autoimmune manifestations when pathological autoantibodies from the mother cross the placenta and reach the circulation of the fetus. In recent years, strategies that exploit the transplacental delivery of antigen/IgG complexes or of Fc-fused proteins have been validated in mouse models of human diseases to impose antigen-specific tolerance, particularly in the case of Fc-fused factor VIII (FVIII) domains in hemophilia A mice or pre-pro-insulin (PPI) in the case of preclinical models of type 1 diabetes (T1D). The present review summarizes the mechanisms underlying the FcRn-mediated transcytosis of IgGs, the physiopathological relevance of this phenomenon, and the repercussion for drug delivery and shaping of the immune system during its ontogeny.