Biology subjects

Toes, R. E. M.

Publications and source records attributed to Toes, R. E. M..

7 recordsLinked to original sources

bioRxiv2026

Clonal autoantibodies identify microbial antigen as trigger of autoreactive B cells in systemic sclerosis

ObjectivesTransformative observations demonstrate unprecedented success of B cell-depleting interventions in many human autoimmune diseases, calling for a deeper understanding of the triggers leading to B cell-mediated autoimmunity and its perpetuation in human disease. Here, we investigated whether the autoreactive B cell response targeting human topoisomerase 1 (TOP1), a hallmark of systemic sclerosis, could cross-react with TOP1 of microbial origin. MethodsHomologies between human and microbial TOP1 were analyzed using Foldseek. TOP1-reactive monoclonal antibodies from patient-derived, human TOP1-reactive B cell receptors were generated and assessed for reactivity against human TOP1 and TOP1 from a prototypic yeast, Saccharomyces cerevisiae (S. cerevisiae). Reactivity of polyclonal serum IgG from anti-TOP1 autoantibody (ATA)+, anti-centromere autoantibody (ACA)+ SSc patients and healthy donors (HDs) was tested. Finally, B cell lines were generated expressing human ATA to study B cell activation upon antigenic stimulation. ResultsStructural homologues of human TOP1 were found in many microbes, particularly in fungi. Taking TOP1 from S. cerevisiae as a prototype, microbial TOP1 was recognized by polyclonal patient IgG and by several monoclonal ATAs. Importantly, S. cerevisiae TOP1 also activated B cells expressing a patient-derived, human TOP1-reactive B cell receptor. Patients affected by interstitial lung disease most frequently showed recognition of microbial TOP1. ConclusionsThese findings identify fungi as potential drivers of immune dysregulation in human autoimmunity, specifically in SSc, highlighting microbial antigen cross-reactive cells as important therapeutic targets. Moreover, these data provide first functional evidence for a breach of B cell tolerance against human TOP1 triggered by cross-reactivity to fungal TOP1.

immunology↗

bioRxiv2025

Symmetrical Dimethylarginine as the Central Antigenic Determinant of Anti-Smith Autoantibodies in Systemic Lupus Erythematosus

ObjectiveSystemic lupus erythematosus (SLE) is a chronic autoimmune disease causing multi-organ damage. The most specific autoantibody response in SLE, present in 20-30% of patients, targets the Sm-protein and has been shown to recognize a linear Sm-derived B cell epitope containing a post-translational modification (PTM) of arginine termed symmetrical dimethyl arginine (sDMA). As autoantibodies to other PTM-modified proteins are often promiscuous, we aimed to determine the specificity and cross-reactivity of anti-Sm antibodies. MethodsSpecificity and promiscuity/cross-reactivity of anti-Sm IgG were measured by ELISA in SLE patients and healthy donors using peptides containing either sDMA or unmodified arginine. Inhibition and cross-reactivity were determined using competitive ELISA and affinity purification. Recognition of endogenous EBNA1 by anti-Sm IgG was performed by Western blot using lysates of EBV-bearing lymphoblastoid cell lines. ResultsThe sDMA residue is recognized by anti-Sm+ SLE patients regardless of the peptide amino acid sequence, with a modest impact of amino acids flanking sDMA on recognition. Most notably, IgGs targeting sDMA comprise the overall majority ([~]90%) of the anti-Sm antibody repertoire and are highly cross-reactive between SmD3108-122 and several sDMA-containing viral-derived epitopes, including full-length EBNA1. ConclusionOur data implicate that the majority of anti-Sm IgGs target the sDMA residue irrespective of its Sm-context, thus representing a prototypic anti-PTM response. These antibodies are highly promiscuous, recognizing several sDMA-modified targets, including naturally occurring viral sDMA-expressing epitopes. These findings suggest a new mechanism by which molecular mimicry of sDMA-modified viral proteins could contribute to a breach of tolerance in anti-Sm+ SLE patients. KEY MESSAGESO_ST_ABSWhat is already known on this topicC_ST_ABSO_LISymmetrical dimethylarginine (sDMA) residues are present on RGG/RG repeat regions within the SmD1, SmD3, and SmB/B subunits of the Sm protein complex in vivo. C_LIO_LIThe introduction of sDMA is essential for recognition of minimal antigenic epitopes spanning amino acids #95-119 on the SmD1 and #108-122 on the SmD3 subunits by a specific subset of anti-Sm autoantibodies. C_LI What this study addsO_LIWe show that autoantibodies targeting sDMA residues are common in anti-Sm+ SLE patients and represent the majority of the anti-Sm autoantibody repertoire. C_LIO_LIWe demonstrate that anti-sDMA autoantibodies are highly cross-reactive and can also bind to sDMA residues on other targets, including several virus-derived epitopes, thus representing a prototypic anti-modified protein antibody (AMPA) response directed against post-translationally modified proteins through symmetrical dimethylation of arginine. C_LI How this study might affect research, practice, or policyO_LIThe high cross-reactivity of Sm antibodies to various sDMA-containing epitopes, combined with a dominant antigenic specificity, provides mechanistic insight in the potential link between viral infection and tolerance breach in anti-Sm+ SLE patients. C_LI

