The collagen antibody-induced arthritis (CAIA) model is highly effective in inducing arthritis, making it an attractive model for screening therapeutic compounds such as glucocorticoids (GCs). The... Show moreThe collagen antibody-induced arthritis (CAIA) model is highly effective in inducing arthritis, making it an attractive model for screening therapeutic compounds such as glucocorticoids (GCs). The severity of discomfort in this model makes it desirable to administer analgesics, but it is a prerequisite that these do not interfere with the model or tested therapeutics. In the present study, we studied the effect of 1 mg/mL tramadol and 3.5 mg/mL paracetamol (TP) on CAIA in male BALB/cAnNCrl mice and the possible interference of TP analgesia with the activity of the GC drug prednisolone (Pred). Our results showed that TP abolished the Pred-induced amelioration of CAIA, as well as several other Pred-induced effects, such as the reduction in thymus weight and the increase in insulin level. This most likely results from the effects of TP on the hepatic metabolism of this drug, since it strongly increased the Cyp3a11 expression in the liver. Altogether, we conclude that TP analgesia is not suitable for the CAIA model in male BALB/cAnNCrl mice, in particular when evaluating the effects of GCs such as Pred. Show less
Objective: Rheumatoid arthritis (RA) is characterized by the presence of disease-specific autoreactive B cell responses, in particular those generating anti-citrullinated protein antibodies (ACPA).... Show moreObjective: Rheumatoid arthritis (RA) is characterized by the presence of disease-specific autoreactive B cell responses, in particular those generating anti-citrullinated protein antibodies (ACPA). For many years, Epstein-Barr virus (EBV) has been implicated in disease pathogenesis, possibly by facilitating the development and persistence of autoreactive B cells. To test this hypothesis, the presence of EBV episomes in ACPA-expressing B cells was analyzed.Methods: ACPA-expressing B cells derived from peripheral blood (PB) of seven EBV-seropositive RA patients, and synovial fluid (SF) of one additional EBV-seropositive RA patient, were isolated by flow cytometry. PB cells were expanded for 11-12 days, after which supernatant was harvested and analyzed for cyclic citrullinated-peptide (CCP)2 reactivity. SF cells were isolated directly in a lysis buffer. DNA was isolated and qPCR reactions were performed to determine the EBV status of the cells. EBV-immortalized B cell lymphoblastoid-cell lines (EBV blasts) served as standardized controls.Results: Two hundred ninety-six PB and 60 SF ACPA-expressing B cells were isolated and divided over 16 and 3 pools containing 10-20 cells, respectively. Supernatants of all 16 cultured PB pools contained CCP2-Ig. DNA of all pools was used for qPCR analysis. While EBV-blast analysis showed sensitivity to detect EBV DNA in single B cells, no EBV DNA was detected in any of the ACPA-expressing B cell pools.Conclusion: ACPA-expressing B cells are not enriched for EBV-DNA-containing clones. These results do not support the hypothesis that EBV infection of autoreactive B cells causes or maintains autoreactive B cell populations in RA. Instead, other mechanisms might explain the association between positive EBV serology and RA. Show less
The hallmark autoantibodies in rheumatoid arthritis are characterized by variable domain glycans (VDGs). Their abundant occurrence results from the selective introduction of N-linked glycosylation... Show moreThe hallmark autoantibodies in rheumatoid arthritis are characterized by variable domain glycans (VDGs). Their abundant occurrence results from the selective introduction of N-linked glycosylation sites during somatic hypermutation, and their presence is predictive for disease development. However, the functional consequences of VDGs on autoreactive B cells remain elusive. Combining crystallography, glycobiology, and functional B cell assays allowed us to dissect key characteristics of VDGs on human B cell biology. Crystal structures showed that VDGs are positioned in the vicinity of the antigen-binding pocket, and dynamic modeling combined with binding assays elucidated their impact on binding. We found that VDG-expressing B cell receptors stay longer on the B cell surface and that VDGs enhance B cell activation. These results provide a rationale on how the acquisition of VDGs might contribute to the breach of tolerance of autoreactive B cells in a major human autoimmune disease. Show less
The hallmark autoantibodies in rheumatoid arthritis are characterized by variable domain glycans (VDGs). Their abundant occurrence results from the selective introduction of N-linked glycosylation... Show moreThe hallmark autoantibodies in rheumatoid arthritis are characterized by variable domain glycans (VDGs). Their abundant occurrence results from the selective introduction of N-linked glycosylation sites during somatic hyper-mutation, and their presence is predictive for disease development. However, the functional consequences of VDGs on autoreactive B cells remain elusive. Combining crystallography, glycobiology, and functional B cell assays allowed us to dissect key characteristics of VDGs on human B cell biology. Crystal structures showed that VDGs are positioned in the vicinity of the antigen-binding pocket, and dynamic modeling combined with binding assays elucidated their impact on binding. We found that VDG-expressing B cell receptors stay longer on the B cell surface and that VDGs enhance B cell activation. These results provide a rationale on how the acquisition of VDGs might contribute to the breach of tolerance of autoreactive B cells in a major human autoimmune disease. Show less
