Edda Fiebiger - Academia.edu (original) (raw)

Papers by Edda Fiebiger

Cell reports, Jan 3, 2015

Epidemiologic studies discovered an inverse association between immunoglobulin E (IgE)-mediated a... more Epidemiologic studies discovered an inverse association between immunoglobulin E (IgE)-mediated allergies and cancer, implying tumor-protective properties of IgE. However, the underlying immunologic mechanisms remain poorly understood. Antigen cross-presentation by dendritic cells (DCs) is of key importance for anti-tumor immunity because it induces the generation of cytotoxic CD8(+) T lymphocytes (CTLs) with specificity for tumor antigens. We demonstrate that DCs use IgE and FcεRI, the high-affinity IgE receptor, for cross-presentation and priming of CTLs in response to free soluble antigen at low doses. Importantly, IgE/FcεRI-mediated cross-presentation is a distinct receptor-mediated pathway because it does not require MyD88 signals or IL-12 induction in DCs. Using passive immunization with tumor antigen-specific IgE and DC-based vaccination experiments, we demonstrate that IgE-mediated cross-presentation significantly improves anti-tumor immunity and induces memory responses in ...

Inflammatory bowel diseases, 2014

The chemokine CCL25, and its receptor CCR9, constitute a unique chemokine/receptor pair, which re... more The chemokine CCL25, and its receptor CCR9, constitute a unique chemokine/receptor pair, which regulates trafficking of T lymphocytes to the small intestine under physiological conditions and is an attractive target for small bowel Crohn's disease drug development. We have previously shown that CCL25/CCR9 interactions regulate the recovery from acute dextran sulfate sodium-induced colonic inflammation. In this study, we explored whether these interactions also regulate chronic colitis development in 2 independent murine models of experimental colitis. Histological flow cytometry and qPCR analyses were performed to evaluate the role of CL25 and CCR9 in chronic colonic inflammation induced by serial exposures to dextran sulfate sodium salts or by adoptive transfer of CD45RB(hi) CD4(+) T cell into lymphopenic mice devoid of CCL25/CCR9 interactions. Chronic dextran sulfate sodium exposure results in exacerbated colitis in mice deficient for either CCR9 or CCL25 when compared with wi...

The American Journal of Gastroenterology, 2014

Recent experimental evidence suggests that environmental microbial factors early in life determin... more Recent experimental evidence suggests that environmental microbial factors early in life determine susceptibility to allergic diseases through inappropriate chemotaxis and local activation of CD1drestricted, invariant chain natural killer T (iNKT) cells. In this study, we analyzed the involvement of these pathways in pediatric patients with eosinophilic esophagitis (EoE) before and after dietary allergen elimination.

Journal of Experimental Medicine, 2002

Here, we describe a new approach designed to monitor the proteolytic activity of maturing phagoso... more Here, we describe a new approach designed to monitor the proteolytic activity of maturing phagosomes in live antigen-presenting cells. We find that an ingested particle sequentially encounters distinct protease activities during phagosomal maturation. Incorporation of active proteases into the phagosome of the macrophage cell line J774 indicates that phagosome maturation involves progressive fusion with early and late endocytic compartments. In contrast, phagosome biogenesis in bone marrow-derived dendritic cells (DCs) and macrophages preferentially involves endocytic compartments enriched in cathepsin S. Kinetics of phagosomal maturation is faster in macrophages than in DCs. Furthermore, the delivery of active proteases to the phagosome is significantly reduced after the activation of DCs with lipopolysaccharide. This observation is in agreement with the notion that DCs prevent the premature destruction of antigenic determinants to optimize T cell activation. Phagosomal maturation is therefore a tightly regulated process that varies according to the type and differentiation stage of the phagocyte.

Journal of Experimental Medicine, 2002

Secretion of proteases is critical for degradation of the extracellular matrix during an inflamma... more Secretion of proteases is critical for degradation of the extracellular matrix during an inflammatory response. Cathepsin (Cat) S and L are the major elastinolytic cysteine proteases in mouse macrophages. A 65 amino acid segment of the p41 splice variant (p41 65aa ) of major histocompatibility complex class II-associated invariant chain (Ii) binds to the active site of CatL and permits the maintenance of a pool of mature enzyme in endosomal compartments of macrophages and dendritic cells (DCs). Here we show that interaction of p41 65aa with mature CatL allows extracellular accumulation of the active enzyme. We detected mature CatL as a complex with p41 65aa in culture supernatants from antigen-presenting cells (APCs). Extracellular accumulation of mature CatL is up-regulated by inflammatory stimuli as observed in interferon (IFN)-␥ -treated macrophages and lipopolysaccharide (LPS)-activated DCs. Despite the neutral pH of the extracellular milieu, released CatL associated with p41 65aa is catalytically active as demonstrated by active site labeling and elastin degradation assays. We propose that p41 65aa stabilizes CatL in the extracellular environment and induces a local increase in the concentration of matrix-degrading enzymes during inflammation. Through its interaction with CatL, Ii may therefore control the migratory response of APCs and/or the recruitment of effectors of the inflammatory response.

