IgG sialylation occurs in B cells pre antibody secretion.

Sialic acids as terminal sugar residues on cell surface or secreted proteins have many functional roles. In particular, the presence or absence of α2,6-linked sialic acid residues at the immunoglobulin G (IgG) Fc fragment can switch IgG effector functions from pro- to anti-inflammatory activity. IgG glycosylation is considered to take place inside the plasma blast/plasma cell while the molecule travels through the endoplasmic reticulum and Golgi apparatus before being secreted. However, more recent studies have suggested that IgG sialylation may occur predominantly post-antibody secretion. To what extent this extracellular IgG sialylation process contributes to overall IgG sialylation remains unclear, however. By generating bone marrow chimeric mice with a B cell-specific deletion of ST6Gal1, the key enzyme required for IgG sialylation, we now show that sialylation of the IgG Fc fragment exclusively occurs within B cells pre-IgG secretion. We further demonstrate that B cells expressing ST6Gal1 have a developmental advantage over B cells lacking ST6Gal1 expression and thus dominate the plasma cell pool and the resulting serum IgG population in mouse models in which both ST6Gal1-sufficient and -deficient B cells are present.

Changes in the N-glycosylation of porcine immune globulin G during postnatal development.

N-glycosylation influences the effectiveness of immune globulin G (IgG) and thus the immunological downstream responses of immune cells. This impact arises from the presence of N-glycans within the Fc region, which not only alters the conformation of IgG but also influences its steric hindrance. Consequently, these modifications affect the interaction between IgG and its binding partners within the immune system. Moreover, this posttranslational modification vary according to the physiological condition of each individual. In this study, we examined the N-glycosylation of IgG in pigs from birth to five months of age. Our analysis identified a total of 48 distinct N-glycan structures. Remarkably, we observed defined changes in the composition of these N-glycans during postnatal development. The presence of agalactosylated and sialylated structures increases in relation to the number of N-glycans terminated by galactose residues during the first months of life. This shift may indicate a transition from passively transferred antibodies from the colostrum of the sow to the active production of endogenous IgG by the pig's own immune system.

Exploiting the neonatal crystallizable fragment receptor to treat kidney disease.

The neonatal Fc receptor (FcRn) was initially discovered as the receptor that allowed passive immunity in newborns by transporting maternal IgG through the placenta and enterocytes. Since its initial discovery, FcRn has been found to exist throughout all stages of life and in many different cell types. Beyond passive immunity, FcRn is necessary for intrinsic albumin and IgG recycling and is important for antigen processing and presentation. Given its multiple important roles, FcRn has been utilized in many disease treatments including a new class of agents that were developed to inhibit FcRn for treatment of a variety of autoimmune diseases. Certain cell populations within the kidney also express high levels of this receptor. Specifically, podocytes, proximal tubule epithelial cells, and vascular endothelial cells have been found to utilize FcRn. In this review, we summarize what is known about FcRn and its function within the kidney. We also discuss how FcRn has been used for therapeutic benefit, including how newer FcRn inhibiting agents are being used to treat autoimmune diseases. Lastly, we will discuss what renal diseases may respond to FcRn inhibitors and how further work studying FcRn within the kidney may lead to therapies for kidney diseases.

A Guide to the Quantitation of Protein Secretion Dynamics at the Single-Cell Level.

Protein secretion is a key cellular functionality, particularly in immunology, where cells can display large heterogeneity in this crucial activity in addition to binary secretion behavior. However, few methods enable quantitative secretion rate measurements at the single-cell level, and these methods are mostly based on microfluidics systems. Here, we describe such a microfluidic single-cell method for precisely measuring protein secretion rates in detail, building on the published droplet-based microfluidic platform DropMap. We give an updated, detailed guide toward quantifying protein secretion rates, discussing its setup and limitations. We illustrate the protocol on two key immunological analytes, immunoglobulin G, and interferon-γ.

Differential effects of FcRn antagonists on the subcellular trafficking of FcRn and albumin.

