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Numerous autoimmune diseases are characterised by the presence of autoantibodies, some of which help in diagnosis, others of which contribute to pathogenesis. In many of these diseases, B cell targeted therapies are of benefit. This has led to intensive exploration of B cell subset and mechanisms of B cell activation, yet studies of B cell repertoire in health and disease are still in their infancy. In this issue of the journal, Ota et al report on B cell receptor (BCR) heavy chain sequences from 595 donors, from individuals with autoimmune diseases and healthy donors.1 The BCR sequences were mapped from bulk RNA-sequencing data of isolated B cell subsets.
The mechanisms that lead to the generation of a productive heavy chain and light chain in each B cell allow a limited amount of DNA to yield an estimated 1018 different antibodies.2 3 This inherent diversity of BCRs has made their study challenging. To accurately understand an individual’s BCR repertoire, large numbers of cells from diverse B cell subpopulations must be analysed. Moreover, in humans, the only readily available tissue is blood. Despite these limitations, recent advances in sequencing technology and bioinformatics have allowed for a better examination of BCR repertoires in disease states.
What does one learn from such studies? Random rearrangement of gene segments generates a high frequency of BCRs capable of recognising self-antigens. Central tolerance mechanisms like receptor editing and apoptosis operate to reduce the frequency of autoreactive B cells in the mature B cell repertoire. Receptor editing involves immunoglobulin light chain gene recombination. This rearrangement event replaces one light chain with another, modifying antigenic specificity and leading to a non-autoreactive B cell (reviewed in reference 4). Notwithstanding the existence of these mechanisms, a significant percentage of circulating mature B cells remains autoreactive.5 6 Some autoreactive B cells contribute to immune homeostasis by producing IgM autoantibodies which clear apoptotic debris,7 while others have the potential to become pathogenic. An analysis of BCRs in B cell subsets representing different stages of B cell maturation allows one to assess early tolerance checkpoints.
Understanding the frequency and sequence specificity of somatic hypermutation (SHM) in antigen-activated B cells may reveal pathways to production of IgG expressing (class switched memory) B cells, and plasmablasts, which can occur through either an extrafollicular (EF) or a germinal centre (GC) pathway.8 It is generally accepted that although SHM can occur outside of the GC, the accumulation of SHM in plasma cells produced as part of an EF response is less than what is seen in plasmablasts from a GC response; thus, it is possible to impute differentiation pathways based on the frequency of SHM. Of note, SHM can cause autoreactive B cells to mutate away from autoreactivity,9 but can also cause non-autoreactive BCRs to become autoreactive. Evidence suggests that the latter process may occur in autoimmune diseases to generate autoreactive B cells.10 11 Interestingly, early studies of hybridomas, obtained from anti-dsDNA antibodies from systemic lupus erythematosus (SLE) patients, showed a high rate of mutation, suggesting a GC origin.12 On the other hand, a fast reduction in the titres of anti-dsDNA antibodies after treatment with rituximab13 suggests an EF origin of the plasma cells producing these antibodies. Precise analysis of SHM of autoreactive B cells may help in understanding selection and proliferation of autoreactive clones with pathogenic potential. For example, peripheral tolerance checkpoints prevent autoreactive GC B cells from differentiating into class-switched plasmablasts6 14 15; less is known about tolerance checkpoints for plasmablasts generated through an EF pathway.
The paper by Ota et al 1 presents evidence that the usage of different V(D)J segments differs across B cell subsets. This was observed in both healthy individuals and individuals with autoimmune disease. This observation highlights the importance of studying BCR sequencing in specific and carefully selected B cell subpopulations. One limitation of the current study is the lack of separation between transitional B cells and mature naïve B cells. Analyses of mixed subsets can interfere with the interpretation of results, as it is not possible to discriminate if changes are due to differences in the composition of each subset, changes within a particular subset or changes in the frequency of each subset.
In this study, the authors also observed reduced CDR3 length in naïve B cells of patients with autoimmune disease, associated with an interferon signature, without being able to determine the mechanistic cause of this alteration. Single-cell RNA sequencing is a promising tool for simultaneously determining the full transcriptome and BCR sequence of a B cell, however, it is currently limited to a few thousand cells per population and few individuals. Future improvements in this area may allow for a more extensive analysis of single B cells. Alternative approaches, such as independently running BCR sequencing and transcriptomic analyses, can still provide valuable insights into B cell biology, as demonstrated by Ota et al but it has some limitations, as it is not clear if exactly the same cell or a specific subset within the population present any given alteration, additionally, is not possible to study paired heavy and light chains.
It is also important to link BCR sequence to antigenic specificity. Currently, this can be done by isolating B cells of a known antigenic specificity prior to obtaining BCR sequences or when paired heavy and light chain sequences are available by employing antibody expression systems, which are both labour-intensive and time-consuming or by computational methods. One indirect method to identify autoreactive clones is through the analysis of antibodies encoded by IGVH4-34, specifically the 9G4 idiotype. Ota et al found increased IGHV4-34 usage and an increased fraction of sequences with amino acid motifs associated with the 9G4 idiotype. In healthy individuals, B cells of the 9G4 idiotype are excluded from GC reactions.16 Based on this observation, it is tempting to speculate that increased usage of 9G4 represents altered pathways of maturation in patients with autoimmune diseases. Identification of autoreactive sequences remains challenging, but it is a crucial step to deepen our understanding of the selection, expansion and regulation of autoreactive B cells in autoimmune diseases.
Understanding the effect of immunosuppressive drugs or biological therapeutics on the B cell repertoire is still limited, although there are reports suggesting differential effects on B cell composition and repertoire for different treatments. For example, mycophenolate and rituximab differentially affect B cell composition and repertoire; B cells that persist after rituximab are predominately isotype switched and clonally expanded.17 There is also limited information about changes in the repertoire associated with inflammatory cytokines levels. Decreased B cell activation threshold, induced by proinflammatory cytokines, might lead to greater diversity of expanded clones. These are all questions that can be explored through BCR sequence analysis.
There is still much to learn about the complex interactions between autoimmune diseases, inflammatory states, treatments and alterations in the B cell repertoire. Integrative papers like the one published by Ota et al in this issue and ongoing improvements in sequencing and computational technologies will undoubtedly contribute to our understanding of these intricate biological processes.
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References
Footnotes
Handling editor Josef S Smolen
Contributors YA-F and BD contributed to the planning and to the writing of the manuscript.
Funding This work was supported by the NIH grant: Heterogeneous pathways to autoantibody production: Implications for prognosis and therapeutic targeting; NIH U19 AI144306
Competing interests None declared.
Provenance and peer review Commissioned; externally peer reviewed.