Cross-reactivity of IgM anti-modified protein antibodies in rheumatoid arthritis despite limited mutational load

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.

Cross-reactivity of anti-modified protein antibodies is also present in predisease and individuals without rheumatoid arthritis

“…These results indicate that cross-reactivity towards different PTMs emerges when AMPA responses become detectable and provide evidence that cross-reactivity towards different PTMs is an intrinsic characteristic of AMPA responses. This finding is in line with the observation that (germline) ACPA-IgM can be cross-reactive towards other PTMs as well 6. Although cross-reactivity seems to be an intrinsic feature of AMPA, it is tempting to speculate that the most cross-reactive B cells are selected during progression towards RA, explaining the increase of the ACPA epitope recognition profile in time towards disease onset.…”

Anti-citrullinated protein antibodies with diverse specificities ameliorate collagen antibody-induced arthritis in a time-dependent manner

“…Thus, it is possible that the immune system generates “ACPA natural autoantibodies” that subsequently undergo T cell-promoted affinity maturation. In line with this, it has been reported that patient-derived, pentameric IgM ACPAs bind to a variety of post-translationally modified self-antigens, including citAgs, and that these properties are conserved within their germline ancestry 55 . Transgenic mice expressing IgM natural antibodies 56 , or mice injected with anti-dsDNA IgM antibodies 57 , were protected from nephritic damage in spontaneous models of lupus.…”

TLR7 and IgM: Dangerous Partners in Autoimmunity

“…This class of antibodies binds autoantigens and consequently affects its functions or leads to cell elimination [ 26 ]. Several recent studies have demonstrated that autoreactive IgM can be causative for autoimmune symptoms as anti-BP180 IgM leads to IgM pemphigoid [ 65 ], anti-PT IgM antibodies interfere with blood coagulation in severe COVID-19 patients [ 51 , 78 ], or AMPA-IgM as disease driver in RA [ 73 ]. In conclusion, these studies and others indicate that autoreactive IgM is capable of causing autoimmune diseases in the absence of IgG autoantibodies ( Figure 2 ).…”

“…Whether AMPA IgG require excessive somatic hypermutation (SHM) in order to bind modified proteins in a cross-reactive manner has been debated intensely. Interestingly, Reijm et al [ 73 ] have suggested that AMPA IgG might originate from AMPA IgM possessing germline configuration. The authors further show that AMPA IgM detected in RA patients can readily bind modified self-proteins and thus induce pathology.…”