Rheumatoid arthritis (RA) is an autoimmune disease affecting joints which is hallmarked by the presence of autoantibodies against citrrulianted protein (ACPA). This thesis describes the phenotypic... Show moreRheumatoid arthritis (RA) is an autoimmune disease affecting joints which is hallmarked by the presence of autoantibodies against citrrulianted protein (ACPA). This thesis describes the phenotypic and functional characteristics of ACPA-expressing autoreactive B cells which suggest potential pathologic roles of these B cells in RA pathogenesis. The thesis also describes strategies to specifically deplete ACPA-expressing B cells to improve current RA therapeutic options. Show less
Holborough-Kerkvliet, M.D.; Kroos, S.; Wetering, R. van de; Toes, R.E.M. 2023
Autoimmune diseases are heterogeneous pathologies characterized by a breakdown of immunological tolerance to self, resulting in a chronic and aberrant immune response to self-antigens. The scope... Show moreAutoimmune diseases are heterogeneous pathologies characterized by a breakdown of immunological tolerance to self, resulting in a chronic and aberrant immune response to self-antigens. The scope and extent of affected tissues can vary greatly per autoimmune disease and can involve multiple organs and tissue types. The patho-genesis of most autoimmune diseases remains unknown but it is widely accepted that a complex interplay be-tween (autoreactive) B and T cells in the context of breached immunological tolerance drives autoimmune pathology. The importance of B cells in autoimmune disease is exemplified by the successful use of B cell tar-geting therapies in the clinic. For example, Rituximab, a depleting anti-CD20 antibody, has shown favorable results in reducing the signs and symptoms of multiple autoimmune diseases, including Rheumatoid Arthritis, Anti-Neutrophil Cytoplasmic Antibody associated vasculitis and Multiple Sclerosis. However, Rituximab depletes the entire B cell repertoire, leaving patients susceptible to (latent) infections. Therefore, multiple ways to target autoreactive cells in an antigen-specific manner are currently under investigation. In this review, we will lay out the current state of antigen-specific B cell inhibiting or depleting therapies in the context of autoimmune diseases. Show less
IntroductionInborn errors of immunity (IEI) are an expanding group of rare diseases whose field has been boosted by next-generation sequencing (NGS), revealing several new entities, accelerating... Show moreIntroductionInborn errors of immunity (IEI) are an expanding group of rare diseases whose field has been boosted by next-generation sequencing (NGS), revealing several new entities, accelerating routine diagnoses, expanding the number of atypical presentations and generating uncertainties regarding the pathogenic relevance of several novel variants. MethodsResearch laboratories that diagnose and provide support for IEI require accurate, reproducible and sustainable phenotypic, cellular and molecular functional assays to explore the pathogenic consequences of human leukocyte gene variants and contribute to their assessment. We have implemented a set of advanced flow cytometry-based assays to better dissect human B-cell biology in a translational research laboratory. We illustrate the utility of these techniques for the in-depth characterization of a novel (c.1685G>A, p.R562Q) de novo gene variant predicted as probably pathogenic but with no previous insights into the protein and cellular effects, located in the tyrosine kinase domain of the Bruton's tyrosine kinase (BTK) gene, in an apparently healthy 14-year-old male patient referred to our clinic for an incidental finding of low immunoglobulin (Ig) M levels with no history of recurrent infections. Results and discussionA phenotypic analysis of bone marrow (BM) revealed a slightly high percentage of pre-B-I subset in BM, with no blockage at this stage, as typically observed in classical X-linked agammaglobulinemia (XLA) patients. The phenotypic analysis in peripheral blood also revealed reduced absolute numbers of B cells, all pre-germinal center maturation stages, together with reduced but detectable numbers of different memory and plasma cell isotypes. The R562Q variant allows Btk expression and normal activation of anti-IgM-induced phosphorylation of Y551 but diminished autophosphorylation at Y223 after anti IgM and CXCL12 stimulation. Lastly, we explored the potential impact of the variant protein for downstream Btk signaling in B cells. Within the canonical nuclear factor kappa B (NF-kappa B) activation pathway, normal I kappa B alpha degradation occurs after CD40L stimulation in patient and control cells. In contrast, disturbed I kappa B alpha degradation and reduced calcium ion (Ca2(+)) influx occurs on anti-IgM stimulation in the patient's B cells, suggesting an enzymatic impairment of the mutated tyrosine kinase domain. Show less