The homeostasis of IgG is maintained by the neonatal Fc receptor, FcRn. Consequently, antagonism of FcRn to reduce endogenous IgG levels is an emerging strategy for treating antibody-mediated autoimmune disorders using either FcRn-specific antibodies or an engineered Fc fragment. For certain FcRn-specific antibodies, this approach has resulted in reductions in the levels of serum albumin, the other major ligand transported by FcRn. Cellular and molecular analyses of a panel of FcRn antagonists have been carried out to elucidate the mechanisms leading to their differential effects on albumin homeostasis. These analyses have identified 2 processes underlying decreases in albumin levels during FcRn blockade: increased degradation of FcRn and competition between antagonist and albumin for FcRn binding. These findings have potential implications for the design of drugs to modulate FcRn function.

USP7 promotes IgA class switching through stabilizing RUNX3 for germline transcription activation.

Class switch recombination (CSR) diversifies the effector functions of antibodies and involves complex regulation of transcription and DNA damage repair. Here, we show that the deubiquitinase USP7 promotes CSR to immunoglobulin A (IgA) and suppresses unscheduled IgG switching in mature B cells independent of its role in DNA damage repair, but through modulating switch region germline transcription. USP7 depletion impairs Sα transcription, leading to abnormal activation of Sγ germline transcription and increased interaction with the CSR center via loop extrusion for unscheduled IgG switching. Rescue of Sα transcription by transforming growth factor ß (TGF-ß) in USP7-deleted cells suppresses Sγ germline transcription and prevents loop extrusion toward IgG CSR. Mechanistically, USP7 protects transcription factor RUNX3 from ubiquitination-mediated degradation to promote Sα germline transcription. Our study provides evidence for active transcription serving as an anchor to impede loop extrusion and reveals a functional interplay between USP7 and TGF-ß signaling in promoting RUNX3 expression for efficient IgA CSR.

Clostridioides difficile toxin B subverts germinal center and antibody recall responses by stimulating a drug-treatable CXCR4-dependent mechanism.

Recurrent Clostridioides difficile infection (CDI) results in significant morbidity and mortality. We previously established that CDI in mice does not protect against reinfection and is associated with poor pathogen-specific B cell memory (Bmem), recapitulating our observations with human Bmem. Here, we demonstrate that the secreted toxin TcdB2 is responsible for subversion of Bmem responses. TcdB2 from an endemic C. difficile strain delayed immunoglobulin G (IgG) class switch following vaccination, attenuated IgG recall to a vaccine booster, and prevented germinal center formation. The mechanism of TcdB2 action included increased B cell CXCR4 expression and responsiveness to its ligand CXCL12, accounting for altered cell migration and a failure of germinal center-dependent Bmem. These results were reproduced in a C. difficile infection model, and a US Food and Drug Administration (FDA)-approved CXCR4-blocking drug rescued germinal center formation. We therefore provide mechanistic insights into C. difficile-associated pathogenesis and illuminate a target for clinical intervention to limit recurrent disease.

Single-B cell analysis correlates high-lactate secretion with stress and increased apoptosis.

While cellular metabolism was proposed to be a driving factor of the activation and differentiation of B cells and the function of the resulting antibody-secreting cells (ASCs), the study of correlations between cellular metabolism and functionalities has been difficult due to the absence of technologies enabling the parallel measurement. Herein, we performed single-cell transcriptomics and introduced a direct concurrent functional and metabolic flux quantitation of individual murine B cells. Our transcriptomic data identified lactate metabolism as dynamic in ASCs, but antibody secretion did not correlate with lactate secretion rates (LSRs). Instead, our study of all splenic B cells during an immune response linked increased lactate metabolism with acidic intracellular pH and the upregulation of apoptosis. T cell-dependent responses increased LSRs, and added TLR4 agonists affected the magnitude and boosted LSRhigh B cells in vivo, while resulting in only a few immunoglobulin-G secreting cells (IgG-SCs). Therefore, our observations indicated that LSRhigh cells were not differentiating into IgG-SCs, and were rather removed due to apoptosis.

In-Membrane Enrichment and Peptic Digestion to Facilitate Analysis of Monoclonal Antibody Glycosylation.