Therapeutic vaccination against cutaneous T cell lymphoma (CTCL) requires the characterization of... more Therapeutic vaccination against cutaneous T cell lymphoma (CTCL) requires the characterization of cancer cell-specific CTL epitopes. Despite reported evidence for tumor-reactive cytotoxicity in CTCL patients, the nature of the recognized determinants remains elusive. The clonotypic TCR of CTCL cells is a promising candidate tumor-specific Ag. In this study, we report that the clonotypic and framework regions of the TCRs expressed

Suggestive evidence indicates that immunoglobulin E (IgE)-dependent activation of mononuclear pha... more Suggestive evidence indicates that immunoglobulin E (IgE)-dependent activation of mononuclear phagocytes plays an important pathogenic role in allergic tissue inflammation. Prevailing opinion holds that low affinity IgE receptors are the relevant IgE-binding structures on monocytes/ macrophages and that functional events occurring after cross-linking of membrane-bound IgE on these cells are mediated by these receptors. Here we demonstrate that Ix-ripheral blood monocytes can bind monomeric IgE via the high affinity IgE receptor (FceRI) and that FceRI expression on these ceils is upregulated in atopic persons. Further, we demonstrate that, upon monocyte adherence to substrate, bridging of monocyte FcdLl is followed by cell activation. We propose that direct interaction of multivalent allergen with Fc~RI+-bound IgE on mononuclear phagocytes results in cell signaling via Fcd~I and that the biological consequences of this event may critically influence the outcome of allergic reactions.

Blood, 2015

Mice reconstituted with a human immune system provide a tractable in vivo model to assess human i... more Mice reconstituted with a human immune system provide a tractable in vivo model to assess human immune cell function. To date, reconstitution of murine strains with human hematopoietic stem cells (HSCs) from patients with monogenic immune disorders have not been reported. One obstacle precluding the development of immune-disease specific "humanized" mice is that optimal adaptive immune responses in current strains have required implantation of autologous human thymic tissue. To address this issue, we developed a mouse strain that lacks murine major histocompatibility complex class II (MHCII) and instead expresses human MHCII DR1. These mice displayed improved adaptive immune responses when reconstituted with human HSCs including enhanced T cell reconstitution, delayed-type hypersensitivity responses, and class-switch recombination. Following immune reconstitution of this novel strain with HSCs from a patient with immune dysregulation, polyendocrinopathy, enteropathy, X-lin...

The Journal of experimental medicine, Jan 30, 2015

Immunoglobulin ε (IgE) antibodies are the primary mediators of allergic diseases, which affect mo... more Immunoglobulin ε (IgE) antibodies are the primary mediators of allergic diseases, which affect more than 1 in 10 individuals worldwide. IgE specific for innocuous environmental antigens, or allergens, binds and sensitizes tissue-resident mast cells expressing the high-affinity IgE receptor, FcεRI. Subsequent allergen exposure cross-links mast cell-bound IgE, resulting in the release of inflammatory mediators and initiation of the allergic cascade. It is well established that precise glycosylation patterns exert profound effects on the biological activity of IgG. However, the contribution of glycosylation to IgE biology is less clear. Here, we demonstrate an absolute requirement for IgE glycosylation in allergic reactions. The obligatory glycan was mapped to a single N-linked oligomannose structure in the constant domain 3 (Cε3) of IgE, at asparagine-394 (N394) in human IgE and N384 in mouse. Genetic disruption of the site or enzymatic removal of the oligomannose glycan altered IgE s...

Cell reports, Jan 3, 2015

Epidemiologic studies discovered an inverse association between immunoglobulin E (IgE)-mediated a... more Epidemiologic studies discovered an inverse association between immunoglobulin E (IgE)-mediated allergies and cancer, implying tumor-protective properties of IgE. However, the underlying immunologic mechanisms remain poorly understood. Antigen cross-presentation by dendritic cells (DCs) is of key importance for anti-tumor immunity because it induces the generation of cytotoxic CD8(+) T lymphocytes (CTLs) with specificity for tumor antigens. We demonstrate that DCs use IgE and FcεRI, the high-affinity IgE receptor, for cross-presentation and priming of CTLs in response to free soluble antigen at low doses. Importantly, IgE/FcεRI-mediated cross-presentation is a distinct receptor-mediated pathway because it does not require MyD88 signals or IL-12 induction in DCs. Using passive immunization with tumor antigen-specific IgE and DC-based vaccination experiments, we demonstrate that IgE-mediated cross-presentation significantly improves anti-tumor immunity and induces memory responses in ...

