Biology subjects

Bondt, A.

Publications and source records attributed to Bondt, A..

7 recordsLinked to original sources

bioRxiv2026

IgG4 antibody repertoire diversity is stable over several years in health and autoimmune disease

Despite its relative anti-inflammatory nature, IgG4 is involved in a variety of pathological conditions such as autoimmune disease and cancer. To investigate whether the IgG4 repertoire presents any abnormalities in diseases where IgG4 autoantibodies predominate, we charted longitudinal IgG4 repertoires in health, in an IgG4-mediated autoimmune disease (AID; muscle-specific kinase myasthenia gravis (MuSK MG)), and in IgG1/IgG3 mediated AIDs (acetylcholine receptor (AChR) MG and Lambert-Eaton myasthenic syndrome (LEMS). In total, we assessed 218 samples of 69 donors using mass spectrometry-based antibody repertoire analysis, covering a timespan of up to eight years. Despite being superficially inconspicuous in clonal diversity and abundances, we detected a slightly elevated number of IgG4 clones in MuSK MG patients compared to healthy donors. Notably, this observation did not coincide with drastic repertoire changes during treatment, suggesting that a large portion of the IgG4 repertoire is not significantly affected by common MuSK MG treatment regimens. Comparing the IgG4 antibody repertoires of all donors, we found that they were exceptionally unique for each individual. Against what would be expected in the case of a constantly adapting antibody repertoire, a large portion of the IgG4 clones remained stable without any further maturation. This observation even extended to patients receiving B cell depleting or IgG modulating treatments. Our findings suggest that a dominating fraction of the IgG4 pool may be produced by long-lived plasma cells, which are unaffected by disease or medical interventions.

immunology↗

bioRxiv2025

Structural promiscuity in the human circulatory IgA1 clonal repertoire

Immunoglobulin A (IgA) is the most abundant antibody in humans, with high concentrations in both mucosae/secretions and circulation. While mucosal IgA has been studied extensively, characterization of human IgA in serum and its distinctive functions lags behind. Circulatory IgA is regularly assumed to be monomeric, despite some reports describing a minor population of J-chain-coupled dimers. Here, we first charted the compositional landscape of human serum IgA in individual healthy donors. In addition to the expected predominating monomers, we consistently observed J-coupled dimers, even representing [~]30% of one donors total serum IgA. To determine whether these structurally distinct populations were also clonally distinct, we employed mass spectrometry-based IgA1 clonal profiling in sera of two donors. Our data revealed the majority of IgA1 clones are present solely as monomers, with a smaller number exclusively dimeric. Strikingly, a third population of IgA1 clones is present in circulation as both monomers and J-coupled dimers. In fact, these shared, structurally-promiscuous IgA1 clones dominated both individuals serum IgA1 clonal repertoires. Our findings suggest likely every J+ IgA-secreting cell can co-produce monomers and J-coupled dimers, but what exactly determines this ratio requires further investigation. This finding is important, as IgA monomers and J-coupled dimers have distinct characteristics in antigen binding, receptor activation, and clearance from circulation, with several reports highlighting, for instance, the enhanced neutralization capacity of dimeric IgA. As we show here, the human immune system is not merely capable of producing both forms, but apparently prefers doing so in parallel. Significance StatementHuman immunoglobulin IgA occurs in diverse assemblies, mainly monomers (mIgA) and J-chain coupled dimers (dIgA). The general view is that these forms are produced at different sites, i.e., circulatory and mucosal, with the former assumed to represent mIgA. Inherently, it would not be expected that these populations have any overlap in their clonal repertoire. Here, we report the first analysis at the protein level of assembly-specific IgA1 clonal repertoires in sera of healthy individual donors. Strikingly, we find a substantial subset of clones that co-occur as both mIgA1 and dIgA1 assemblies, hinting at shared B-cell origins. This marks a paradigm shift in current understanding of human IgA, where these assemblies are classically viewed as structurally, functionally, and clonally distinct.