The number of therapeutic monoclonal antibodies (mAbs) is growing rapidly due to their widespread use for treating various diseases and health conditions. Assessing the glycosylation profile of mAbs during production is essential to ensuring their safety and efficacy. This research aims to rapidly isolate and digest mAbs for liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification of glycans and monitoring of glycosylation patterns, potentially during manufacturing. Immobilization of an Fc region-specific ligand, oFc20, in a porous membrane enables the enrichment of mAbs from cell culture supernatant and efficient elution with an acidic solution. Subsequent digestion of the mAb eluate occurred in a pepsin-modified membrane within 5 min. The procedure does not require alkylation and desalting, greatly shortening the sample preparation time. Subsequent LC-MS/MS analysis identified 11 major mAb N-glycan proteoforms and assessed the relative peak areas of the glycosylated peptides. This approach is suitable for the glycosylation profiling of various human IgG mAbs, including biosimilars and different IgG subclasses. The total time required for this workflow is less than 2 h, whereas the conventional enzymatic release and labeling of glycans can take much longer. Thus, the integrated membranes are suitable for facilitating the analysis of mAb glycosylation patterns.

Chromatography affinity resin with photosynthetically-sourced protein A ligand.

Green, photosynthesizing plants can be proficiently used as cost-effective, single-use, fully biodegradable bioreactors for environmentally-friendly production of a variety of valuable recombinant proteins. Being near-infinitely scalable and most energy-efficient in generating biomass, plants represent profoundly valid alternatives to conventionally used stationary fermenters. To validate this, we produced a plastome-engineered tobacco bioreactor line expressing a recombinant variant of the protein A from Staphylococcus aureus, an affinity ligand widely useful in antibody purification processes, reaching accumulation levels up to ~ 250 mg per 1 kg of fresh leaf biomass. Chromatography resin manufactured from photosynthetically-sourced recombinant protein A ligand conjugated to agarose beads demonstrated the innate pH-driven ability to bind and elute IgG-type antibodies and allowed one-step efficient purification of functional monoclonal antibodies from the supernatants of the producing hybridomas. The results of this study emphasize the versatility of plant-based recombinant protein production and illustrate its vast potential in reducing the cost of diverse biotechnological applications, particularly the downstream processing and purification of monoclonal antibodies.

Enhancement of NETosis by ACE2-cross-reactive anti-SARS-CoV-2 RBD antibodies in patients with COVID-19.

BACKGROUND: High levels of neutrophil extracellular trap (NET) formation or NETosis and autoantibodies are related to poor prognosis and disease severity of COVID-19 patients. Human angiotensin-converting enzyme 2 (ACE2) cross-reactive anti-severe acute respiratory syndrome coronavirus 2 spike protein receptor-binding domain (SARS-CoV-2 RBD) antibodies (CR Abs) have been reported as one of the sources of anti-ACE2 autoantibodies. However, the pathological implications of CR Abs in NET formation remain unknown. METHODS: In this study, we first assessed the presence of CR Abs in the sera of COVID-19 patients with different severity by serological analysis. Sera and purified IgG from CR Abs positive COVID-19 patients as well as a mouse monoclonal Ab (mAb 127) that can recognize both ACE2 and the RBD were tested for their influence on NETosis and the possible mechanisms involved were studied. RESULTS: An association between CR Abs levels and the severity of COVID-19 in 120 patients was found. The CR Abs-positive sera and IgG from severe COVID-19 patients and mAb 127 significantly activated human leukocytes and triggered NETosis, in the presence of RBD. This NETosis, triggered by the coexistence of CR Abs and RBD, activated thrombus-related cells but was abolished when the interaction between CR Abs and ACE2 or Fc receptors was disrupted. We also revealed that CR Abs-induced NETosis was suppressed in the presence of recombinant ACE2 or the Src family kinase inhibitor, dasatinib. Furthermore, we found that COVID-19 vaccination not only reduced COVID-19 severity but also prevented the production of CR Abs after SARS-CoV-2 infection. CONCLUSIONS: Our findings provide possible pathogenic effects of CR Abs in exacerbating COVID-19 by enhancing NETosis, highlighting ACE2 and dasatinib as potential treatments, and supporting the benefit of vaccination in reducing disease severity and CR Abs production in COVID-19 patients.

