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

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.

Dynamics of circulating TNF during adalimumab treatment using a drug-tolerant TNF assay.

Patients with rheumatoid arthritis (RA) can be successfully treated with tumor necrosis factor (TNF) inhibitors, including the monoclonal antibody adalimumab. Once in remission, a proportion of patients can successfully discontinue treatment, indicating that blocking TNF is no longer required for disease control. To explore the dynamics of circulating TNF during adalimumab treatment, we developed a competition enzyme-linked immunosorbent assay that can quantify TNF in the presence of large amounts of TNF inhibitor, i.e., a "drug-tolerant" assay. In 193 consecutive adalimumab-treated patients with RA, we demonstrated that circulating TNF increased in average of >50-fold upon treatment and reached a stable concentration in time for most patients. A similar increase in TNF was found in 30 healthy volunteers after one dose of adalimumab. This implies that TNF in circulation during anti-TNF treatment is not primarily associated with disease activity. During treatment, TNF was in complex with adalimumab and could be recovered as inactive 3:1 adalimumab-TNF complexes. No quantitative association was found between TNF and adalimumab concentrations. Low TNF concentrations at week 4 were associated with a higher frequency of antidrug antibodies (ADAs) at subsequent time points, less frequent methotrexate use at baseline, and less frequent remission after 52 weeks. Also in healthy volunteers, early low TNF concentrations are associated with ADAs. In conclusion, longitudinal TNF concentrations are mostly stable during adalimumab treatment and may therefore not predict successful treatment discontinuation. However, early low TNF is strongly associated with ADA formation and may be used as timely predictor of nonresponse toward adalimumab treatment.