Our immune system is supposed to protect us from infections, but it can also attack our own tissues if not properly controlled. This can lead to autoimmune diseases like rheumatoid arthritis (RA).... Show moreOur immune system is supposed to protect us from infections, but it can also attack our own tissues if not properly controlled. This can lead to autoimmune diseases like rheumatoid arthritis (RA). People with RA have antibodies to the body’s own proteins (self), but it is not known how they arise. The data described in this thesis show that these antibodies cross-react with self-proteins carrying different post-translational modifications, suggesting that multiple proteins may be involved in the initial loss of the immune system’s ability to discriminate between self- and foreign-proteins. As another unique feature, these antibodies carry additional sugars at an unexpected site in the molecule. Our data show that these sugars (variable domain glycans) can prevent binding to potential self-proteins, affect complement activation, and set the threshold for immune B-cell activation. In addition, our data show that the abundance of these sugars increases toward disease onset and predicts the development of chronic, persistent disease or the chance of subsequent remission. Taken together, these sugars may help B cells to escape the tight control mechanism in our body that are in place to prevent the development autoimmunity. This new “sugar mechanism” could be beneficial for diagnosis and future treatment. Show less
Pasmans, H.; Berkowska, M.A.; Diks, A.M.; Mooij, B. de; Groenland, R.J.; Rond, L. de; ... ; Buisman, A.M. 2022
Introduction: Current human papillomavirus (HPV) vaccines consist of virus-like particles (VLPs) which are based on the L1 protein, but they are produced by different expression systems and use... Show moreIntroduction: Current human papillomavirus (HPV) vaccines consist of virus-like particles (VLPs) which are based on the L1 protein, but they are produced by different expression systems and use different adjuvants. We performed in-depth immunophenotyping of multiple innate and adaptive immune cells after vaccination with bivalent versus nonavalent HPV vaccines. Method: Twenty pre-menopausal HPV-seronegative women were enrolled and randomized to receive three-doses of either the bivalent or the nonavalent HPV vaccine. Blood samples were collected at multiple time points from baseline up to 7 months after first vaccination. Four extensive EuroFlow flow cytometry antibody panels were used to monitor various immune cell subsets. Additionally, HPV-specific memory B- and T cells were determined by ELISPOT and HPV-specific antibody levels were measured by a VLP-based multiplex immunoassay. Results: In both cohorts, the numbers of plasma cells expanded in the first week after both primary and tertiary vaccination. HPV16 and HPV18-specific antibody levels and memory B and T-cell responses were higher in the bivalent than in the nonavalent vaccinees one month post third vaccination. For HPV31 and HPV45-specific antibody levels this pattern was reversed. Monocytes showed an expansion one day after vaccination in both cohorts but were significantly higher in the bivalent vaccine cohort. Large heterogeneity in responses of the other cell subsets was observed between donors. Conclusion: This pilot study showed a consistent response of monocytes and plasma cells after vaccination and a considerable variation in other circulating immune cells in both types of HPV vaccines between donors. Show less
For many years, hematopoietic stem cell kinetics and/or cell signaling pathway activity has been studied through fluorescent in vitro or in vivo models. However, inaccurate measurement of the... Show moreFor many years, hematopoietic stem cell kinetics and/or cell signaling pathway activity has been studied through fluorescent in vitro or in vivo models. However, inaccurate measurement of the fluorescent proteins or lack of knowlegde about the genetic design of these models lead to incomplete conclusions. In the present thesis, in vivo fluorescent models are improved and new models are proposed together with analysis protocols to ensure precise measurement of fluorescent protein dynamics. Show less
Mol, J. de; Kuiper, J.; Tsiantoulas, D.; Foks, A.C. 2021
Aging is considered to be an important risk factor for several inflammatory diseases. B cells play a major role in chronic inflammatory diseases by antibody secretion, antigen presentation and T... Show moreAging is considered to be an important risk factor for several inflammatory diseases. B cells play a major role in chronic inflammatory diseases by antibody secretion, antigen presentation and T cell regulation. Different B cell subsets have been implicated in infections and multiple autoimmune diseases. Since aging decreases B cell numbers, affects B cell subsets and impairs antibody responses, the aged B cell is expected to have major impacts on the development and progression of these diseases. In this review, we summarize the role of B cells in health and disease settings, such as atherosclerotic disease. Furthermore, we provide an overview of age-related changes in B cell development and function with respect to their impact in chronic inflammatory diseases. Show less
