Biology subjects

Rispens, T.

Publications and source records attributed to Rispens, T..

10 recordsLinked to original sources

bioRxiv2026

Assessment of polymeric immunoglobulin A and M via detection of the joining chain

Polymeric immunoglobulin M (IgM) and A (IgA) play key roles in systemic and mucosal immunity, yet quantitative assessment of their polymeric forms has been hampered by the lack of robust, high-throughput assays. Polymerization of both isotypes implies incorporation of the joining chain (J chain), making direct detection of integrated J chain an attractive surrogate marker. Here, we report the generation and characterization of a novel panel of monoclonal antibodies targeting human J chain. Binding analyses revealed distinct antibody clusters with differential preferences for IgA-J and/or IgM-J. We developed sensitive ELISAs that allow reliable quantification of J-chain- containing IgM and IgA in recombinant preparations and complex biological samples such as serum and saliva. For IgM, assay performance in serum required mild dissociation of the IgM- CD5L complex, enabling accurate detection of integrated J chain. For IgA, clone 9G10 showed remarkable specificity for IgA-J, with minimal cross-reactivity to IgM. Application of these assays demonstrates that on average, 10% of circulating IgA is J-chain-containing, with proportional contributions of IgA1 and IgA2, and enables high-throughput measurement of antigen-specific polymeric IgA responses, exemplified by SARS-CoV-2 vaccination. These tools provide a long-needed platform to study polymeric antibody dynamics in health, infection, vaccination, and B-cell-driven diseases.

immunology↗

bioRxiv2026

FCRL5 is a fucose-sensitive IgG-Fc receptor with binding properties distinct from classical Fcγ receptors

Fc receptor-like protein 5 (FCRL5) is a low-affinity IgG receptor expressed on B cells, with emerging therapeutic relevance due to its expression on multiple myeloma cells, and a potential role in regulating B cell responses. Previous reports on the FCRL5-IgG interaction vary widely in reported affinities, binding differences across IgG subclasses, and molecular requirements for maximal binding. Furthermore, the impact of Fc-engineering strategies, as used in (therapeutic) monoclonal antibodies, remains poorly understood. Here, we provide a comprehensive biochemical analysis of the FCRL5-IgG interaction. We demonstrate that FCRL5 is a true IgG Fc-receptor, binding with very low affinity (60-80 {micro}M). FCRL5 binds IgG in a manner involving primarily the two N-terminal domains of FCRL5, and the third domain for maximal binding, but with distinct essential residues in the IgG Fc-tail. Surface plasmon resonance analysis of the binding of FCRL5 to the various IgG subclasses revealed a preference for IgG1 and IgG4. Interestingly, various Fc-engineered IgG variants commonly used for silencing or enhancing of Fc receptor binding do not impact FCRL5 binding. Screening the binding of a set of IgG antibodies carrying defined sets of Fc-mutations to FCRL5 revealed E293 as a key binding determinant and led to the discovery of E293R as a mutation that selectively abrogates FCRL5 binding while preserving binding to other classical Fc{gamma}Rs. Lastly, we show that FCRL5 has considerable preference for binding afucosylated IgG. Together, our results define the essential characteristics of the IgG-FCRL5 interaction and demonstrate the potential of both naturally occurring IgG variants as well as therapeutically explored bioengineered IgG formats to differentially engage FCRL5.

immunology↗

bioRxiv2025

Transcriptome-Proteome analysis of human naive and memory B cell subsets reveal isotype and subclass-specific phenotypes

Antibodies produced by B cells aid in recognition and clearance of pathogens and is the cornerstone of vaccination strategies. Humans produce nine different antibody isotypes and their effector functions differ according to the type of antigen and route of exposure. Phenotypic variation between isotype-switched B cell subsets is expected but not studied in detail. To obtain a molecular definition of isotype-defined cell identity, we performed proteomics and transcriptomics on isotype-defined populations of human naive and memory B cells (MBCs): CD27-IgM+IgD+, CD27+CD38lo/-IgM+IgD+, CD27+CD38lo/-IgM+IgD-, and IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4 MBCs (CD27+CD38lo/-Ig+). Combined proteome and transcriptome analysis revealed that mRNA and protein expression profiles separate isotype-defined B cell subsets according to their differentiation status. mRNA and protein expression levels correlated reasonably well for many genes. IgG4-switched B cells were most distinct from naive B cells in terms of mRNA as well as protein expression profiles. Besides a distinct expression profile of cytokine and Fc receptors, we identified a high expression of IgE-coding mRNA in IgG4-switched B cells. SDR16C5 was identified as uniquely upregulated in IgG4-switched B cells. Taken together, this study highlights the distinct phenotypic profile of IgG4-switched B cells.

