Immunoglobulin E (IgE) and its corresponding Fc epsilon receptors (FcϵRs) are essential components of the immune system. The constant, crystallizable fragment (Fc) region of IgE binds with high affinity to its specific receptor, FcϵRI, anchoring IgE molecules to the surface of effector cells such as mast cells and basophils. Once bound, IgE uses its antigen-binding fragment (Fab) to recognize specific antigens. Antigen-induced crosslinking of cell-bound IgE triggers activation of these effector cells. Over fifty years ago, intensive research identified IgE as a key mediator of allergic reactions. Subsequent studies have demonstrated that the production of antigen-specific IgE and its interactions with innate immune cells are critical not only for allergic responses but also for certain non-atopic immune processes. N-glycosylation, a crucial post-translational modification, has been shown to strongly influence the stability and function of IgG antibodies. Similarly, glycosylation is vital for maintaining the structure and biological activity of IgE. Individual variations in IgE glycosylation patterns regulate its functional properties, contributing to the diversity and complexity of IgE-mediated immune responses. Given the emerging role of IgE in non-atopic diseases, understanding how site-specific glycosylation variations affect IgE function is essential for characterizing disease-specific molecular signatures and identifying new therapeutic targets. Comprehensive glycoproteomic analyses of IgE from diverse pathological conditions may clarify how glycosylation influences disease progression, identify Fc glycans associated with pathology, and elucidate their biological roles.

Neonatal hemochromatosis (NH) is a unique disease with an unknown etiology, characterized by neonatal liver failure (NLF) and associated with iron deposition in the fetal liver and extrahepatic organs, except the organs in the reticuloendothelial system. The pathologic mechanism of an alloimmune attack of pathogenic maternal IgGs on the fetal liver occurs through the generation of membrane attack complex (MAC), demonstrating the interplay between maternal adaptive immunity and fetal innate immune responses. Pathogenic alloimmune IgGs can induce various inflammatory responses, ranging from hyperacute fetal liver inflammation to chronic liver injury. However, the precise mechanism by which IgG triggers distinct inflammatory responses following its binding to fetal liver antigens remains to be elucidated. Remarkably, the IgG subclass and glycosylation machinery of IgG are essential for the disparities in IgG-associated inflammatory responses. Hence, modulation of IgG glycosylation can increase or decrease IgG activity, which may severely affect human health. It is well recognized that disease-associated changes in the IgG glycome composition may function as diagnostic biomarkers or contribute to disease pathogenesis. Specific IgG glycans may elicit complex immune responses during the antenatal and postnatal periods and may facilitate future endeavors in developing novel diagnostic and therapeutic interventions for NH. Hence, analyzing glycoprofiles of class/subclass IgG in maternal and fetal blood could potentially distinguish pathogenic IgGs with pro-inflammatory properties from normal IgGs with anti-inflammatory features. These insights may unveil new strategies for developing novel diagnostic and therapeutic approaches for early detection and pre-and postnatal management of NH.

The mechanism of the association of S. aureus skin colonization with food allergy in atopic dermatitis (AD) is unknown. Interleukin-4 (IL-4) plays an important role in food allergy. We found elevated serum IL-4 concentrations in AD patients with S. aureus skin colonization and food allergy. Using an AD mouse model, we demonstrated that epicutaneous application of antigen together with superantigen-producing S. aureus, or staphylococcal enterotoxin B (SEB), caused a heightened systemic antigen-specific T helper-2 (Th2) response and elevated serum IL-4 concentrations. T cell-derived IL-4 acted on intestinal epithelial cells to enhance intestinal permeability and anaphylaxis to enteral antigen challenge. CD40-dependent SEB binding to keratinocytes triggered IL-33 release, which caused T cells to produce IL-3 that elicited a basophil influx in skin-draining lymph nodes (dLNs). Basophil-derived IL-4 augmented Th2 cell polarization by antigen-bearing dendritic cells from skin dLNs. These results suggest therapeutic interventions that might attenuate food allergy in AD patients.