immunology↗

bioRxiv2025

Enhanced recognition of topoisomerase 1 upon DNA binding by a subset of anti-topoisomerase 1 autoantibodies in systemic sclerosis

ObjectivesSystemic sclerosis (SSc) is a deleterious disease. Its clinical management is complicated by strong interpatient heterogeneity. Progressive organ fibrosis is linked to autoantibodies against topoisomerase 1 (TOP1). Here, we hypothesized that the molecular interaction between anti-TOP1 autoantibodies (ATAs) and TOP1 could be a relevant determinant of SSc pathogenesis. DNA binding by TOP1 might affect its interaction with ATAs. Therefore, we studied the effect of DNA binding by TOP1 on ATA recognition. MethodsATA monoclonal antibodies (ATA mAbs) were generated from patient-derived TOP1-reactive B cells. Reactivity of ATA mAbs and antibodies in patient plasma towards TOP1 and TOP1-DNA cleavage complexes (TOP1cc) was determined by ELISA and mass photometry. Immunostimulatory properties of ATA mAbs in complex with TOP1/TOP1cc were assessed by stimulation of monocytic THP-1 cells. ResultsDNA binding by TOP1 differentially affected the recognition of TOP1 by ATA mAbs. A subset of mAbs showed enhanced binding to TOP1cc, whereas others recognized TOP1 and TOP1cc to a similar extent. ATAs with enhanced TOP1cc recognition were observed in plasma of ATA+ SSc patients and correlated with ATA levels and interstitial lung disease. These TOP1cc-enhanced ATAs variably affected the enzymatic function of TOP1 and circulated predominantly as IgG1 and IgM. The interaction of TOP1cc-enhanced ATA mAbs with TOP1cc strongly increased IL-8 production by THP-1 cells. ConclusionsThe differential recognition of TOP1 upon DNA binding by ATAs demonstrates heterogeneity in the ATA B cell response, possibly impacting on disease-relevant processes in severe SSc.

immunology↗

bioRxiv2024

IFN-associated B cell hyperactivity is highly enriched in SLE patients harboring Sm/RNP antibodies

Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by an array of autoantibodies, in particular anti-nuclear antibodies (ANA). The disease is also hallmarked by an expansion of plasmablasts (PB) in peripheral blood. How these relate to autoantibody production is not clear. Here, we aimed to understand B cell alterations in SLE and their relationship to immunoglobulin levels and autoantibody production. We demonstrate that a subgroup of SLE patients is characterized by a high frequency of PB relative to memory B cells (high PB/M). Patients with this phenotype more frequently had high disease activity. Despite low overall frequencies of memory B cells, these patients exhibited an increased activation in the switched CD27+ memory compartment and a strong IFN signature in PB. Repertoire analysis revealed a highly polyclonal expansion and enrichment for IgG1 expressing PB in patients with a high PB/M ratio which was reflected in increased serum IgG levels. Importantly, the hyperactive B cell phenotype was highly enriched in patients harboring Sm/RNP autoantibodies (OR: 9.17 (2.97-26.0)). In summary, we show for the first time a direct relationship between IFN and PB expansion in a subgroup of SLE patients, highly enriched in those harboring Sm/RNP antibodies. These results provide insight into the pathways leading to B cell hyperactivity and autoantibody production which may guide the tailoring of B cell- and IFN-targeted therapies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/624119v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1f74053org.highwire.dtl.DTLVardef@1a01da4org.highwire.dtl.DTLVardef@fa27f4org.highwire.dtl.DTLVardef@155140f_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2024