Regulation of BCR-mediated Ca2+ mobilization by MIZ1-TMBIM4 safeguards IgG1+ GC B cell-positive selection.

The transition from immunoglobulin M (IgM) to affinity-matured IgG antibodies is vital for effective humoral immunity. This is facilitated by germinal centers (GCs) through affinity maturation and preferential maintenance of IgG+ B cells over IgM+ B cells. However, it is not known whether the positive selection of the different Ig isotypes within GCs is dependent on specific transcriptional mechanisms. Here, we explored IgG1+ GC B cell transcription factor dependency using a CRISPR-Cas9 screen and conditional mouse genetics. We found that MIZ1 was specifically required for IgG1+ GC B cell survival during positive selection, whereas IgM+ GC B cells were largely independent. Mechanistically, MIZ1 induced TMBIM4, an ancestral anti-apoptotic protein that regulated inositol trisphosphate receptor (IP3R)-mediated calcium (Ca2+) mobilization downstream of B cell receptor (BCR) signaling in IgG1+ B cells. The MIZ1-TMBIM4 axis prevented mitochondrial dysfunction-induced IgG1+ GC cell death caused by excessive Ca2+ accumulation. This study uncovers a unique Ig isotype-specific dependency on a hitherto unidentified mechanism in GC-positive selection.

Reducing neonatal Fc receptor binding enhances clearance and brain-to-blood ratio of TfR-delivered bispecific amyloid-ß antibody.

Recent development of amyloid-ß (Aß)-targeted immunotherapies for Alzheimer's disease (AD) have highlighted the need for accurate diagnostic methods. Antibody-based positron emission tomography (PET) ligands are well suited for this purpose as they can be directed toward the same target as the therapeutic antibody. Bispecific, brain-penetrating antibodies can achieve sufficient brain concentrations, but their slow blood clearance remains a challenge, since it prolongs the time required to achieve a target-specific PET signal. Here, two antibodies were designed based on the Aß antibody bapineuzumab (Bapi) - one monospecific IgG (Bapi) and one bispecific antibody with an antigen binding fragment (Fab) of the transferrin receptor (TfR) antibody 8D3 fused to one of the heavy chains (Bapi-Fab8D3) for active, TfR-mediated transport into the brain. A variant of each antibody was designed to harbor a mutation to the neonatal Fc receptor (FcRn) binding domain, to increase clearance. Blood and brain pharmacokinetics of radiolabeled antibodies were studied in wildtype (WT) and AD mice (AppNL-G-F). The FcRn mutation substantially reduced blood half-life of both Bapi and Bapi-Fab8D3. Bapi-Fab8D3 showed high brain uptake and the brain-to-blood ratio of its FcRn mutated form was significantly higher in AppNL-G-F mice than in WT mice 12 h after injection and increased further up to 168 h. Ex vivo autoradiography showed specific antibody retention in areas with abundant Aß pathology. Taken together, these results suggest that reducing FcRn binding of a full-sized bispecific antibody increases the systemic elimination and could thereby drastically reduce the time from injection to in vivo imaging.

A Multifunctionalized Potyvirus-Derived Nanoparticle That Targets and Internalizes into Cancer Cells.

Plant viral nanoparticles (VNPs) are attractive to nanomedicine researchers because of their safety, ease of production, resistance, and straightforward functionalization. In this paper, we developed and successfully purified a VNP derived from turnip mosaic virus (TuMV), a well-known plant pathogen, that exhibits a high affinity for immunoglobulins G (IgG) thanks to its functionalization with the Z domain of staphylococcal Protein A via gene fusion. We selected cetuximab as a model IgG to demonstrate the versatility of this novel TuMV VNP by developing a fluorescent nanoplatform to mark tumoral cells from the Cal33 line of a tongue squamous cell carcinoma. Using confocal microscopy, we observed that fluorescent VNP-cetuximab bound selectively to Cal33 and was internalized, revealing the potential of this nanotool in cancer research.