biochemistry↗

bioRxiv2025

Fab-arm exchange affects each and all endogenous serum IgG4 evenly

Antibodies are key molecular elements of the human immune system and account for an increasingly large proportion of therapeutics. Next to the well-studied and explored IgG1, several other classes (e.g. IgA, IgM) and sub-classes (e.g., IgG2, IgG3 and IgG4) exist in humans. In particular IgG4 is worth a closer examination as it has unique natural properties and is regularly used as a scaffold in biologicals. IgG4 stands out from the other IgG by its ability to dissociate and form two half-molecules which can interchange with those of other clones to form novel bivalent antibodies. Detailed analysis of endogenous IgG4 so far has been hampered by a lack of analytical methods to dissect and analyze IgG4 molecules with clonal resolution. Here, we present an LC-MS-based approach enabling the analysis of IgG4 repertoires with clonal resolution, which we used to monitor endogenous serum IgG4 repertoires from seven healthy donors. Most strikingly, our data reveal the combinatorial explosion in diversity of the serum IgG4 clonal repertoire. This phenomenon is explained by the stochastic behavior of Fab-arm exchange, making virtually each IgG4 molecule in serum bispecific. Although the endogenous IgG4 clonal repertoire is therefore extremely diverse, we demonstrate that this IgG4 repertoire persists over time within an individual healthy donor for more than one year. This newly established method now enables repertoire analysis for IgG4, which plays a critical role in a plethora of disease settings including allergy, autoimmunity and vaccination.

biochemistry↗

bioRxiv2024

Longitudinal fluctuations in protein concentrations and higher-order structures in the plasma proteome of kidney failure patients subjected to a kidney transplant

Using proteomics and complexome profiling we evaluated over a period of a year longitudinal variations in the plasma proteome of kidney failure patients, prior to and after a kidney transplantation, comparing this data with two healthy controls. The post-transplant period was complicated by numerous bacterial infections, resulting in dramatic changes in the plasma proteome, mostly related to an acute phase condition. As positive acute phase proteins, being elevated upon inflammation, we observed the well-described C-reactive protein (CRP) and Serum Amyloid A (SAA1 and SAA2), but our analyses added to that Fibrinogen (FGA, FGB and FGG), Haptoglobin (HP), Leucine-rich alpha-2-glycoprotein (LRG1), Lipopolysaccharide- binding protein (LBP), Alpha-1-antitrypsin (SERPINA1), Alpha-1-antichymotrypsin (SERPINA3), Protein S100 (S100A8, S100A9), Complement protein C4, C4b-binding protein alpha chain (C4BPA), Complement factor B (CFB) and Monocyte differentiation antigen CD14. As negative acute phase proteins, being downregulated upon inflammation, we identified the well-documented Serotransferrin (TF) and Transthyretin (TTR), but add on to that Kallistatin (SERPINA4), Heparin cofactor 2 (SERPIND1), Inter-alpha-trypsin inhibitor heavy chain H1 and H2 (ITIH1, ITIH2). For a patient with the most severe acute phase response, we furthermore performed plasma complexome profiling by SEC-LC-MS on all longitudinal samples. We observe that several plasma proteins displaying alike concentration patterns, co- elute and putatively form macromolecular complexes. These include a) FGA, FGB and FGG (as expected, b) ITIH1 and ITIH2, c) HP together with Hemoglobin (HB), d) the small acute phase biomarker proteins SAA1 and SAA2 with the Apolipoproteins A-I, A-II, A-IV (APOA1, APOA2, APOA4). By complexome profiling we expose how SAA1 and SAA2 become incorporated into high-density lipid particles, thereby replacing partly APOA1 and APOA4. Overall, our data highlight that the combination of in-depth longitudinal plasma proteome and complexome profiling can shed further light on the correlated variations in the abundance of several plasma proteins upon inflammatory events.