Fc proteoforms of ACPA IgG discriminate autoimmune responses in plasma and synovial fluid of rheumatoid arthritis patients and associate with disease activity

Autoantibodies and their post-translational modifications (PTMs) are insightful markers of autoimmune diseases providing diagnostic and prognostic clues, thereby informing clinical decisions. However, current autoantibody analyses focus mostly on IgG1 glycosylation representing only a subpopulation of the actual IgG proteome. Here, by taking rheumatoid arthritis (RA) as prototypic autoimmune disease, we sought to circumvent these shortcomings and illuminate the importance of (auto)antibody proteoforms employing a novel comprehensive mass spectrometry (MS)-based analytical workflow. Profiling of anti-citrullinated protein antibodies (ACPA) IgG and total IgG in paired samples of plasma and synovial fluid revealed a clear distinction of autoantibodies from total IgG and between biofluids. This discrimination relied on comprehensive subclass-specific PTM profiles including previously neglected features such as IgG3 CH3 domain glycosylation, allotype ratios, and non-glycosylated IgG. Intriguingly, specific proteoforms were found to correlate with markers of inflammation and disease accentuating the need of such approaches in clinical investigations and calling for further mechanistic studies to comprehend the role of autoantibody proteoforms in defining autoimmune responses.

immunology↗

bioRxiv2022

Benchmarking computational methods for B-cell receptor reconstruction from single-cell RNA-seq data

Multiple methods have recently been developed to reconstruct full-length B-cell receptors (BCRs) from single-cell RNA-seq (scRNA-seq) data. This need emerged from the expansion of scRNA-seq techniques, the increasing interest in antibody-based drug development and the importance of BCR repertoire changes in cancer and autoimmune disease progression. However, a comprehensive assessment of performance-influencing factors like the sequencing depth, read length or the number of somatic hypermutations (SHMs) as well as guidance regarding the choice of methodology are still lacking. In this work, we evaluated the ability of six available methods to reconstruct full-length BCRs using one simulated and three experimental SMART-seq datasets. In addition, we validated that the BCRs assembled in silico recognize their intended targets when expressed as monoclonal antibodies. We observed that methods like BALDR, BASIC and BRACER showed the best overall performance across the tested datasets and conditions whereas only BASIC demonstrated acceptable results on very short read libraries. Furthermore, the de novo assembly-based methods BRACER and BALDR were the most accurate in reconstructing BCRs harboring different degrees of SHMs in the variable domain, while TRUST4, MiXCR and BASIC were the fastest. Finally, we propose guidelines to select the best method based on the given data characteristics.

bioinformatics↗

bioRxiv2021

ACPA-IgG variable domain glycosylation increases before the onset of rheumatoid arthritis and stabilizes thereafter; a cross-sectional study encompassing over 1500 samples

ObjectiveThe autoimmune response in rheumatoid arthritis (RA) is marked by anti-citrullinated protein antibodies (ACPA). A remarkable feature of ACPA-IgG is the abundant expression of N-linked glycans in the variable domain. Nonetheless, the presence of ACPA variable domain glycans (VDG) across disease stages and its response to therapy is poorly described. To understand its dynamics, we investigated the abundance of ACPA-IgG VDG in 1574 samples from individuals in different clinical disease stages. MethodsUsing liquid chromatography, we analyzed ACPA-IgG VDG profiles of 7 different cohorts from Japan, Canada, the Netherlands and Sweden. We assessed 184 healthy, 228 pre-symptomatic, 277 arthralgia, 305 patients at RA-onset and 117 RA-patients 4, 8 and 12 months after disease onset. Additionally, we measured VDG of 234 samples from RA-patients that did or did not achieve long-term drug-free remission (DFR) during up to 16 years follow-up. ResultsOur data show that ACPA-IgG VDG significantly increases (p<0.0001) towards disease-onset and associates with ACPA-levels and epitope spreading pre-diagnosis. A slight increase in VDG was observed in established RA and a moderate influence of treatment. Individuals who later achieved DFR displayed reduced ACPA-IgG VDG already at RA-onset. ConclusionThe abundance of ACPA-IgG VDG rises towards RA-onset and correlates with maturation of the ACPA-response. Although, ACPA-IgG VDG levels are rather stable in established disease, a lower degree at RA-onset correlates with DFR. Even though the underlying biological mechanisms are still elusive, our data support the concept that VDG relates to an expansion of the ACPA-response pre-disease and contributes to disease-development.

immunology↗