Controlling nanoparticle-induced endothelial leakiness with the protein corona.

Understanding nanoparticle-cell interaction is essential for advancing research in nanomedicine and nanotoxicology. Apart from the transcytotic pathway mediated by cellular recognition and energetics, nanoparticles (including nanomedicines) may harness the paracellular route for their transport by inducing endothelial leakiness at cadherin junctions. This phenomenon, termed as NanoEL, is correlated with the physicochemical properties of the nanoparticles in close association with cellular signalling, membrane mechanics, as well as cytoskeletal remodelling. However, nanoparticles in biological systems are transformed by the ubiquitous protein corona and yet the potential effect of the protein corona on NanoEL remains unclear. Using confocal fluorescence microscopy, biolayer interferometry, transwell, toxicity, and molecular inhibition assays, complemented by molecular docking, here we reveal the minimal to significant effects of the anionic human serum albumin and fibrinogen, the charge neutral immunoglobulin G as well as the cationic lysozyme on negating gold nanoparticle-induced endothelial leakiness in vitro and in vivo. This study suggests that nanoparticle-cadherin interaction and hence the extent of NanoEL may be partially controlled by pre-exposing the nanoparticles to plasma proteins of specific charge and topology to facilitate their biomedical applications.

Functional role of immunoglobulin G as an oxytocin-carrier protein.

It has been long-time known that oxytocin in plasma is bound to a carrier protein, a common feature of circulating peptide hormones, however, the nature of such protein was uncertain. A recent study revealed that about 60% of oxytocin present in plasma is bound to immunoglobulin G (IgG) and that oxytocin-binding IgG plays a role of a functional oxytocin carrier protein. Here, we review the historical background and methodology leading to this discovery. Moreover, we review the data showing the functional role of oxytocin-binding IgG in the modulation of oxytocin signaling relevant to the regulation of motivated behavior and several neuropsychiatric disorders. Furthermore, the possible role of gut microbiota in the origin of such IgG is discussed and the relevant new therapeutic strategies for the enhancement of oxytocin signaling are presented.

The IgG-specific endoglycosidases EndoS and EndoS2 are distinguished by conformation and antibody recognition.

The IgG-specific endoglycosidases EndoS and EndoS2 from Streptococcus pyogenes can remove conserved N-linked glycans present on the Fc region of host antibodies to inhibit Fc-mediated effector functions. These enzymes are therefore being investigated as therapeutics for suppressing unwanted immune activation, and have additional application as tools for antibody glycan remodeling. EndoS and EndoS2 differ in Fc glycan substrate specificity due to structural differences within their catalytic glycosyl hydrolase domains. However, a chimeric EndoS enzyme with a substituted glycosyl hydrolase from EndoS2 loses catalytic activity, despite high structural homology between the two enzymes, indicating either mechanistic divergence of EndoS and EndoS2, or improperly-formed domain interfaces in the chimeric enzyme. Here, we present the crystal structure of the EndoS2-IgG1 Fc complex determined to 3.0 Å resolution. Comparison of complexed and unliganded EndoS2 reveals relative reorientation of the glycosyl hydrolase, leucine-rich repeat and hybrid immunoglobulin domains. The conformation of the complexed EndoS2 enzyme is also different when compared to the earlier EndoS-IgG1 Fc complex, and results in distinct contact surfaces between the two enzymes and their Fc substrate. These findings indicate mechanistic divergence of EndoS2 and EndoS. It will be important to consider these differences in the design of IgG-specific enzymes, developed to enable customizable antibody glycosylation.

Identification of Four Mouse FcRn Splice Variants and FcRn-Specific Vesicles.