immunology↗

bioRxiv2024

Characterization of a heterogenous activated B cell compartment arising early after antigen exposure preceding long-lived memory B cell formation

Once formed, plasma cells and memory B cells (MBCs) are difficult to eradicate, posing a problem in the context of unwanted antibody responses. Characterizing early B cell differentiation stages after antigen encounter is thus crucial to target and prevent unwanted antibody formation. Here, we unravelled in-depth antigen-specific B cell responses longitudinally after SARS-CoV-2 mRNA vaccination in healthy individuals using multiparameter spectral flow cytometry. The early antigen-specific B cell response was dominated by spike-specific IgG+ CD27+ CD71+ activated B cells (ActBCs), previously assigned as germinal center-derived and DN2 extrafollicular B cells. Within the early IgG+ ActBC compartment, six distinct clusters were identified with specific contraction dynamics, whereby some of these clusters were more closely related to pre-ASCs and others more to long-lived MBCs. Some of the highly contracting ActBC clusters expressed CD11c, a marker previously used to define atypical B cells. The transient presence of different ActBC clusters could also be observed in total B cells when gated in an antigen- independent manner. Our results thus delineate the early stages of the antigen-specific B cell response, with a further dissection of the CD71+ ActBC compartment. Detection of ActBC clusters early after antigen encounter in total B cells opens avenues for future evaluation of their potential to serve as a proxy for antigen-reactive B cells in autoimmunity or other unwanted B cell responses.

immunology↗

bioRxiv2023

Deep profiling of antigen-specific B cells from different pathogens identifies novel compartments in the IgG memory B cell and antibody-secreting cell lineages

A better understanding of the bifurcation of human B cell differentiation into memory B cells (MBC) and antibody-secreting cells (ASC) and identification of MBC and ASC precursors is crucial to optimize vaccination strategies or block undesired antibody responses. To unravel the dynamics of antigen-induced B cell responses, we compared circulating B cells reactive to SARS-CoV-2 (Spike, RBD and Nucleocapsid) in COVID-19 convalescent individuals to B cells specific to Influenza-HA, RSV-F and TT, induced much longer ago. High-dimensional spectral flow cytometry indicated that the decision point between ASC- and MBC-formation lies in the CD43+CD71+IgG+ Activated B cell compartment, showing properties indicative of recent germinal center activity and recent antigen encounter. Within this Activated B cells compartment, CD86+ B cells exhibited close phenotypical similarity with ASC, while CD86- B cells were closely related to IgG+ MBCs. Additionally, different activation stages of the IgG+ MBC compartment could be further elucidated. The expression of CD73 and CD24, regulators of survival and cellular metabolic quiescence, discerned activated MBCs from resting MBCs. Activated MBCs (CD73-CD24lo) exhibited phenotypical similarities with CD86- IgG+ Activated B cells and were restricted to SARS-CoV-2 specificities, contrasting with the resting MBC compartment (CD73-/CD24hi) that exclusively encompassed antigen-specific B cells established long ago. Overall, these findings identify novel stages for IgG+ MBC and ASC formation and bring us closer in defining the decision point for MBC or ASC differentiation. ImportanceIn this study, researchers aimed to better understand human B cell differentiation and their role in establishing long-lived humoral immunity. Using high-dimensional flow cytometry, they studied B cells reactive to three SARS-CoV-2 antigens in individuals convalescent for COVID-19, and compared their phenotypes to B cells reactive to three distinct protein antigens derived from vaccines or viruses encountered months to decades before. Their findings showed that Activated B cells reflect recent germinal center graduates that may have diverse fates; with some feeding the pool of antibody-secreting cells and others fueling the resting memory B cell compartment. Activated B cells gradually differentiate into resting memory B cells through an activated MBC phase. Increased expression of the cellular metabolic regulators CD73 and CD24 in resting memory B cells distinguishes them from the activated memory B cells phase, and is likely involved in sustaining a durable memory of humoral immunity. These findings are crucial for the development of vaccines that provide lifelong protection and may show potential to define reactive B cells in diseases where the cognate-antigen is still unknown such as in autoimmunity, cancers, or novel viral outbreaks.