Antibodies play a vital role in the immune system, with distinct isotypes having unique tropisms and performing specialized functions. Of these isotypes, IgE is the least abundant in circulation yet plays a critical role in defense against parasitic infection and allergic reactions. IgE is also heavily N-linked glycosylated, a posttranslational modification that influences receptor interactions of effector responses. The importance of glycosylation on IgG function is well established, and the roles of IgE glycans are emerging. This review examines the relationship between IgE glycosylation and its biological function. IgE glycosylation, specifically the oligomannosidic glycan, is necessary for IgE binding to its high-affinity receptor FcεRI on mast cells and basophils. Recent evidence suggests that terminal sialic acid residues on complex biantennary glycans significantly enhance IgE's allergic potential, with sialylation of IgE demonstrating reduced capacity to trigger degranulation and anaphylaxis. Glycosylation also influences IgE's interaction with its low-affinity receptor FcεRII/CD23, affecting serum clearance and antigen presentation. Beyond allergy, this review also covers IgE's impacts on its roles in autoimmunity, parasite defense, and protection against venoms. Current therapeutic approaches targeting IgE include monoclonal antibodies like omalizumab, with emerging therapeutics looking to target systemic IgE production mechanisms also covered. Although the understanding of IgG glycosylation is known, there is much to uncover in terms of IgE glycosylation, which may open new avenues for developing more precise interventions that modulate its effector functions.

IgG antibodies are the basis for many successful therapeutics. A single, N-linked glycan is present on the Fc on all IgGs, and the composition of that glycan exerts marked influence over effector functions of the IgG. We and others have shown terminal sialylation of the Fc glycan confers anti-inflammatory activity to the IgG1 subclass and is thought to be responsible for the anti-inflammatory activity of high-dose intravenous immunoglobulin. However, whether sialylation results in anti-inflammatory activity for other IgG subclasses is unknown. We found that IgG1 and IgG3, but not IgG2 nor IgG4, suppressed autoantibody-mediated inflammation in vivo when sialylated. This activity was dependent upon human DC-SIGN or its murine ortholog, SIGN-R1. Fc γ receptor-binding profiles for asialylated and sialylated IgG subclasses reinforced these similarities, with IgG1 and IgG3 sharing binding patterns. Amino acid sequence alignments identified two uniquely conserved amino acid residues at positions 234 and 327 of the CH2 domain of all human IgG1 and IgG3 allotypes that were distinct from IgG2 and IgG4. Indeed, molecular modeling of the shared residues in IgG1 and IgG3 revealed a hydrophobic-heavy interchain interaction that was not present in IgG2 nor IgG4. Introduction of those IgG1/3 residues into an IgG4-Fc resulted in anti-inflammatory activity in vivo when sialylated. Conversely, a reciprocal sialylated IgG1 mutant with IgG2/4 residues lacked anti-inflammatory activity. These results define the amino acid requirements of anti-inflammatory sialylated IgG and enable rational amino acid and glycan engineering across all therapeutic IgG subclasses.