The Journal of experimental medicine, Jan 17, 2005

The human high affinity receptor for IgE (FcepsilonRI) is a cell surface structure critical for t... more The human high affinity receptor for IgE (FcepsilonRI) is a cell surface structure critical for the pathology of allergic reactions. Human FcepsilonRI is expressed as a tetramer (alphabetagamma(2)) on basophils or mast cells and as trimeric (alphagamma(2)) complex on antigen-presenting cells. Expression of the human alpha subunit can be down-regulated by a splice variant of FcepsilonRIbeta (beta(var)). We demonstrate that FcepsilonRIalpha is the core subunit with which the other subunits assemble strictly cotranslationally. In addition to alphabetagamma(2) and alphagamma(2), we demonstrate the presence of alphabeta and alphabeta(var)gamma(2) complexes that are stable in the detergent Brij 96. The role of individual FcepsilonRI subunits for the formation of functional, immunoglobulin E-binding FcepsilonRI complexes during endoplasmic reticulum (ER) assembly can be defined as follows: beta and gamma support ER insertion, signal peptide cleavage and proper N-glycosylation of alpha, whe...

Molecular biology of the cell, 2004

The mammalian endoplasmic reticulum (ER)-to-cytosol degradation pathway for disposal of misfolded... more The mammalian endoplasmic reticulum (ER)-to-cytosol degradation pathway for disposal of misfolded proteins is an attractive target for therapeutic intervention in diseases that are characterized by impaired protein degradation. The ability to do so is hampered by the small number of specific inhibitors available and by our limited understanding of the individual steps involved in this pathway. Cells that express a class I major histocompatibility complex (MHC) heavy chain-enhanced green fluorescent protein (EGFP) fusion protein and the human cytomegalovirus protein US11, which catalyzes dislocation of the class I MHC EGFP reporter, show only little fluorescence. Treatment with proteasome inhibitors increases their fluorescence by stabilizing EGFP-tagged MHC class I molecules. We used this change in signal intensity as a readout to screen a chemical library of 16,320 compounds and identified two structurally related compounds (eeyarestatin I and II) that interfered with the degradati...

Background: Circulating autoantibodies against FcεRI, IgE, or both occur in approximately one thi... more Background: Circulating autoantibodies against FcεRI, IgE, or both occur in approximately one third of patients with chronic idiopathic urticaria (CIU), but not all autoantibodies initiate histamine release. Objective: We sought to classify patients with CIU into subsets on the basis of serum bioactivity and immunoreactivity and to examine the relationship between newly defined subtype and disease severity. Methods: Sera from patients with CIU (n = 78), dermographism (n = 15), and cholinergic urticaria (n = 10) and sera from healthy subjects (n = 39) were analyzed by means of Western blot analysis for anti-FcεRI autoantibodies and for histamine release from basophils and dermal mast cells. In vivo reactivity of autologous serum was tested by means of intradermal injection, and CIU severity was determined on the basis of clinical interview. Results: We classified sera from patients with CIU into 5 subsets: immunoreactive histamine-releasing anti-FcεRI autoantibodies (n = 20 [26%]); immunoreactive anti-FcεRI autoantibodies without histamine-releasing activity (n = 12 [15%]); anti-IgE-like autoantibodies (n = 7 [9%]); serum containing a mast cell-specific histamine-releasing factor (n = 7 [9%]); and sera with no identifiable factor (n = 32 [41%]). Patients with serum histamine-releasing activity had more severe urticaria than patients without such activity. Positive skin test responses to autologous sera were associated with histamine-releasing anti-FcεRI autoantibodies but not with non-histamine-releasing anti-FcεRI autoantibodies. Neither healthy subjects nor patients with dermographism or cholinergic urticaria had histamine-releasing anti-FcεRI autoantibodies. Conclusion: These data support the specificity of functional

Frontiers in Immunology, 2014

The ability of dendritic cells (DCs) to cross-present tumor antigens has long been a focus of int... more The ability of dendritic cells (DCs) to cross-present tumor antigens has long been a focus of interest to physicians, as well as basic scientists, that aim to establish efficient cell-based cancer immune therapy. A prerequisite for exploiting this pathway for therapeutic purposes is a better understanding of the mechanisms that underlie the induction of tumor-specific cytotoxic T-lymphocyte (CTL) responses when initiated by DCs via cross-presentation. The ability of humans DC to perform cross-presentation is of utmost interest, as this cell type is a main target for cell-based immunotherapy in humans. The outcome of a crosspresentation event is guided by the nature of the antigen, the form of antigen uptake, and the subpopulation of DCs that performs presentation. Generally, CD8α + DCs are considered to be the most potent cross-presenting DCs. This paradigm, however, only applies to soluble antigens. During adaptive immune responses, immune complexes form when antibodies interact with their specific epitopes on soluble antigens. Immunoglobulin G (IgG) immune complexes target Fc-gamma receptors on DCs to shuttle exogenous antigens efficiently into the cross-presentation pathway. This receptor-mediated cross-presentation pathway is a well-described route for the induction of strong CD8 + T cell responses. IgGmediated cross-presentation is intriguing because it permits the CD8 − DCs, which are commonly considered to be weak cross-presenters, to efficiently cross-present. Engaging multiple DC subtypes for cross-presentation might be a superior strategy to boost CTL responses in vivo. We here summarize our current understanding of how DCs use IgG-complexed antigens for the efficient induction of CTL responses. Because of its importance for human cell therapy, we also review the recent advances in the characterization of cross-presentation properties of human DC subsets.

