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# Autoreactive B cells in autoimmune diseases: Mechanisms, functions and clinical implications

Aurore Collet, Thomas Guerrier, Sebastien Sanges, Aurelien Chepy, Vincent Sobanski, David Launay, Sylvain Dubucquoi

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## To cite this version:

Aurore Collet, Thomas Guerrier, Sebastien Sanges, Aurelien Chepy, Vincent Sobanski, et al., Autoreactive B cells in autoimmune diseases: Mechanisms, functions and clinical implications. Autoimmunity Reviews, 2025, Autoimmunity Reviews, 24 (8), pp.103851. 10.1016/j.autrev.2025.103851. hal-05302210

HAL Id: hal-05302210

https://lilloa.hal.science/hal-05302210v1

Submitted on 7 Oct 2025

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Review

# Autoreactive B cells in autoimmune diseases: Mechanisms, functions and clinical implications

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Aurore Collet<sup>a,b,*</sup>, Thomas Guerrier<sup>a</sup>, Sébastien Sanges<sup>a,c</sup>, Aurélien Chépy<sup>a,c</sup>, Vincent Sobanski<sup>a,c,d</sup>, David Launay<sup>a,c,1</sup>, Sylvain Dubucquoi<sup>a,b,1</sup>

ARTICLE INFO

Keywords:

Autoreactive B cell

Autoimmune disease

Autoantibody

Tolerance

A B S T R A C T

Autoreactive B cells play a key role in the pathophysiology of autoimmune diseases (AIDs). Still, due to their low frequency in the circulating blood, they are less well characterized than the global B cell pool. This review aims at describing the tools that allow the study of autoreactive B cells, deciphering their features and functions, and discussing the therapeutic implications that arise from these findings.

The capacity to detect and analyze autoreactive B cells has been significantly improved by diverse techniques such as ELISpot and flow cytometry, shedding light on their roles in immune dysregulation. It reveals the multifaceted features of autoreactive B cells in terms of phenotypic and functional characteristics, that vary between different AIDs, and from one autoantigen to another. This heterogeneity may be influenced by factors such as the nature of the targeted autoantigen, or the costimulatory signals involved. These observations highlight that autoreactive B cells contribute not only to autoantibody production, but also to the perpetuation of autoimmunity through additional mechanisms, including antigen presentation and cytokine secretion. Recent therapeutic approaches have been developed to target autoreactive B cells, allowing an antigen-specific depletion of B cells, without causing widespread immunosuppression. Challenges remain, such as understanding the precise mechanisms by which the B cell tolerance breakdown occur, and the pathways by which autoreactive B cells activate (i.e. germinal centre or extrafollicular pathway). Ongoing research into the mechanisms regulating autoreactive B cells will be crucial for designing more targeted and effective therapies, leading to better outcomes for AID patients.

1. Introduction

Autoimmune diseases (AIDs) arise when immunological tolerance is

compromised, leading the immune system to erroneously target self-tissues. A central driver of this breakdown is the emergence and persistence of autoreactive B cells, which recognize and respond to self-

Abbreviations: aAb, autoantibody; Ab, antibody; ACPA, anti-CCP autoantibody; ADCC, Antibody-dependent cellular cytotoxicity; AID, Autoimmune disease; ANA, anti-nuclear antibody; APC, antigen presenting cells; APS, anti-phospholipid syndrome; ASC, antibody-secreting cell; BCR, B cell receptor; BiAATEs, Bispecific AutoAntigen-T cell Engagers; CAAR-T cell, chimeric autoantibody receptor T cell; CCP, cyclic-citrulinated peptides; CDC, Complement-dependent cytotoxicity; CITE-seq, Cellular Indexing of Transcriptomes and Epitopes by sequencing; COL-II, type-II collagen; DN, double negative; Dsg3, desmoglein 3; EAE, experimental autoimmune encephalomyelitis; ER, endoplasmic reticulum; ELISpot, Enzyme-Linked ImmunoSpot; ENA, extractable nuclear antigens; FITC, fluorescein isothiocyanate; GC, germinal center; HA, hemagglutinin; HC, healthy control; ILD, interstitial lung disease; LIBRAseq, Linking B cell Receptor to Antigen Specificity through Sequencing; MBC, memory B cell; PB, plasmablast; PC, plasma cell; PTM, post-translational modification; PV, pemphigus vulgaris; RA, rheumatoid arthritis; RF, rheumatoid factor; RTX, rituximab; scRNA-seq, single-cell RNA sequencing; SHM, somatic hypermutation; SLA, soluble liver antigen; SLE, systemic lupus erythematosus; SS, Sjögren's syndrome; T1D, type-1 diabetes; Topo, topoisomerase 1; TT, tetanus-toxoid.