IgE binds FcεRI monomerically with high affinity.1-4 A recent manuscript interrogated interactions between IgE and FcεRI using sandwich ELISA and flow cytometry,5 methods which quantify the amount of IgE bound to FcεRI, but not interaction strength. The authors found more asialylated IgE bound FcεRI than sialylated IgE at saturating levels in both assays. Therefore, we asked whether removing sialic acid enabled binding of multiple IgE to FcεRI. We treated ⍺-OVA mIgE (Figure 1A) with neuraminidase and buffer (asialylated mIgE) or buffer-only (sialylated mIgE) as described.6 Digestions were confirmed by gel electrophoresis followed by lectin blotting with sialic acid-specific Sambucus Nigra Lectin (SNA) (Figure 1B). Sialylated and asialylated mIgE bound and saturated OVA similarly (Figure 1C). The mIgE preparations were tested in real-time human FcεRI (hFcεRI) binding assays by biolayer interferometry (BLI, Figure 1D). FcεRI binding was similar between sialylated (pink) and asialylated (yellow) mIgE, and modeling of both interactions was consistent with a 1:1 interaction (not shown). Next, we tested sialylated and asialylated mIgE in a model of passive cutaneous anaphylaxis (PCA), which is insensitive to serum half-life. Indeed, ears sensitized with sialylated mIgE exhibited robust inflammation, while inflammation in ears treated with asialylated IgE was markedly attenuated (Figure 1E).6 To extend these results, we generated sialylated and asialylated ⍺-OVA-human IgE (hIgE) as above (Figure 1F,G). The hIgE preparations were examined in ELISA and BLI experiments and found to interact similarly with OVA and hFcεRI (Figure 1H,I). The hIgE preparations were used to sensitize human LAD2 mast cells, and flow cytometry-based analysis revealed similar IgE loading on the cells (Figure 1J). We obtained similar results using hybridoma-derived ⍺-TNP mIgE following digestion and purification conditions reported elsewhere5 (Figure 2A,B, Figure S1). We next examined passive systemic anaphylaxis (PSA) 2 h after sensitization with sialylated or asialylated IgE. Mice sensitized with sialylated ⍺-TNP IgE had significant temperature loss, while those sensitized with asialylated IgE did not (Figure 2C). However, asialylated ⍺-TNP IgE serum titers 2 h after sensitization were significantly reduced compared to sialylated IgE (Figure 2D). Therefore, we blocked the asialoglycoprotein receptor (ASGPR) during PSA as previously described.5 This result phenocopied our previous result, with robust anaphylaxis in mice sensitized with sialylated ⍺-TNP IgE, but not asialylated IgE (Figure 2E). Serum ⍺-TNP mIgE titers were similar between sialylated and asialylated preparations 2 h after sensitization with ASGR blockade (Figure 2F). Indeed, PSA induced 24 h after sensitization was robust in mice sensitized with sialylated but not asialylated mIgE (Figure 2G), while no serum sialylated or asialylated IgE was detected at this timepoint (Figure 2H). Also, no differences in peritoneal mast cell surface IgE loading were observed 2 h following intraperitoneally sensitized with sialylated or asialylated ⍺-TNP IgE (Figure 2I). As ASGPR blockade impacted asialylated IgE serum titers 2 h after sensitization, we examined the interaction of sialylated and asialylated IgE with ASGR by BLI. Sialylated hIgE did not bind ASGPR, while asiaylated IgE bound ASGR with modest affinity (Figure 2J), albeit magnitudes lower than monomeric IgE-FcεRI binding. Together, our results indicate that the presence of sialic acid on IgE minimally impacts FcεRI interactions, allergen binding, or mast cell loading, but does influence the magnitude of anaphylaxis. The work was supported by NIH NIAID awards R01AI139669 to RMA and R01AI167933 to MEC. SB, CPP, and BBR conducted generated data, MEC and RMA obtained funding, supervised the research, and wrote the manuscript with SB. The authors declare no conflict of interests. If published, the manuscript will be submitted into PubMed Central, an open access source. Appendix S1 Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Immunoglobulin G (IgG) antibodies in the form of high-dose intravenous immunoglobulin (IVIG) exert immunomodulatory activity and are used in this capacity to treat inflammatory and autoimmune diseases. Reductionist approaches have revealed that terminal sialylation of the single asparagine-linked (N-linked) glycan at position 297 of the IgG1 Fc bestows antiinflammatory activity, which can be recapitulated by introduction of an F241A point mutation in the IgG1 Fc (FcF241A). Here, we examined the antiinflammatory activity of CHO-K1 cell-produced FcF241A in vivo in models of autoimmune inflammation and found it to be independent of sialylation. Intriguingly, sialylation markedly improved the half-life and bioavailability of FcF241A via impaired interaction with the asialoglycoprotein receptor ASGPR. Further, FcF241A suppressed inflammation through the same molecular pathways as IVIG and sialylated IgG1 Fc and required the C-type lectin SIGN-R1 in vivo. This contrasted with FcAbdeg (efgartigimod), an engineered IgG1 Fc with enhanced neonatal Fc receptor (FcRn) binding, which reduced total serum IgG concentrations, independent of SIGN-R1. When coadministered, FcF241A and FcAbdeg exhibited combinatorial antiinflammatory activity. Together, these results demonstrated that the antiinflammatory activity of FcF241A requires SIGN-R1, similarly to that of high-dose IVIG and sialylated IgG1, and can be used in combination with other antiinflammatory therapeutics that rely on divergent pathways, including FcAbdeg.

Antibody responses against highly conserved epitopes on the stalk domain of influenza virus hemagglutinin (HA) confer broad protection; however, such responses are limited. To effectively induce stalk-specific immunity against conserved HA epitopes, sequential immunization strategies have been developed based on chimeric HA (cHA) constructs featuring different head domains but the same stalk regions. Immunogenicity studies in small animal models, as well as in humans, revealed that cHA immunogens elicit stalk-specific IgG responses with broad specificity against heterologous influenza virus strains. However, the mechanisms by which these antibodies confer in vivo protection and the contribution of their Fc effector function remain unclear. To characterize the role of Fc-FcγR (Fcγ receptor) interactions to the in vivo protective activity of IgG antibodies elicited in participants in a phase I trial of a cHA vaccine candidate, we performed passive transfer studies of vaccine-elicited IgG antibodies in mice humanized for all classes of FcγRs, as well as in mice deficient for FcγRs. IgG antibodies elicited upon cHA vaccination completely protected FcγR humanized mice against lethal influenza virus challenge, while no protection was evident in FcγR-deficient mice, suggesting a major role for FcγR pathways in the protective function of vaccine-elicited IgG antibodies. These findings have important implications for influenza vaccine development, guiding the design of vaccination approaches with the capacity to elicit IgG responses with optimal Fc effector function.

