Philip J. Titcombe

B cell tolerance to self-antigen is critical to preventing antibody-mediated autoimmunity. Previous work using B cell antigen receptor transgenic animals suggested that self-antigen-specific B cells are either deleted from the repertoire, enter a state of diminished function termed anergy, or are ignorant to the presence of self-antigen. These mechanisms have not been assessed in a normal polyclonal repertoire because of an inability to detect rare antigen-specific B cells. Using a novel detection and enrichment strategy to assess polyclonal self-antigen-specific B cells, we find no evidence of deletion or anergy of cells specific for antigen not bound to membrane, and tolerance to these types of antigens appears to be largely maintained by the absence of T cell help. In contrast, a combination of deleting cells expressing receptors with high affinity for antigen with anergy of the undeleted lower affinity cells maintains tolerance to ubiquitous membrane-bound self-antigens.

OBJECTIVE: Antibodies against posttranslationally modified proteins are a hallmark of rheumatoid arthritis (RA), but the emergence and pathogenicity of these autoantibodies are still incompletely understood. The aim of this study was to analyze the antigen specificities and mutation patterns of monoclonal antibodies (mAb) derived from RA synovial plasma cells and address the question of antigen cross-reactivity. METHODS: IgG-secreting cells were isolated from RA synovial fluid, and the variable regions of the immunoglobulins were sequenced (n = 182) and expressed in full-length mAb (n = 93) and also as germline-reverted versions. The patterns of reactivity with 53,019 citrullinated peptides and 49,211 carbamylated peptides and the potential of the mAb to promote osteoclastogenesis were investigated. RESULTS: Four unrelated anti-citrullinated protein autoantibodies (ACPAs), of which one was clonally expanded, were identified and found to be highly somatically mutated in the synovial fluid of a patient with RA. The ACPAs recognized >3,000 unique peptides modified by either citrullination or carbamylation. This highly multireactive autoantibody feature was replicated for Ig sequences derived from B cells from the peripheral blood of other RA patients. The plasma cell-derived mAb were found to target distinct amino acid motifs and partially overlapping protein targets. They also conveyed different effector functions as revealed in an osteoclast activation assay. CONCLUSION: These findings suggest that the high level of cross-reactivity among RA autoreactive B cells is the result of different antigen encounters, possibly at different sites and at different time points. This is consistent with the notion that RA is initiated in one context, such as in the mucosal organs, and thereafter targets other sites, such as the joints.

OBJECTIVE: Anti-citrullinated protein antibodies (ACPAs) have proven highly useful as biomarkers for rheumatoid arthritis (RA). However, composition and functionality of the associated autoreactive B cell repertoire have not been directly assessed. We aimed to selectively investigate citrullinated autoantigen-specific B cell receptors (BCRs) involved in RA and initiate studies on their pathogenicity. METHODS: Blood samples were obtained from patients in a University of Minnesota cohort with ACPA-positive RA (n = 89). Tetramer sets bearing citrullinated filaggrin peptide cfc1 or citrullinated α-enolase peptide were constructed to specifically capture autoreactive B cells from the unaltered, polyclonal repertoire in RA patients. Citrullinated peptide tetramer-bound B cells were subjected to flow cytometric cell sorting and single-cell IGH, IGK, and IGL gene sequencing for B cell lineage determinations. BCR gene sequences were also expressed as recombinant monoclonal antibodies (mAb) for direct evaluation of citrullinated autoantigen binding and effector functionality. RESULTS: Using citrullinated peptide tetramer enrichment to investigate single autoreactive blood B cells, we identified biased V-region gene usage and conserved junction arrangements in BCRs from RA patients. Parsimonious clustering of related immunoglobulin gene nucleotide sequences revealed clonal expansions of rare individual B cell clades, in parallel with divergent sequence mutations. Correspondingly, recombinant mAb generated from such BCR lineages demonstrated citrulline-dependent cross-reactivity extending beyond the citrullinated peptides used for B cell capture. A pair of citrullinated autoantigen-specific mAb with cross-reactive binding profiles also promoted arthritis in mice. CONCLUSION: Our findings suggest that broad ACPA specificities in RA arise from a restricted repertoire of evolving citrulline-multispecific B cell clades with pathogenic potential.