Background Anti-modified protein antibodies (AMPA) targeting citrullinated, acetylated and/or carbamylated self-antigens are hallmarks of rheumatoid arthritis (RA). Although AMPA-IgG cross... Show moreBackground Anti-modified protein antibodies (AMPA) targeting citrullinated, acetylated and/or carbamylated self-antigens are hallmarks of rheumatoid arthritis (RA). Although AMPA-IgG cross-reactivity to multiple post-translational modifications (PTMs) is evident, it is unknown whether the first responding B cells, expressing IgM, display similar characteristics or if cross-reactivity is crucially dependent on somatic hypermutation (SHM). We now studied the reactivity of (germline) AMPA-IgM to further understand the breach of B cell tolerance and to identify if cross-reactivity depends on extensive SHM. Moreover, we investigated whether AMPA-IgM can efficiently recruit immune effector mechanisms. Methods Polyclonal AMPA-IgM were isolated from RA patients and assessed for cross-reactivity towards PTM antigens. AMPA-IgM B cell receptor sequences were obtained by single cell isolation using antigen-specific tetramers. Subsequently, pentameric monoclonal AMPA-IgM, their germline counterparts and monomeric IgG variants were generated. The antibodies were analysed on a panel of PTM antigens and tested for complement activation. Results Pentameric monoclonal and polyclonal AMPA-IgM displayed cross-reactivity to multiple antigens and different PTMs. PTM antigen recognition was still present, although reduced, after reverting the IgM into germline. Valency of AMPA-IgM was crucial for antigen recognition as PTM-reactivity significantly decreased when AMPA-IgM were expressed as IgG. Furthermore, AMPA-IgM was 15- to 30-fold more potent in complement-activation compared to AMPA-IgG. Conclusions We provide first evidence that AMPA-IgM are cross-reactive towards different PTMs, indicating that PTM (cross-)reactivity is not confined to IgG and does not necessarily depend on extensive somatic hypermutation. Moreover, our data indicate that a diverse set of PTM antigens could be involved in the initial tolerance breach in RA and suggest that AMPA-IgM can induce complement-activation and thereby inflammation. Show less
This thesis contains a variety of information about the natural and vaccine induced immunity against the human papillomavirus. The spontaneously induced HPV-specific humoral response after... Show moreThis thesis contains a variety of information about the natural and vaccine induced immunity against the human papillomavirus. The spontaneously induced HPV-specific humoral response after infection was assessed in population-based studies. The vaccine-induced changes in HPV-seroprevalence among the HPV unvaccinated Dutch population aged 0-89 years, where we compared the HPV-seroprevalence before the introduction of the HPV vaccine with data of approximately six years post-implementation of the national HPV vaccination program. Also, the HPV immune status of the Dutch Caribbean population just after introduction of HPV vaccination was determined. Moreover, the longitudinal relation between the hr-HPV antibody levels and the prevalence of HPV infections in three-dose vaccinated girls were studied. And more insight was gained into humoral and cellular immune responses after just a one-dose of the HPV vaccine. At last, the kinetics of innate and adaptive immune responses directly after vaccination different HPV vaccines were investigated. In the coming years some important changes are expected regarding HPV screening and vaccination. The effectiveness of the one-dose schedule will become clear as clinical trials end. In the Netherlands, a sex-neutral vaccination will be implemented soon. These changes will need to be monitored to provide scientific answers about the effectiveness and immunogenicity. Show less
The immune response against posttranslationally modified (PTM) antigens, in particular the generation of anti-citrullinated protein antibodies (ACPA), is a very specific hallmark of rheumatoid... Show moreThe immune response against posttranslationally modified (PTM) antigens, in particular the generation of anti-citrullinated protein antibodies (ACPA), is a very specific hallmark of rheumatoid arthritis. The factors that initiate this immune response and the triggers that stimulate the transition from asymptomatic autoimmunity to autoimmune disease are so far unknown. Genetic risk factors and the maturation of the ACPA response prior to the onset of arthritis indicate an important role for helper T cells in this process. Antigens that trigger this process, however, remain to be defined. Notably, recent data demonstrate that ACPA do not only recognize citrullinated protein antigens. Other posttranslational protein modifications such as homocitrulline and acetyllysine are also recognized. This cross-reactivity towards different PTM antigens was found for various monoclonal ACPA and broadens the spectrum of antigens that can stimulate and activate ACPA-expressing B cells. Also, it suggests that such B cells could receive help from autoreactive but also from non-autoreactive T cells. This review summarizes these recent findings and provides insight into their potential relevance for the disease rheumatoid arthritis. Show less