Altered tryptophan catabolism has been identified in inflammatory diseases like rheumatoid arthritis (RA) and spondyloarthritis (SpA), but the causal mechanisms linking tryptophan metabolites to disease are unknown. Using the collagen-induced arthritis (CIA) model, we identified alterations in tryptophan metabolism, and specifically indole, that correlated with disease. We demonstrated that both bacteria and dietary tryptophan were required for disease and that indole supplementation was sufficient to induce disease in their absence. When mice with CIA on a low-tryptophan diet were supplemented with indole, we observed significant increases in serum IL-6, TNF, and IL-1β; splenic RORγt+CD4+ T cells and ex vivo collagen-stimulated IL-17 production; and a pattern of anti-collagen antibody isotype switching and glycosylation that corresponded with increased complement fixation. IL-23 neutralization reduced disease severity in indole-induced CIA. Finally, exposure of human colonic lymphocytes to indole increased the expression of genes involved in IL-17 signaling and plasma cell activation. Altogether, we propose a mechanism by which intestinal dysbiosis during inflammatory arthritis results in altered tryptophan catabolism, leading to indole stimulation of arthritis development. Blockade of indole generation may present a unique therapeutic pathway for RA and SpA.

Single-cell profiling of prenatal samples reveals multiple macrophage types and states, including microglia-like cells in non-neuronal tissues.

Self-antigen-specific T cells are prevalent in the mature adaptive immune system but are regulated through multiple mechanisms of tolerance. However, inflammatory conditions such as tissue injury may allow these T cells to break tolerance and trigger autoimmunity. To understand how the T cell repertoire responds to the presentation of self-antigen under highly stimulatory conditions, we use peptide:major histocompatibility complex (MHC) class II tetramers to track the behavior of endogenous CD4+ T cells with specificity to a lung-expressed self-antigen in mouse models of immune-mediated lung injury. Acute injury results in the exclusive expansion of CD4+ regulatory T cells (Tregs) that is dependent on self-antigen recognition and interleukin-2 (IL-2). Conversely, conventional CD4+ T cells of the same self-antigen specificity remain unresponsive even following Treg ablation. Thus, the self-antigen-specific CD4+ T cell repertoire is poised to serve a regulatory function during acute tissue damage to limit further damage and the possibility of autoimmunity.

Self antigen-specific T cells are prevalent in the mature adaptive immune system, but are regulated through multiple mechanisms of tolerance. However, inflammatory conditions such as tissue injury may provide these T cells with an opportunity to break tolerance and trigger autoimmunity. To understand how the T cell repertoire responds to the presentation of self antigen under highly stimulatory conditions, we used peptide:MHCII tetramers to track the behavior of endogenous CD4+ T cells with specificity to a lung-expressed self antigen in mouse models of immune-mediated lung injury. Acute injury resulted in the exclusive expansion of regulatory T cells (Tregs) that was dependent on self antigen recognition and IL-2. Conversely, conventional T cells of the same self antigen specificity remained unresponsive, even following Treg ablation. Thus, the self antigen-specific T cell repertoire is poised to serve a regulatory function during acute tissue damage to limit further damage and the possibility of autoimmunity.

Antibodies play a critical role in linking the adaptive immune response to the innate immune system. In humans, antibodies are categorized into five classes, IgG, IgM, IgA, IgE, and IgD, based on constant region sequence, structure, and tropism. In serum, IgG is the most abundant antibody, comprising 75% of antibodies in circulation, followed by IgA at 15%, IgM at 10%, and IgD and IgE are the least abundant. All human antibody classes are post-translationally modified by sugars. The resulting glycans take on many divergent structures and can be attached in an N-linked or O-linked manner, and are distinct by antibody class, and by position on each antibody. Many of these glycan structures on antibodies are capped by sialic acid. It is well established that the composition of the N-linked glycans on IgG exert a profound influence on its effector functions. However, recent studies have described the influence of glycans, particularly sialic acid for other antibody classes. Here, we discuss the role of glycosylation, with a focus on terminal sialylation, in the biology and function across all antibody classes. Sialylation has been shown to influence not only IgG, but IgE, IgM, and IgA biology, making it an important and unappreciated regulator of antibody function.

