ObjectiveAutoantibodies 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.

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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 are hallmarked by autoreactive B and plasma cell responses that are directly or indirectly involved in disease pathogenesis. These B-cell responses show large variability between diseases, both in terms of the secreted autoantibody repertoire and the dynamics and characteristics of the underlying B-cell responses. Hence, different mechanisms have been proposed to explain the emergence of autoreactive B cells in an otherwise self-tolerant immune system. Notably, most mechanistic insights have been obtained from murine studies using models harboring genetic modifications of B and T cells. Given recent technological advances that have rendered autoreactive human B cells accessible for analysis, we here discuss the phenomenon of extensive N-glycosylation of the B-cell receptor (BCR) variable domain of a prototypic human autoreactive B-cell response and its potential role in the generation of autoimmunity. Anti-citrullinated protein antibodies (ACPA) hallmark the most disease-specific autoimmune response in Rheumatoid Arthritis (RA). Remarkably, ACPA-IgG are heavily N-glycosylated in the variable domain due to somatic mutations that generate abundant N-glycosylation consensus sequences. These sites, obtained from full-length BCR sequences of ACPA-expressing B cells from 12 ACPA-positive RA patients, were here analyzed in detail. Sites that required a single nucleotide mutation to be generated were defined as single somatic hypermutation (s-SHM) sites, whereas sites requiring multiple mutations were defined as m-SHM sites. IgG sequences of 12 healthy donors were used as control. Computational modeling of the germinal center reaction (CLONE algorithm) was used with the germline counterparts of ACPA-IgG heavy chain (HC) sequences to simulate the germinal center response. Our analyses revealed an abundance of N-glycosylation sites in ACPA-IgG HC that frequently required multiple mutations and predominated in specific positions. Based on these data, and taking into account recent insights into the dynamics of the ACPA-response during disease development, we here discuss the hypothesis that N-glycosylation sites in ACPA-IgG variable domains could lead to alternative, possibly antibody affinity-independent selection forces. Presumably, this occurs during germinal center responses allowing these B cells to escape from putative tolerance checkpoints, thereby driving autoreactive B cell development in the pathogenesis of RA.

Primary cutaneous follicle center lymphoma (PCFCL) is a rare mature B-cell lymphoma with an unknown etiology. PCFCL resembles follicular lymphoma (FL) by cytomorphologic and microarchitectural criteria. FL B cells are selected for N-linked glycosylation motifs in their B-cell receptors (BCRs) that are acquired during continuous somatic hypermutation. The stimulation of mannosylated BCR by lectins on the tumor microenvironment is therefore a candidate driver in FL pathogenesis. We investigated whether the same mechanism could play a role in PCFCL pathogenesis. Full-length functional variable, diversity, and joining gene sequences of 18 PCFCL and 8 primary cutaneous diffuse large B-cell lymphoma, leg-type were identified by unbiased Anchoring Reverse Transcription of Immunoglobulin Sequences and Amplification by Nested PCR and BCR reconstruction from RNA sequencing data. Low BCR variation demonstrated negligible ongoing somatic hypermutation in PCFCL and primary cutaneous diffuse large B-cell lymphoma, leg-type, and indicated that the PCFCL microarchitecture does not act as a functional germinal center. Similar to FL but in contrast to primary cutaneous diffuse large B-cell lymphoma, leg-type, BCR genes of 15 PCFCLs (83%) had acquired N-linked glycosylation motifs. These motifs were located at the BCR positions converted to N-linked glycosylation motifs in normal B-cell repertoires with low prevalence but mostly at different positions than those found in FL. The cutaneous localization of PCFCL might suggest a role for lectins from commensal skin bacteria in PCFCL lymphomagenesis.