ObjectiveAutoantibodies against antigens carrying distinct post-translational modifications (PTMs), such as citrulline, homocitrulline or acetyllysine, are hallmarks of rheumatoid arthritis (RA).... Show moreObjectiveAutoantibodies against antigens carrying distinct post-translational modifications (PTMs), such as citrulline, homocitrulline or acetyllysine, are hallmarks of rheumatoid arthritis (RA). The relation between these anti-modified protein antibody (AMPA)-classes is poorly understood as is the ability of different PTM-antigens to activate B-cell receptors (BCRs) directed against citrullinated proteins (CP). Insights into the nature of PTMs able to activate such B cells are pivotal to understand the 'evolution' of the autoimmune response conceivable underlying the disease. Here, we investigated the cross-reactivity of monoclonal AMPA and the ability of different types of PTM-antigens to activate CP-reactive BCRs.MethodsBCR sequences from B cells isolated using citrullinated or acetylated antigens were used to produce monoclonal antibodies (mAb) followed by a detailed analysis of their cross-reactivity towards PTM-antigens. Ramos B-cell transfectants expressing CP-reactive IgG BCRs were generated and their activation on stimulation with PTM-antigens investigated.ResultsMost mAbs were highly cross-reactive towards multiple PTMs, while no reactivity was observed to the unmodified controls. B cells carrying CP-reactive BCRs showed activation on stimulation with various types of PTM-antigens.ConclusionsOur study illustrates that AMPA exhibit a high cross-reactivity towards at least two PTMs indicating that their recognition pattern is not confined to one type of modification. Furthermore, our data show that CP-reactive B cells are not only activated by citrullinated, but also by carbamylated and/or acetylated antigens. These data are vital for the understanding of the breach of B-cell tolerance against PTM-antigens and the possible contribution of these antigens to RA-pathogenesis. Show less
Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by systemic and persistent inflammation of the joints. Around 50-80% of the RA patients harbour either one or multiple types... Show moreRheumatoid arthritis (RA) is a chronic autoimmune disease characterized by systemic and persistent inflammation of the joints. Around 50-80% of the RA patients harbour either one or multiple types of autoantibodies, under which also Anti-Citrullinated Protein Antibodies (ACPA). ACPA are highly specific for RA and are strongly associated with the severity of arthritis. The studies described in this thesis aim to explore the structure and biological function of ACPA and the B cells producing these ACPA.The studies highlight the importance of the synovial environment for the survival of ACPA B cells. Moreover, we demonstrate that the variable region of ACPA-IgG differs from that of ACPA-IgM by the introduction of glycans, and that glycans in the constant part of antibodies can be modulated by influences from the micro-environment. This may affect the biological functions of the antibodies. Furthermore, we show that ACPA can bind to so-called Fc gamma receptor I on immune cells which could be a potential effector mechanisms of ACPA. These findings demonstrate that the structure and environment of ACPA play an important role in the ACPA immune response and provide multiple arguments for the active contribution of ACPA in the chronic inflammation of RA. Show less
Volkov, M.; Schie, K.A. van; Woude, D. van der 2019
Cardiovascular disease is a major global burden and atherosclerosis is the main underlying pathological process. Despite better management of cholesterol levels, there remains a significant... Show moreCardiovascular disease is a major global burden and atherosclerosis is the main underlying pathological process. Despite better management of cholesterol levels, there remains a significant residual risk of developing atherosclerosis and cardiovascular events. Hence, novel pathways and targets should be identified to optimize atherosclerosis therapy. Despite dyslipidemia, the immune system is also heavily involved in the pathophysiology of atherosclerosis. Protective immune responses in the acute setting of increased cholesterol levels eventually turn into debilitating responses when the immune system is chronically stimulated. Hence, we aimed to identify new therapeutic targets to dampen the immune response in atherosclerosis. More specifically, we focused our efforts on modulating the B lymphocyte response, for which there was a scarcity of data. In this thesis we describe novel ways to modulate the B cell response in atherosclerosis. We have found that there are specific B cell subsets that have different effects on the progress of atherosclerosis. For instance, removal of TIM-1+ B cells resulted in increased atherosclerosis, while removal of BTLA+ follicular B cells reduced atherosclerosis. In conclusion, this thesis provides promising immunological targets for the treatment of atherosclerosis. Show less