The American Journal of Gastroenterology, 2014

Recent experimental evidence suggests that environmental microbial factors early in life determin... more Recent experimental evidence suggests that environmental microbial factors early in life determine susceptibility to allergic diseases through inappropriate chemotaxis and local activation of CD1drestricted, invariant chain natural killer T (iNKT) cells. In this study, we analyzed the involvement of these pathways in pediatric patients with eosinophilic esophagitis (EoE) before and after dietary allergen elimination.

Soluble IgE receptors are potential in vivo modulators of IgE-mediated immune responses and are t... more Soluble IgE receptors are potential in vivo modulators of IgE-mediated immune responses and are thus important for our basic understanding of allergic responses. We here characterize a novel soluble version of the IgE-binding alpha-chain of Fcepsilon-RI (sFceRI), the high affinity receptor for IgE. sFceRI immunoprecipitates as a protein of ,40 kDa and contains an intact IgE-binding site. In human serum, sFceRI is found as a soluble free IgE receptor as well as a complex with IgE. Using a newly established ELISA, we show that serum sFceRI levels correlate with serum IgE in patients with elevated IgE. We also show that serum of individuals with normal IgE levels can be found to contain high levels of sFceRI. After IgE-antigenmediated crosslinking of surface FceRI, we detect sFceRI in the exosome-depleted, soluble fraction of cell culture supernatants. We further show that sFceRI can block binding of IgE to FceRI expressed at the cell surface. In summary, we here describe the alpha-chain of FceRI as a circulating soluble IgE receptor isoform in human serum.

The EMBO Journal, 2002

The human cytomegalovirus gene products US2 and US11 induce proteasomal degradation of MHC class ... more The human cytomegalovirus gene products US2 and US11 induce proteasomal degradation of MHC class I heavy chains. We have generated an enhanced green uorescent protein±class I heavy chain (EGFP±HC) chimeric molecule to study its dislocation and degradation in US2-and US11-expressing cells. The EGFP±HC fusion is stable in control cells, but is degraded rapidly in US2-or US11-expressing cells. Proteasome inhibitors induce in a time-dependent manner the accumulation of EGFP±HC molecules in US2-and US11-expressing cells, as assessed biochemically and by cyto¯uorimetry of intact cells. Pulse±chase analysis and subcellular fractionation show that EGFP±HC proteins are dislocated from the endoplasmic reticulum and can be recovered as degly-cosylated¯uorescent intermediates in the cytosol. These results raise the possibility that dislocation of glycoproteins from the ER may not require their full unfolding.

Molecular Immunology, 2009

The family of activating immune receptors stabilizes via the 3-helix assembly principle. A charge... more The family of activating immune receptors stabilizes via the 3-helix assembly principle. A charged basic transmembrane residue interacts with two charged acidic transmembrane residues and forms a 3-helix interface to stabilize receptor complexes in the lipid bilayer. One family member, the high affinity receptor for IgE, FcεRI, is a key regulator of immediate allergic responses. Tetrameric FcεRI consists of the IgE-binding α-chain, the multimembrane spanning β-chain and a dimer of the γ-subunit (FcεRγ). Comparative analysis of these seven transmembrane regions indicates that FcεRI does not meet the charge requirements for the 3-helix assembly mechanism. We performed alanine mutagenesis to show that the only basic amino acid in the transmembrane regions, βK97, is not involved in FcεRI stabilization or surface up-regulation, a hallmark function of the β-chain. Even a βK97E mutant is functional despite four negatively charged acidic amino acids in the transmembrane regions. Using truncation mutants, we demonstrate that the first uncharged transmembrane domain of the β-chain contains the interface for receptor stabilization. In vitro translation experiments depict the first transmembrane region as the internal signal peptide of the β-chain. We also show that this β-chain domain can function as a cleavable signal peptide when used as a leader peptide for a Type I protein.

Journal of Clinical Immunology, 2013

Careful regulation of the body's immunoglobulin G (IgG) and albumin concentrations is necessitate... more Careful regulation of the body's immunoglobulin G (IgG) and albumin concentrations is necessitated by the importance of their respective functions. As such, the neonatal Fc receptor (FcRn), as a single receptor, is capable of regulating both of these molecules and has become an important focus of investigation. In addition to these essential protection functions, FcRn possesses a number of other functions that are equally as critical and are increasingly coming to attention. During the very first stages of life, FcRn mediates the passive transfer of IgG from mother to offspring both before and after birth. In the adult, FcRn regulates the persistence of both IgG and albumin in the serum as well as the movement of IgG, and any bound cargo, between different compartments of the body via transcytosis across polarized cells. FcRn is also expressed by hematopoietic cells; consistent with this, FcRn regulates MHC class II presentation and MHC class I crosspresentation by dendritic cells. As such, FcRn plays an important role in immune surveillance throughout adult life. The increasing appreciation for FcRn in both homeostatic and pathological conditions is generating an intense interest in the potential for therapeutic modulation of FcRn binding to IgG and albumin.