Rheumatoid Arthritis (RA) is an autoimmune disease targeting the synovial joints. Several factors have been shown to be implicated in the onset and disease progression, among also genetic factors.... Show moreRheumatoid Arthritis (RA) is an autoimmune disease targeting the synovial joints. Several factors have been shown to be implicated in the onset and disease progression, among also genetic factors. HLA class II molecules have been implicated in the so called 'second hit' leading to disease onset. The implication of HLA class II molecules suggests that CD4+ T cells are involved in the disease development. In this thesis, several aspects of CD4+ T cells within RA are investigated to get a glimpse of what their role could be in disease onset. 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
Leeuwen, T. van; Araman, C.; Pournara, L.P.; Kampstra, A.S.B.; Bakkum, T.; Marqvorsen, M.H.S.; ... ; Kasteren, S.I. van 2021
Proteolysis is fundamental to many biological processes. In the immune system, it underpins the activation of the adaptive immune response: degradation of antigenic material into short peptides and... Show moreProteolysis is fundamental to many biological processes. In the immune system, it underpins the activation of the adaptive immune response: degradation of antigenic material into short peptides and presentation thereof on major histocompatibility complexes, leads to activation of T-cells. This initiates the adaptive immune response against many pathogens. Studying proteolysis is difficult, as the oft-used polypeptide reporters are susceptible to proteolytic sequestration themselves. Here we present a new approach that allows the imaging of antigen proteolysis throughout the processing pathway in an unbiased manner. By incorporating bioorthogonal functionalities into the protein in place of methionines, antigens can be followed during degradation, whilst leaving reactive sidechains open to templated and non-templated post-translational modifications, such as citrullination and carbamylation. Using this approach, we followed and imaged the post-uptake fate of the commonly used antigen ovalbumin, as well as the post-translationally citrullinated and/or carbamylated auto-antigen vinculin in rheumatoid arthritis, revealing differences in antigen processing and presentation. Show less
Leeuwen, T. van; Araman, C.; Pournara, L.P.; Kampstra, A.S.B.; Bakkum, T.; Marqvorsen, M.H.S.; ... ; Kasteren, S.I. van 2021
Proteolysis is fundamental to many biological processes. In the immune system, it underpins the activation of the adaptive immune response: degradation of antigenic material into short peptides and... Show moreProteolysis is fundamental to many biological processes. In the immune system, it underpins the activation of the adaptive immune response: degradation of antigenic material into short peptides and presentation thereof on major histocompatibility complexes, leads to activation of T-cells. This initiates the adaptive immune response against many pathogens. Studying proteolysis is difficult, as the oft-used polypeptide reporters are susceptible to proteolytic sequestration themselves. Here we present a new approach that allows the imaging of antigen proteolysis throughout the processing pathway in an unbiased manner. By incorporating bioorthogonal functionalities into the protein in place of methionines, antigens can be followed during degradation, whilst leaving reactive sidechains open to templated and non-templated post-translational modifications, such as citrullination and carbamylation. Using this approach, we followed and imaged the post-uptake fate of the commonly used antigen ovalbumin, as well as the post-translationally citrullinated and/or carbamylated auto-antigen vinculin in rheumatoid arthritis, revealing differences in antigen processing and presentation. 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
Kampstra, A.S.B.; Heemst, J. van; Janssen, G.M.; Ru, A.H. de; Lummel, M. van; Veelen, P.A. van; Toes, R.E.M. 2019
Human CD4+ T lymphocytes play an important role in inducing potent immune responses. T cells are activated and stimulated by peptides presented in human leucocyte antigen (HLA)-class II molecules.... Show moreHuman CD4+ T lymphocytes play an important role in inducing potent immune responses. T cells are activated and stimulated by peptides presented in human leucocyte antigen (HLA)-class II molecules. These HLA-class II molecules typically present peptides of between 12 and 20 amino acids in length. The region that interacts with the HLA molecule, designated as the peptide-binding core, is highly conserved in the residues which anchor the peptide to the molecule. In addition, as these peptides are the product of proteolytic cleavages, certain conserved residues may be expected at the N- and C-termini outside the binding core. To study whether similar conserved residues are present in different cell types, potentially harbouring different proteolytic enzymes, the ligandomes of HLA-DRB1*03:01/HLA-DRB>1 derived from two different cell types (dendritic cells and EBV-transformed B cells) were identified with mass spectrometry and the binding core and N- and C-terminal residues of a total of 16,568 peptides were analysed using the frequencies of the amino acids in the human proteome. Similar binding motifs were found as well as comparable conservations in the N- and C-terminal residues. Furthermore, the terminal conservations of these ligandomes were compared to the N- and C-terminal conservations of the ligandome acquired from dendritic cells homozygous for HLA-DRB1*04:01. Again, comparable conservations were evident with only minor differences. Taken together, these data show that there are conservations in the terminal residues of peptides, presumably the result of the activity of proteases involved in antigen processing. Show less