immunology↗

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

Single-cell analysis reveals dynamics of human B cell differentiation and identifies novel B and antibody-secreting cell intermediates

Differentiation of B cells into antibody-secreting cells (ASCs) is a key process to generate protective humoral immunity. A detailed understanding of the cues controlling ASC differentiation is important to devise strategies to modulate antibody formation. Here, we dissected differentiation trajectories of human naive B cells into ASCs using single-cell RNA sequencing. By comparing transcriptomes of B cells at different stages of differentiation from an in vitro model with ex vivo B cells and ASCs, we uncovered a novel pre-ASC population present ex vivo in lymphoid tissues. For the first time, a germinal-center-like population is identified in vitro from human naive B cells and possibly progresses into a memory B cell population through an alternative route of differentiation, thus recapitulating in vivo human GC reactions. Our work allows further detailed characterization of human B cell differentiation into ASCs or memory B cells in both healthy and diseased conditions.

immunology↗

bioRxiv2022

The BNT162b2 mRNA SARS-CoV-2 vaccine induces transient afucosylatedIgG1 in naive but not antigen-experienced vaccinees

The onset of severe SARS-CoV-2 infection is characterized by the presence of afucosylated IgG1 responses against the viral spike (S) protein, which can trigger exacerbated inflammatory responses. Here, we studied IgG glycosylation after BNT162b2 SARS-CoV-2 mRNA vaccination to explore whether vaccine-induced S protein expression on host cells also generates afucosylated IgG1 responses. SARS-CoV-2 naive individuals initially showed a transient afucosylated anti-S IgG1 response after the first dose, albeit to a lower extent than severely ill COVID-19 patients. In contrast, previously infected, antigen-experienced individuals had low afucosylation levels, which slightly increased after immunization. Afucosylation levels after the first dose correlated with low fucosyltransferase 8 (FUT8) expression levels in a defined plasma cell subset. Remarkably, IgG afucosylation levels after primary vaccination correlated significantly with IgG levels after the second dose. Further studies are needed to assess efficacy, inflammatory potential, and protective capacity of afucosylated IgG responses. One sentence summaryA transient afucosylated IgG response to the BNT162b2 mRNA vaccine was observed in naive but not in antigen-experienced individuals, which predicted antibody titers upon the second dose.

immunology↗

bioRxiv2020

Divergent SARS-CoV-2-specific T and B cell responses in severe but not mild COVID-19

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic. Understanding both the immunological processes providing specific immunity and potential immunopathology underlying the pathogenesis of this disease may provide valuable insights for potential therapeutic interventions. Here, we quantified SARS-CoV-2 specific immune responses in patients with different clinical courses. Compared to individuals with a mild clinical presentation, CD4+ T cell responses were qualitatively impaired in critically ill patients. Strikingly, however, in these patients the specific IgG antibody response was remarkably strong. The observed disparate T and B cell responses could be indicative of a deregulated immune response in critically ill COVID-19 patients.

immunology↗

bioRxiv2020

Unravelling the effect of a potentiating anti-Factor H antibody on atypical hemolytic uremic syndrome associated factor H variants

The complement system plays an important role in our innate immune system. Complement activation results in clearance of pathogens, immune complex and apoptotic cells. The host is protected from complement-mediated damage by several complement regulators. Factor H (FH) is the most important fluid-phase regulator of the alternative pathway of the complement system. Heterozygous mutations in FH are associated with complement-related diseases such as atypical hemolytic uremic syndrome (aHUS) and age-related macular degeneration. We recently described an agonistic anti-FH monoclonal antibody that can potentiate the regulatory function of FH. This antibody could serve as a potential new drug for aHUS patients and alternative to C5 blockade by Eculizumab. However, it is unclear whether this antibody can potentiate FH mutant variants in addition to wild type FH. Here, the functionality and potential of the agonistic antibody in the context of pathogenic aHUS-related FH mutant proteins was investigated. The binding affinity of recombinant WT FH, and the FH variants, W1183L, V1197A, R1210C, and G1194D to C3b was increased upon addition of the potentiating antibody and similarly, the decay accelerating activity of all mutants is increased. The potentiating anti-FH antibody is able to restore the surface regulatory function of most of the tested FH mutants to WT FH levels. In conclusion, our potentiating anti-FH is broadly active and able to enhance both WT FH function as well as most aHUS-associated FH variants tested in this study.

immunology↗