Cell reports, Jan 3, 2015

Epidemiologic studies discovered an inverse association between immunoglobulin E (IgE)-mediated a... more Epidemiologic studies discovered an inverse association between immunoglobulin E (IgE)-mediated allergies and cancer, implying tumor-protective properties of IgE. However, the underlying immunologic mechanisms remain poorly understood. Antigen cross-presentation by dendritic cells (DCs) is of key importance for anti-tumor immunity because it induces the generation of cytotoxic CD8(+) T lymphocytes (CTLs) with specificity for tumor antigens. We demonstrate that DCs use IgE and FcεRI, the high-affinity IgE receptor, for cross-presentation and priming of CTLs in response to free soluble antigen at low doses. Importantly, IgE/FcεRI-mediated cross-presentation is a distinct receptor-mediated pathway because it does not require MyD88 signals or IL-12 induction in DCs. Using passive immunization with tumor antigen-specific IgE and DC-based vaccination experiments, we demonstrate that IgE-mediated cross-presentation significantly improves anti-tumor immunity and induces memory responses in ...

Inflammatory bowel diseases, 2014

The chemokine CCL25, and its receptor CCR9, constitute a unique chemokine/receptor pair, which re... more The chemokine CCL25, and its receptor CCR9, constitute a unique chemokine/receptor pair, which regulates trafficking of T lymphocytes to the small intestine under physiological conditions and is an attractive target for small bowel Crohn's disease drug development. We have previously shown that CCL25/CCR9 interactions regulate the recovery from acute dextran sulfate sodium-induced colonic inflammation. In this study, we explored whether these interactions also regulate chronic colitis development in 2 independent murine models of experimental colitis. Histological flow cytometry and qPCR analyses were performed to evaluate the role of CL25 and CCR9 in chronic colonic inflammation induced by serial exposures to dextran sulfate sodium salts or by adoptive transfer of CD45RB(hi) CD4(+) T cell into lymphopenic mice devoid of CCL25/CCR9 interactions. Chronic dextran sulfate sodium exposure results in exacerbated colitis in mice deficient for either CCR9 or CCL25 when compared with wi...

The American Journal of Gastroenterology, 2014

Recent experimental evidence suggests that environmental microbial factors early in life determin... more Recent experimental evidence suggests that environmental microbial factors early in life determine susceptibility to allergic diseases through inappropriate chemotaxis and local activation of CD1drestricted, invariant chain natural killer T (iNKT) cells. In this study, we analyzed the involvement of these pathways in pediatric patients with eosinophilic esophagitis (EoE) before and after dietary allergen elimination.

Journal of Experimental Medicine, 2002

Here, we describe a new approach designed to monitor the proteolytic activity of maturing phagoso... more Here, we describe a new approach designed to monitor the proteolytic activity of maturing phagosomes in live antigen-presenting cells. We find that an ingested particle sequentially encounters distinct protease activities during phagosomal maturation. Incorporation of active proteases into the phagosome of the macrophage cell line J774 indicates that phagosome maturation involves progressive fusion with early and late endocytic compartments. In contrast, phagosome biogenesis in bone marrow-derived dendritic cells (DCs) and macrophages preferentially involves endocytic compartments enriched in cathepsin S. Kinetics of phagosomal maturation is faster in macrophages than in DCs. Furthermore, the delivery of active proteases to the phagosome is significantly reduced after the activation of DCs with lipopolysaccharide. This observation is in agreement with the notion that DCs prevent the premature destruction of antigenic determinants to optimize T cell activation. Phagosomal maturation is therefore a tightly regulated process that varies according to the type and differentiation stage of the phagocyte.

Journal of Experimental Medicine, 2002

Secretion of proteases is critical for degradation of the extracellular matrix during an inflamma... more Secretion of proteases is critical for degradation of the extracellular matrix during an inflammatory response. Cathepsin (Cat) S and L are the major elastinolytic cysteine proteases in mouse macrophages. A 65 amino acid segment of the p41 splice variant (p41 65aa ) of major histocompatibility complex class II-associated invariant chain (Ii) binds to the active site of CatL and permits the maintenance of a pool of mature enzyme in endosomal compartments of macrophages and dendritic cells (DCs). Here we show that interaction of p41 65aa with mature CatL allows extracellular accumulation of the active enzyme. We detected mature CatL as a complex with p41 65aa in culture supernatants from antigen-presenting cells (APCs). Extracellular accumulation of mature CatL is up-regulated by inflammatory stimuli as observed in interferon (IFN)-␥ -treated macrophages and lipopolysaccharide (LPS)-activated DCs. Despite the neutral pH of the extracellular milieu, released CatL associated with p41 65aa is catalytically active as demonstrated by active site labeling and elastin degradation assays. We propose that p41 65aa stabilizes CatL in the extracellular environment and induces a local increase in the concentration of matrix-degrading enzymes during inflammation. Through its interaction with CatL, Ii may therefore control the migratory response of APCs and/or the recruitment of effectors of the inflammatory response.

