Autoantibodies against post-translationally modified proteins (anti-modified protein antibodies or AMPAs) are a hallmark of rheumatoid arthritis (RA). A variety of classes of AMPAs against different ...modifications on proteins, such as citrullination, carbamylation and acetylation, have now been described in RA. At present, there is no conceptual framework explaining the concurrent presence or mutual relationship of different AMPA responses in RA. Here, we aimed to gain understanding of the co-occurrence of AMPA by postulating that the AMPA response shares a common 'background' that can evolve into different classes of AMPAs.
Mice were immunised with modified antigens and analysed for AMPA responses. In addition, reactivity of AMPA purified from patients with RA towards differently modified antigens was determined.
Immunisation with carbamylated proteins induced AMPAs recognising carbamylated proteins and also acetylated proteins. Similarly, acetylated proteins generated (autoreactive) AMPAs against other modifications as well. Analysis of anti-citrullinated protein antibodies from patients with RA revealed that these also display reactivity to acetylated and carbamylated antigens. Similarly, anti-carbamylated protein antibodies showed cross-reactivity against all three post-translational modifications.
Different AMPA responses can emerge from exposure to only a single type of modified protein. These findings indicate that different AMPA responses can originate from a common B-cell response that diversifies into multiple distinct AMPA responses and explain the presence of multiple AMPAs in RA, one of the hallmarks of the disease.
Abstract
Background
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.
Rheumatoid arthritis (RA) is a chronic autoimmune disease affecting 1% of the world population. RA is associated with the presence of autoantibodies, of which anti-citrullinated protein antibodies ...(ACPA) are most prominent. ACPA are produced by citrullinated antigen-binding B cells that have presumably survived tolerance checkpoints. So far, it is unclear how and when such autoreactive B cells emerge. Light chain (LC) rearrangement and mutation rates can be informative with regard to selection steps during B-cell development. Therefore, we studied LC characteristics of ACPA-expressing B cells and secreted ACPA with the aim to better understand the development of this disease-specific, autoreactive B-cell response. Paired ACPA-IgG and ACPA-depleted IgG were isolated from serum (n = 87) and synovial fluid (SF, n = 21) of patients with established RA. We determined the LC composition for each fraction by ELISA using kappa(Igκ)- and lambda(Igλ) LC-specific antibodies. Cellular LC expression was determined using flow cytometry. In addition, we used a B-cell receptor (BCR)-specific PCR to obtain LC variable region sequences of citrullinated antigen- and tetanus toxoid (TT)-binding B cells. In serum, we observed an increased frequency of lambda LC in ACPA-IgG (1.64:1) compared to control IgG (2.03:1) and to the κ/λ ratio reported for healthy individuals (2:1). A similar trend towards higher frequencies of lambda LCs was observed for ACPA-IgG in SF (1.84:1). Additionally, the percentage of Igλ-expressing B cells was higher for citrullinated antigen-binding B cells (51%) compared to TT-specific (43%) and total CD19+CD20+ B cells (36%). Moreover, an increased Igλ percentage was observed in BCR-sequences derived from ACPA-expressing (49%) compared to TT-specific B cells (34%). Taken together, we report an enhanced frequency of lambda LCs in the secreted ACPA-IgG repertoire and, on the cellular level, in BCR sequences of ACPA-expressing B cells compared to control. This skewing in the autoreactive B-cell repertoire could reflect a process of active selection.
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 ...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.
Many autoimmune diseases (AIDs) are characterized by the persistence of autoreactive B cell responses, which have been directly implicated in disease pathogenesis. How and why these cells are ...generated or how they are maintained for years is largely unknown. Rheumatoid arthritis (RA) is among the most common AIDs and is characterized by autoantibodies recognizing proteins with posttranslational modifications (PTMs). This PTM-directed autoreactive B cell compartment is ill defined. Here, we visualized the B cell response against the three main types of PTM antigens implicated in RA by spectral flow cytometry. Our results showed extensive cross-reactivity of PTM-directed B cells against all three PTM antigens (citrulline, homocitrulline, and acetyllysine). Unsupervised clustering revealed several distinct memory B cell (mBC) populations. PTM-directed cells clustered with the most recently activated class-switched mBC phenotype, with high CD80, low CD24, and low CD21 expression. Notably, patients also harbored large fractions of PTM-directed plasmablasts (PBs). Both PTM-directed mBCs and PBs showed high expression of CXCR3, a receptor for chemokines present in abundance in arthritic joints. Together, our data provide detailed insight into the biology of B cell autoreactivity and its remarkable, seemingly exhaustless persistence in a prominent human AID.
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