molecular biology↗

bioRxiv2023

CD5L is a canonical component of circulatory IgM

Immunoglobulin M (IgM) is an evolutionary conserved key component of humoral immunity, and the first antibody isotype to emerge during an immune response. IgM is a large (1 MDa), multimeric protein, for which both hexameric and pentameric structures have been described, the latter additionally containing a joining (J) chain. Using a combination of single-particle mass spectrometry and mass photometry, proteomics and immunochemical assays, we here demonstrate that circulatory (serum) IgM exclusively exists as a complex of J-chain-containing pentamers covalently bound to the small CD5 antigen-like (CD5L, 36 kDa) protein. In sharp contrast, secretory IgM in saliva and milk is principally devoid of CD5L. Unlike IgM itself, CD5L is not produced by B cells, implying that it associates with IgM in the extracellular space. We demonstrate that CD5L integration has functional implications, i.e., it diminishes IgM binding to two of its receptors, the Fc{micro}R and the polymeric Immunoglobulin receptor (pIgR). On the other hand, binding to Fc{micro}R as well as complement activation via C1q seem unaffected by CD5L integration. Taken together, we redefine the composition of circulatory IgM as a J-chain containing pentamer, always in complex with CD5L.

molecular biology↗

bioRxiv2022

Humans have distinct repertoires of IgA1

The most abundant immunoglobulin present in the human body is IgA1. It has the highest concentrations at the mucosal lining and in biofluids such as milk and is the second most abundant class of antibodies in serum. We assessed the structural diversity and clonal repertoire of IgA1-containing molecular assemblies longitudinally in human serum and milk from three donors using a mass spectrometry-based approach. IgA-containing molecules purified from serum or milk were assessed by the release and subsequent analysis of their Fab fragments. Our data revealed that serum IgA1 consists of two distinct structural populations, namely monomeric IgA1 ([~] 80%) and dimeric joining (J-) chain coupled IgA1 ([~] 20%). Also, we confirmed that IgA1 in milk is present solely as secretory (S)IgA, consisting of two ([~] 50%), three ([~] 33%) or four ([~] 17%) IgA1 molecules assembled with a J-chain and secretory component (SC). Interestingly, the serum and milk IgA1-Fab repertoires were distinct between monomeric, and J-chain coupled dimeric IgA1. The serum dimeric J-chain coupled IgA1 repertoire contained several abundant clones also observed in the milk IgA1 repertoire. The latter repertoire had little to no overlap with the serum monomeric IgA1 repertoire. This suggests that human IgA1s have (at least) two distinct origins; one of these produces dimeric J-chain coupled IgA1 molecules, shared in human serum and milk, and another produces monomeric IgA1 ending up exclusively in serum.

immunology↗

bioRxiv2022

IgG1 responses following SARS-CoV-2 infection are polyclonal and highly personalized, whereby each donor and each clone displays a distinct pattern of cross-reactivity against SARS-CoV-2 variants

Using a recently introduced efficient mass spectrometry-based approach we monitored individual donors IgG1 clonal responses in molecular detail, examining SARS-CoV-2 spike-protein-specific IgG1 repertoires. We monitored the plasma clonal IgG1 profiles of 8 donors (4 male and 4 female) who had recently experienced an infection by either the wild type Wuhan Hu-1 virus or one of 3 VOCs (Alpha, Beta and Gamma). In these donors we charted the full plasma IgG1 repertoires as well as the IgG1 repertoires targeting the SARS-CoV-2 spike protein trimer as antigen. We observed that shortly after infection in between <0.1% to almost 10% of all IgG1 antibody molecules present in plasma did bind to the spike protein. Each donor displayed a unique plasma IgG1 repertoire, but also each donor displayed a unique and polyclonal antibody response against the SARS-CoV-2 spike-protein variants. Our analyses revealed that certain clones exhibit (alike) binding affinity towards all four tested spike-protein variants, whereas other clones displayed strong unique mutant-specific affinity. We conclude that each infected person generates a unique polyclonal response following infection, whereby some of these clones can bind multiple viral variants, whereas other clones do not display such cross-reactivity. In general, by assessing IgG1 repertoires following infection it becomes possible to identify and select fully matured human plasma antibodies that target specific antigens, and display either high specificity or cross-reactivity versus mutated versions of the antigen, which will aid in selecting antibodies that may be developed into biotherapeutics.

immunology↗