Therapeutic vaccination against cutaneous T cell lymphoma (CTCL) requires the characterization of... more Therapeutic vaccination against cutaneous T cell lymphoma (CTCL) requires the characterization of cancer cell-specific CTL epitopes. Despite reported evidence for tumor-reactive cytotoxicity in CTCL patients, the nature of the recognized determinants remains elusive. The clonotypic TCR of CTCL cells is a promising candidate tumor-specific Ag. In this study, we report that the clonotypic and framework regions of the TCRs expressed

Suggestive evidence indicates that immunoglobulin E (IgE)-dependent activation of mononuclear pha... more Suggestive evidence indicates that immunoglobulin E (IgE)-dependent activation of mononuclear phagocytes plays an important pathogenic role in allergic tissue inflammation. Prevailing opinion holds that low affinity IgE receptors are the relevant IgE-binding structures on monocytes/ macrophages and that functional events occurring after cross-linking of membrane-bound IgE on these cells are mediated by these receptors. Here we demonstrate that Ix-ripheral blood monocytes can bind monomeric IgE via the high affinity IgE receptor (FceRI) and that FceRI expression on these ceils is upregulated in atopic persons. Further, we demonstrate that, upon monocyte adherence to substrate, bridging of monocyte FcdLl is followed by cell activation. We propose that direct interaction of multivalent allergen with Fc~RI+-bound IgE on mononuclear phagocytes results in cell signaling via Fcd~I and that the biological consequences of this event may critically influence the outcome of allergic reactions.

Blood, 2015

Mice reconstituted with a human immune system provide a tractable in vivo model to assess human i... more Mice reconstituted with a human immune system provide a tractable in vivo model to assess human immune cell function. To date, reconstitution of murine strains with human hematopoietic stem cells (HSCs) from patients with monogenic immune disorders have not been reported. One obstacle precluding the development of immune-disease specific "humanized" mice is that optimal adaptive immune responses in current strains have required implantation of autologous human thymic tissue. To address this issue, we developed a mouse strain that lacks murine major histocompatibility complex class II (MHCII) and instead expresses human MHCII DR1. These mice displayed improved adaptive immune responses when reconstituted with human HSCs including enhanced T cell reconstitution, delayed-type hypersensitivity responses, and class-switch recombination. Following immune reconstitution of this novel strain with HSCs from a patient with immune dysregulation, polyendocrinopathy, enteropathy, X-lin...

The Journal of experimental medicine, Jan 30, 2015

Immunoglobulin ε (IgE) antibodies are the primary mediators of allergic diseases, which affect mo... more Immunoglobulin ε (IgE) antibodies are the primary mediators of allergic diseases, which affect more than 1 in 10 individuals worldwide. IgE specific for innocuous environmental antigens, or allergens, binds and sensitizes tissue-resident mast cells expressing the high-affinity IgE receptor, FcεRI. Subsequent allergen exposure cross-links mast cell-bound IgE, resulting in the release of inflammatory mediators and initiation of the allergic cascade. It is well established that precise glycosylation patterns exert profound effects on the biological activity of IgG. However, the contribution of glycosylation to IgE biology is less clear. Here, we demonstrate an absolute requirement for IgE glycosylation in allergic reactions. The obligatory glycan was mapped to a single N-linked oligomannose structure in the constant domain 3 (Cε3) of IgE, at asparagine-394 (N394) in human IgE and N384 in mouse. Genetic disruption of the site or enzymatic removal of the oligomannose glycan altered IgE s...

Cell reports, Jan 3, 2015

Epidemiologic studies discovered an inverse association between immunoglobulin E (IgE)-mediated a... more Epidemiologic studies discovered an inverse association between immunoglobulin E (IgE)-mediated allergies and cancer, implying tumor-protective properties of IgE. However, the underlying immunologic mechanisms remain poorly understood. Antigen cross-presentation by dendritic cells (DCs) is of key importance for anti-tumor immunity because it induces the generation of cytotoxic CD8(+) T lymphocytes (CTLs) with specificity for tumor antigens. We demonstrate that DCs use IgE and FcεRI, the high-affinity IgE receptor, for cross-presentation and priming of CTLs in response to free soluble antigen at low doses. Importantly, IgE/FcεRI-mediated cross-presentation is a distinct receptor-mediated pathway because it does not require MyD88 signals or IL-12 induction in DCs. Using passive immunization with tumor antigen-specific IgE and DC-based vaccination experiments, we demonstrate that IgE-mediated cross-presentation significantly improves anti-tumor immunity and induces memory responses in ...