Research into the neonatal Fc receptor (FcRn) has increased dramatically ever since Simister and Mostov first purified a rat version of the receptor. Over the years, FcRn has been shown to function not only as a receptor that transfers immunity from mother to fetus but also performs an array of different functions that include transport and recycling of immunoglobulins and albumin in the adult. Due to its important cellular roles, several clinical trials have been designed to either inhibit/enhance FcRn function or develop of non-invasive therapeutic delivery system such as fusion of drugs to IgG Fc or albumin to enhance delivery inside the cells. Here, we report the accidental identification of several FcRn alternatively spliced variants in both mouse and human cells. The four new mouse splice variants are capable of binding immunoglobulins' Fc and Fab portions. In addition, we have identified FcRn-specific vesicles in which immunoglobulins and albumin can be stored and that are involved in the endosomal-lysosomal system. The complexity of FcRn functions offers significant potential to design and develop novel and targeted therapeutics.

Gonadal androgens are associated with decreased type I interferon production by plasmacytoid dendritic cells and increased IgG titres to BNT162b2 following co-vaccination with live attenuated influenza vaccine in adolescents.

mRNA vaccine technologies introduced following the SARS-CoV-2 pandemic have highlighted the need to better understand the interaction of adjuvants and the early innate immune response. Type I interferon (IFN-I) is an integral part of this early innate response that primes several components of the adaptive immune response. Women are widely reported to respond better than men to tri- and quadrivalent influenza vaccines. Plasmacytoid dendritic cells (pDCs) are the primary cell type responsible for IFN-I production, and female pDCs produce more IFN-I than male pDCs since the upstream pattern recognition receptor Toll-like receptor 7 (TLR7) is encoded by X chromosome and is biallelically expressed by up to 30% of female immune cells. Additionally, the TLR7 promoter contains several putative androgen response elements, and androgens have been reported to suppress pDC IFN-I in vitro. Unexpectedly, therefore, we recently observed that male adolescents mount stronger antibody responses to the Pfizer BNT162b2 mRNA vaccine than female adolescents after controlling for natural SARS-CoV-2 infection. We here examined pDC behaviour in this same cohort to determine the impact of IFN-I on anti-spike and anti-receptor-binding domain IgG titres to BNT162b2. Through flow cytometry and least absolute shrinkage and selection operator (LASSO) modelling, we determined that serum-free testosterone was associated with reduced pDC IFN-I, but contrary to the well-described immunosuppressive role for androgens, the most bioactive androgen dihydrotestosterone was associated with increased IgG titres to BNT162b2. Also unexpectedly, we observed that co-vaccination with live attenuated influenza vaccine boosted the magnitude of IgG responses to BNT162b2. Together, these data support a model where systemic IFN-I increases vaccine-mediated immune responses, yet for vaccines with intracellular stages, modulation of the local IFN-I response may alter antigen longevity and consequently improve vaccine-driven immunity.

Mixed-mode size-exclusion silica resin for polishing human antibodies in flow-through mode.

The polishing step in the downstream processing of therapeutic antibodies removes residual impurities from Protein A eluates. Among the various classes of impurities, antibody fragments are especially challenging to remove due to the broad biomolecular diversity generated by a multitude of fragmentation patterns. The current approach to fragment removal relies on ion exchange or mixed-mode adsorbents operated in bind-and-gradient-elution mode. However, fragments that bear strong similarity to the intact product or whose biophysical features deviate from the ensemble average can elude these adsorbents, and the lack of a chromatographic technology enabling robust antibody polishing is recognized as a major gap in downstream bioprocessing. Responding to this challenge, this study introduces size-exclusion mixed-mode (SEMM) silica resins as a novel chromatographic adsorbent for the capture of antibody fragments irrespective of their biomolecular features. The pore diameter of the silica beads features a narrow distribution and is selected to exclude monomeric antibodies, while allowing their fragments to access the pores where they are captured by the mixed-mode ligands. The static and dynamic binding capacity of the adsorbent ranged respectively between 30-45 and 25-33 gs of antibody fragments per liter of resin. Selected SEMM-silica resins also demonstrated the ability to capture antibody aggregates, which adsorb on the outer layer of the beads. Optimization of the SEMM-silica design and operation conditions - namely, pore size (10 nm) and ligand composition (quaternary amine and alkyl chain) as well as the linear velocity (100 cm/h), ionic strength (5.7 mS/cm), and pH (7) of the mobile phase - afforded a significant reduction of both fragments and aggregates, resulting into a final antibody yield up to 80% and monomeric purity above 97%.