Pino-Molina, L. del; Rodriguez-Ubreva, J.; Canizales, J.T.; Coronet-Diaz, M.; Kulis, M.; Martin-Subero, J.I.; ... ; Lopez-Granados, E. 2019
Common Variable Immunodeficiency (CVID) is characterized by impaired antibody production and poor terminal differentiation of the B cell compartment, yet its pathogenesis is still poorly understood... Show moreCommon Variable Immunodeficiency (CVID) is characterized by impaired antibody production and poor terminal differentiation of the B cell compartment, yet its pathogenesis is still poorly understood. We first reported the occurrence of epigenetic alterations in CVID by high-throughput methylation analysis in CVID-discordant monozygotic twins. Data from a recent whole DNA methylome analysis throughout different stages of normal B cell differentiation allowed us to design a new experimental approach. We selected CpG sites for analysis based on two criteria: one, CpGs with potential association with the transcriptional status of relevant genes for B cell activation and differentiation; and two, CpGs that undergo significant demethylation from naive to memory B cells in healthy individuals. DNA methylation was analyzed by bisulfite pyrosequencing of specific CpG sites in sorted naive and memory B cell subsets from CVID patients and healthy donors. We observed impaired demethylation in two thirds of the selected CpGs in CVID memory B cells, in genes that govern B cell-specific processes or participate in B cell signaling. The degree of demethylation impairment associated with the extent of the memory B cell reduction. The impaired demethylation in such functionally relevant genes as AICDA in switched memory B cells correlated with a lower proliferative rate. Our new results reinforce the hypothesis of altered demethylation during B cell differentiation as a contributing pathogenic mechanism to the impairment of B cell function and maturation in CVID. In particular, deregulated epigenetic control of AICDA could play a role in the defective establishment of a post-germinal center B cell compartment in CVID. Show less
Parasitic helminths modulate host immune responses. While the induction of type 2 immune responses is a widely recognized feature of helminth infections, a network of regulatory immune responses is... Show moreParasitic helminths modulate host immune responses. While the induction of type 2 immune responses is a widely recognized feature of helminth infections, a network of regulatory immune responses is often dominant during the chronic phase of infection. Suppression of the host immune system during helminth infections inhibits anti-parasite immunity, prevents tissue damage due to excessive inflammation and conveys spill-over suppression to inflammatory conditions such as allergy and asthma. The first part of this thesis focuses on the role of regulatory B cells, a prominent member of the immune regulatory network, in protection from allergic asthma by chronic Schistosoma (S.) mansoni infections. It furthermore identifies signals required for schistosome-induced regulatory B cell development. The second part of this thesis describes the protective effect of S. mansoni eggs, and a specific egg-derived glycoprotein, against allergic asthma in the absence of chronic infection. A better understanding how helminthes including S. mansoni modulate host immune responses, and the implications this has for inflammatory diseases such as allergic asthma, may provide valuable leads for the development of novel pharmaceutical agents for the treatment of allergic disorders. Show less
Limiting the pro-inflammatory immune response is critical for the treatment of atherosclerosis. Regulatory B cells (Bregs) can modulate the immune response through interleukin-10 (IL-10). Current... Show moreLimiting the pro-inflammatory immune response is critical for the treatment of atherosclerosis. Regulatory B cells (Bregs) can modulate the immune response through interleukin-10 (IL-10). Current data regarding Bregs and atherosclerosis is scarce and conflicting.\n B cells on atherosclerosis.\n B cells on atherosclerosis.\n B cells in atherosclerosis. Show less
Theunissen, P.M.J.; Sedek, L.; Haas, V. de; Szczepanski, T.; Sluijs, A. van der; Mejstrikova, E.; ... ; EuroFlow Consortium 2017