The Journal of experimental medicine, Jan 17, 2005

The human high affinity receptor for IgE (FcepsilonRI) is a cell surface structure critical for t... more The human high affinity receptor for IgE (FcepsilonRI) is a cell surface structure critical for the pathology of allergic reactions. Human FcepsilonRI is expressed as a tetramer (alphabetagamma(2)) on basophils or mast cells and as trimeric (alphagamma(2)) complex on antigen-presenting cells. Expression of the human alpha subunit can be down-regulated by a splice variant of FcepsilonRIbeta (beta(var)). We demonstrate that FcepsilonRIalpha is the core subunit with which the other subunits assemble strictly cotranslationally. In addition to alphabetagamma(2) and alphagamma(2), we demonstrate the presence of alphabeta and alphabeta(var)gamma(2) complexes that are stable in the detergent Brij 96. The role of individual FcepsilonRI subunits for the formation of functional, immunoglobulin E-binding FcepsilonRI complexes during endoplasmic reticulum (ER) assembly can be defined as follows: beta and gamma support ER insertion, signal peptide cleavage and proper N-glycosylation of alpha, whe...

Molecular biology of the cell, 2004

The mammalian endoplasmic reticulum (ER)-to-cytosol degradation pathway for disposal of misfolded... more The mammalian endoplasmic reticulum (ER)-to-cytosol degradation pathway for disposal of misfolded proteins is an attractive target for therapeutic intervention in diseases that are characterized by impaired protein degradation. The ability to do so is hampered by the small number of specific inhibitors available and by our limited understanding of the individual steps involved in this pathway. Cells that express a class I major histocompatibility complex (MHC) heavy chain-enhanced green fluorescent protein (EGFP) fusion protein and the human cytomegalovirus protein US11, which catalyzes dislocation of the class I MHC EGFP reporter, show only little fluorescence. Treatment with proteasome inhibitors increases their fluorescence by stabilizing EGFP-tagged MHC class I molecules. We used this change in signal intensity as a readout to screen a chemical library of 16,320 compounds and identified two structurally related compounds (eeyarestatin I and II) that interfered with the degradati...

Background: Circulating autoantibodies against FcεRI, IgE, or both occur in approximately one thi... more Background: Circulating autoantibodies against FcεRI, IgE, or both occur in approximately one third of patients with chronic idiopathic urticaria (CIU), but not all autoantibodies initiate histamine release. Objective: We sought to classify patients with CIU into subsets on the basis of serum bioactivity and immunoreactivity and to examine the relationship between newly defined subtype and disease severity. Methods: Sera from patients with CIU (n = 78), dermographism (n = 15), and cholinergic urticaria (n = 10) and sera from healthy subjects (n = 39) were analyzed by means of Western blot analysis for anti-FcεRI autoantibodies and for histamine release from basophils and dermal mast cells. In vivo reactivity of autologous serum was tested by means of intradermal injection, and CIU severity was determined on the basis of clinical interview. Results: We classified sera from patients with CIU into 5 subsets: immunoreactive histamine-releasing anti-FcεRI autoantibodies (n = 20 [26%]); immunoreactive anti-FcεRI autoantibodies without histamine-releasing activity (n = 12 [15%]); anti-IgE-like autoantibodies (n = 7 [9%]); serum containing a mast cell-specific histamine-releasing factor (n = 7 [9%]); and sera with no identifiable factor (n = 32 [41%]). Patients with serum histamine-releasing activity had more severe urticaria than patients without such activity. Positive skin test responses to autologous sera were associated with histamine-releasing anti-FcεRI autoantibodies but not with non-histamine-releasing anti-FcεRI autoantibodies. Neither healthy subjects nor patients with dermographism or cholinergic urticaria had histamine-releasing anti-FcεRI autoantibodies. Conclusion: These data support the specificity of functional

Frontiers in Immunology, 2014

The ability of dendritic cells (DCs) to cross-present tumor antigens has long been a focus of int... more The ability of dendritic cells (DCs) to cross-present tumor antigens has long been a focus of interest to physicians, as well as basic scientists, that aim to establish efficient cell-based cancer immune therapy. A prerequisite for exploiting this pathway for therapeutic purposes is a better understanding of the mechanisms that underlie the induction of tumor-specific cytotoxic T-lymphocyte (CTL) responses when initiated by DCs via cross-presentation. The ability of humans DC to perform cross-presentation is of utmost interest, as this cell type is a main target for cell-based immunotherapy in humans. The outcome of a crosspresentation event is guided by the nature of the antigen, the form of antigen uptake, and the subpopulation of DCs that performs presentation. Generally, CD8α + DCs are considered to be the most potent cross-presenting DCs. This paradigm, however, only applies to soluble antigens. During adaptive immune responses, immune complexes form when antibodies interact with their specific epitopes on soluble antigens. Immunoglobulin G (IgG) immune complexes target Fc-gamma receptors on DCs to shuttle exogenous antigens efficiently into the cross-presentation pathway. This receptor-mediated cross-presentation pathway is a well-described route for the induction of strong CD8 + T cell responses. IgGmediated cross-presentation is intriguing because it permits the CD8 − DCs, which are commonly considered to be weak cross-presenters, to efficiently cross-present. Engaging multiple DC subtypes for cross-presentation might be a superior strategy to boost CTL responses in vivo. We here summarize our current understanding of how DCs use IgG-complexed antigens for the efficient induction of CTL responses. Because of its importance for human cell therapy, we also review the recent advances in the characterization of cross-presentation properties of human DC subsets.