OBJECTIVE: Anti-citrullinated protein antibodies (ACPAs) are a hallmark of seropositive rheumatoid arthritis (RA). Yet, the precise disease-relevant autoantigens that are targeted by ACPAs remains a matter of debate. This study utilized patient-derived monoclonal ACPAs, rather than serum autoantibody analysis, to characterize the multireactivity to different protein modifications and to reveal autoantibody subsets in patients with RA. METHODS: Twelve human monoclonal ACPAs (positive by the second-generation cyclic citrullinated peptide test) were generated from 6 RA patients, and a head-to-head comparison of their reactivities was performed. For profiling, we used a complementary DNA-based protein array (Engine GmbH) and 3 peptide-screening platforms with RA autoantigens (Thermo Fisher Scientific), citrullinated and carbamylated peptides (NimbleGen/Roche), or histone-derived peptides with different posttranslational modifications (JPT Histone Code), covering >207,000 peptides (>7,800 gene products). RESULTS: The fine-specificity profiles of the investigated ACPAs varied, but all of the monoclonal ACPAs displayed multireactivity to a large number of citrullinated peptides/proteins, each characterized by specific binding properties. ACPA subsets could be defined by clone-distinct consensus binding motifs (e.g., Cit-Gly, Gly-Cit, or Arg-Cit-Asp), with the most common ACPA recognition being that of a Gly in the +1 flanking position, but with additional amino acid preferences. For ACPA protein recognition, we observed a preference for citrullinated RNA-binding proteins with high Arg/Gly content. Six of the 12 ACPA clones also bound acetylated lysine (KAc) or homocitrulline peptide motifs, displaying a similar affinity or higher apparent affinity than that for citrullinated peptides. CONCLUSION: ACPAs and anti-modified protein autoantibodies represent overlapping facets of RA autoimmunity and bind to a wide variety of modified proteins, extending well beyond the historically recognized set of RA autoantigens. So far, KAc reactivity has been detected only in the context of anti-carbamylated and anti-citrullinated peptide autoantibody responses, postulating the existence of hierarchies of autoreactivity in RA. Future investigations of ACPA fine specificities and functionality should take into consideration the presence of consensus Cit/Carb/KAc motifs and the multireactivity of these autoantibodies in patients with RA.

The clinical efficacy of B cell targeting therapies highlights the pathogenic potential of B cells in inflammatory diseases. Expression of Fc Receptor like 4 (FcRL4) identifies a memory B cell subset, which is enriched in the joints of patients with rheumatoid arthritis (RA) and in mucosa-associated lymphoid tissue. The high level of RANKL production by this B cell subset indicates a unique pathogenic role. In addition, recent work has identified a role for FcRL4 as an IgA receptor, suggesting a potential function in mucosal immunity. Here, the contribution of FcRL4+ B cells to the specific autoimmune response in the joints of patients with RA was investigated. Single FcRL4+ and FcRL4- B cells were sorted from synovial fluid and tissue from RA patients and their immunoglobulin genes characterized. Levels of hypermutation in the variable regions in both populations were largely consistent with memory B cells selected by an antigen- and T cell-dependent process. Recombinant antibodies were generated based on the IgH and IgL variable region sequences and investigated for antigen specificity. A significantly larger proportion of the recombinant antibodies generated from individual synovial FcRL4+ B cells showed reactivity towards citrullinated autoantigens. Furthermore, both in analyses based on heavy chain sequences and flow cytometric detection, FcRL4+ B cells have significantly increased usage of the IgA isotype. Their low level of expression of immunoglobulin and plasma cell differentiation genes does not suggest current antibody secretion. We conclude that these activated B cells are a component of the local autoimmune response, and through their RANKL expression, can contribute to joint destruction. Furthermore, their expression of FcRL4 and their enrichment in the IgA isotype points towards a potential role for these cells in the link between mucosal and joint inflammation.