How mis-regulated chromatin directly impacts human immune disorders is poorly understood. Speckled Protein 140 (SP140) is an immune-restricted PHD and bromodomain-containing epigenetic "reader," and SP140 loss-of-function mutations associate with Crohn's disease (CD), multiple sclerosis (MS), and chronic lymphocytic leukemia (CLL). However, the relevance of these mutations and mechanisms underlying SP140-driven pathogenicity remains unexplored. Using a global proteomic strategy, we identified SP140 as a repressor of topoisomerases (TOPs) that maintains heterochromatin and macrophage fate. In humans and mice, SP140 loss resulted in unleashed TOP activity, de-repression of developmentally silenced genes, and ultimately defective microbe-inducible macrophage transcriptional programs and bacterial killing that drive intestinal pathology. Pharmacological inhibition of TOP1/2 rescued these defects. Furthermore, exacerbated colitis was restored with TOP1/2 inhibitors in Sp140-/- mice, but not wild-type mice, in vivo. Collectively, we identify SP140 as a TOP repressor and reveal repurposing of TOP inhibition to reverse immune diseases driven by SP140 loss.

Advances in experimental capabilities in the glycosciences offer expanding opportunities for discovery in the broad areas of immunology and microbiology. These two disciplines overlap when microbial infection stimulates host immune responses and glycan structures are central in the processes that occur during all such encounters. Microbial glycans mediate host-pathogen interactions by acting as surface receptors or ligands, functioning as virulence factors, impeding host immune responses, or playing other roles in the struggle between host and microbe. In the context of the host, glycosylation drives cell–cell interactions that initiate and regulate the host response and modulates the effects of antibodies and soluble immune mediators. This perspective reports on a workshop organized jointly by the National Institute of Allergy and Infectious Diseases and the National Institute of Dental and Craniofacial Research in May 2020. The conference addressed the use of emerging glycoscience tools and resources to advance investigation of glycans and their roles in microbe-host interactions, immune-mediated diseases, and immune cell recognition and function. Future discoveries in these areas will increase fundamental scientific understanding and have the potential to improve diagnosis and treatment of infections and immune dysregulation.

Approximately one-third of the world’s population suffers from allergies1. Exposure to allergens crosslinks immunoglobulin E (IgE) antibodies that are bound to mast cells and basophils, triggering the release of inflammatory mediators, including histamine2. Although IgE is absolutely required for allergies, it is not understood why total and allergen-specific IgE concentrations do not reproducibly correlate with allergic disease3–5. It is well-established that glycosylation of IgG dictates its effector function and has disease-specific patterns. However, whether IgE glycans differ in disease states or affect biological activity is completely unknown6. Here we perform an unbiased examination of glycosylation patterns of total IgE from individuals with a peanut allergy and from non-atopic individuals without allergies. Our analysis reveals an increase in sialic acid content on total IgE from individuals with a peanut allergy compared with non-atopic individuals. Removal of sialic acid from IgE attenuates effector-cell degranulation and anaphylaxis in several functional models of allergic disease. Therapeutic interventions—including removing sialic acid from cell-bound IgE with a neuraminidase enzyme targeted towards the IgE receptor FcεRI, and administering asialylated IgE—markedly reduce anaphylaxis. Together, these results establish IgE glycosylation, and specifically sialylation, as an important regulator of allergic disease. A specific type of glycosylation—sialylation—is more common on immunoglobulin E from individuals with a peanut allergys than from non-atopic people, suggesting that it has a role in regulating anaphylaxis.

IgE are absolutely required for initiation of allergy reactions, which affect over 20% of the world's population. IgE are the least prevalent immunoglobulins in circulation with 12-h and 2-day half-lives in mouse and human serum, respectively, but an extended tissue half-life of 3-weeks bound to the surface of mast cells by the high affinity IgE receptor, FcεRI (Gould and Sutton 2008). Although the importance of glycosylation to IgG biology is well established, less is known regarding the contribution of IgE glycosylation to allergic inflammation. IgE has seven and nine N-linked glycosylation sites distributed across human and murine constant chains, respectively. Here we discuss studies that have analyzed IgE glycosylation and its function, and how IgE glycosylation contributions to health and disease.