The American Journal of Gastroenterology, 2014

Recent experimental evidence suggests that environmental microbial factors early in life determin... more Recent experimental evidence suggests that environmental microbial factors early in life determine susceptibility to allergic diseases through inappropriate chemotaxis and local activation of CD1drestricted, invariant chain natural killer T (iNKT) cells. In this study, we analyzed the involvement of these pathways in pediatric patients with eosinophilic esophagitis (EoE) before and after dietary allergen elimination.

Soluble IgE receptors are potential in vivo modulators of IgE-mediated immune responses and are t... more Soluble IgE receptors are potential in vivo modulators of IgE-mediated immune responses and are thus important for our basic understanding of allergic responses. We here characterize a novel soluble version of the IgE-binding alpha-chain of Fcepsilon-RI (sFceRI), the high affinity receptor for IgE. sFceRI immunoprecipitates as a protein of ,40 kDa and contains an intact IgE-binding site. In human serum, sFceRI is found as a soluble free IgE receptor as well as a complex with IgE. Using a newly established ELISA, we show that serum sFceRI levels correlate with serum IgE in patients with elevated IgE. We also show that serum of individuals with normal IgE levels can be found to contain high levels of sFceRI. After IgE-antigenmediated crosslinking of surface FceRI, we detect sFceRI in the exosome-depleted, soluble fraction of cell culture supernatants. We further show that sFceRI can block binding of IgE to FceRI expressed at the cell surface. In summary, we here describe the alpha-chain of FceRI as a circulating soluble IgE receptor isoform in human serum.

The EMBO Journal, 2002

The human cytomegalovirus gene products US2 and US11 induce proteasomal degradation of MHC class ... more The human cytomegalovirus gene products US2 and US11 induce proteasomal degradation of MHC class I heavy chains. We have generated an enhanced green uorescent protein±class I heavy chain (EGFP±HC) chimeric molecule to study its dislocation and degradation in US2-and US11-expressing cells. The EGFP±HC fusion is stable in control cells, but is degraded rapidly in US2-or US11-expressing cells. Proteasome inhibitors induce in a time-dependent manner the accumulation of EGFP±HC molecules in US2-and US11-expressing cells, as assessed biochemically and by cyto¯uorimetry of intact cells. Pulse±chase analysis and subcellular fractionation show that EGFP±HC proteins are dislocated from the endoplasmic reticulum and can be recovered as degly-cosylated¯uorescent intermediates in the cytosol. These results raise the possibility that dislocation of glycoproteins from the ER may not require their full unfolding.

Molecular Immunology, 2009

The family of activating immune receptors stabilizes via the 3-helix assembly principle. A charge... more The family of activating immune receptors stabilizes via the 3-helix assembly principle. A charged basic transmembrane residue interacts with two charged acidic transmembrane residues and forms a 3-helix interface to stabilize receptor complexes in the lipid bilayer. One family member, the high affinity receptor for IgE, FcεRI, is a key regulator of immediate allergic responses. Tetrameric FcεRI consists of the IgE-binding α-chain, the multimembrane spanning β-chain and a dimer of the γ-subunit (FcεRγ). Comparative analysis of these seven transmembrane regions indicates that FcεRI does not meet the charge requirements for the 3-helix assembly mechanism. We performed alanine mutagenesis to show that the only basic amino acid in the transmembrane regions, βK97, is not involved in FcεRI stabilization or surface up-regulation, a hallmark function of the β-chain. Even a βK97E mutant is functional despite four negatively charged acidic amino acids in the transmembrane regions. Using truncation mutants, we demonstrate that the first uncharged transmembrane domain of the β-chain contains the interface for receptor stabilization. In vitro translation experiments depict the first transmembrane region as the internal signal peptide of the β-chain. We also show that this β-chain domain can function as a cleavable signal peptide when used as a leader peptide for a Type I protein.

Journal of Clinical Immunology, 2013

Careful regulation of the body's immunoglobulin G (IgG) and albumin concentrations is necessitate... more Careful regulation of the body's immunoglobulin G (IgG) and albumin concentrations is necessitated by the importance of their respective functions. As such, the neonatal Fc receptor (FcRn), as a single receptor, is capable of regulating both of these molecules and has become an important focus of investigation. In addition to these essential protection functions, FcRn possesses a number of other functions that are equally as critical and are increasingly coming to attention. During the very first stages of life, FcRn mediates the passive transfer of IgG from mother to offspring both before and after birth. In the adult, FcRn regulates the persistence of both IgG and albumin in the serum as well as the movement of IgG, and any bound cargo, between different compartments of the body via transcytosis across polarized cells. FcRn is also expressed by hematopoietic cells; consistent with this, FcRn regulates MHC class II presentation and MHC class I crosspresentation by dendritic cells. As such, FcRn plays an important role in immune surveillance throughout adult life. The increasing appreciation for FcRn in both homeostatic and pathological conditions is generating an intense interest in the potential for therapeutic modulation of FcRn binding to IgG and albumin.