<footnote><sup>a</sup> Univ. Lille, Inserm, CHU Lille, U1286 – INFINITE – Institute for Translational Research in Inflammation, Lille, France</footnote>

<footnote><sup>b</sup> CHU Lille, Institut d'Immunologie, Lille, France</footnote>

<footnote><sup>c</sup> CHU Lille, Département de Médecine interne et Immunologie Clinique, Centre de Référence des Maladies Auto-immunes Systémiques Rares du Nord et Nord-Ouest, Méditerranée et Guadeloupe (CeRAINOM), Lille, France</footnote>

<footnote><sup>d</sup> Institut Universitaire de France (IUF), Paris, France</footnote>

<footnote>* Corresponding author at: CHU de Lille, Centre de Biologie Pathologie Génétique, Institut d'Immunologie, Rue du Pr Jules Leclercq, 59037 Lille Cedex, France.
E-mail address: aurore.collet@chu-lille.fr (A. Collet).</footnote>

<footnote n="1">¹ Contributed equally.</footnote>
antigens (autoantigens). Under normal physiological conditions, central and peripheral tolerance mechanisms — including clonal deletion, re-ceptor editing, and anergy — actively eliminate or inactivate autor-active B cells to prevent autoantibody (aAb) production. In the context of AIDs, these regulatory checkpoints are dysregulated or defective, permitting the survival, activation, and differentiation of autoreactive B cells, which ultimately contribute to pathogenic autoantibody genera-tion and tissue damage [1]. Autoreactive B cells contribute to AIDs through multiple mechanisms, both dependent and independent of Aab production (see Box 1). Thus, B cells — and autoreactive B cells in particular — represent key therapeutic targets in the development of treatments for AIDs [2,3].

Hence, the study of autoreactive B cells, particularly their mecha-nisms of emergence and pathogenic functions, represents a rapidly evolving area of research. However, frequency of each autoantigen-specific B cell is typically very low in the circulation, complicating their detection and characterization [4]. Despite these technical chal-lenges, significant advancements have been made in identifying and characterizing autoreactive B cells. During the late 2000s, the devel-opment of flow cytometry techniques has enabled the detection and sorting of antigen-specific B cells based on their recognition of a labelled antigen [4]. Since then, research efforts have increasingly focused on the precise characterization of autoreactive B cells across various AIDs. With recent technological advances, it is now possible to study autoreactive B cells at the single-cell level, enabling a more complete understanding of their phenotypic and functional diversity [5,6]. Deciphering the char-acteristics and roles of autoreactive B cells in AIDs is crucial for devel-oping biomarkers and targeted therapies. Indeed, the number or the activation state of autoreactive B cells could help to predict disease severity [7] or response to treatment [8]. Moreover, current treatments, such as anti-CD20 or anti-CD19 monoclonal antibodies and anti-CD19 CAR-T cells [2], that target B cells broadly, have shown promise in reducing disease activity and improving clinical outcomes [9]. Howev-er, infectious events associated with these depleting strategies [10] also underscore the need for more specific therapies that target only the pathogenic autoreactive B cells while sparing the remainder of the B cell compartment [3].

In this narrative review, we aim to summarize the current knowledge on autoreactive B cells in AIDs. We will only focus on the character-ization of autoantigen-specific (i.e. autoreactive) B cells), and not on the abnormalities observed in the global B cell pool. We will first discuss the technical tools currently available to identify and characterize autor-active B cells, and their respective advantages and limitations. Then, we will detail the results of the studies exploring the autoreactive B cells emergence mechanisms, phenotype, function, and contribution to dis-ease pathology, taking the example of various AIDs. Thirdly, the

potential application of autoreactive B cells as biomarkers and thera- peutic targets will be reviewed. Finally, we will discuss the remaining unresolved topics, such as the possible different mechanisms of emer-gence or survival of autoreactive B cells between distinct AIDs. By elucidating these aspects, we hope to provide a comprehensive under-standing of the complex interplay between autoreactive B cells and AIDs pathophysiology, paving the way for improved therapeutic interventions.

## 2. Techniques to identify, characterize and isolate autoreactive B cells in blood and tissues

### 2.1. Identifying and/or isolating autoreactive B cells

There are two main ways to identify autoreactive B cells: either (i) by their ability to secrete aAbs, by ELISpot, or (ii) by detecting an autor-active B cell receptor (BCR) on their surface using a labelled auto-antigen, by flow cytometry (Fig. 1).

ELISpot (Enzyme-Linked ImmunoSpot) is a technique used to detect antibody-secreting cells (ASCs), including autoreactive B cells. Its technical principle is detailed in Table 1. While it is a sensitive and precise method for detecting autoreactive B cells, it has several limita-tions. One major drawback is its inability to provide information about the characteristics of the cells producing the aAbs, as the method focuses solely on the secretion function. Once the spots are quantified on the plate, the cells are no longer available for downstream analyses. Another limitation is that it only permits to detect B cells that secrete antibodies, in other words plasmablasts (PB) and plasma cells (PC), which are rare in the circulating blood. One way to circumvent this issue is to increase the ASC number by pre-activating B cells before incubation in the ELI-Spot plate. For example, culture with R848 (a toll-like receptor 7/8 agonist) and IL-2 induces the differentiation of memory B cells (MBCs) into ASCs [11]. Finally, the interpretation of results can be subjective, as the detection and counting of spots can vary between operators, potentially leading to variability in the data.

On the other hand, a comparative advantage over flow cytometry is the lack of necessity to use a pre-labelled antigen, making this technique more accessible for rare autoantigens for which labelled versions are not commercially available.

**Flow cytometry** is the most used technique to identify and sort autoreactive B cells. It is based on the recognition of a labelled auto-antigen by the autoreactive BCR at the surface of the B cells. Thus, it requires to have access to an autoantigen labelled (directly or indirectly) with a fluorochrome.

### Box 1

Potential roles of B cells in autoimmune diseases.

Autoreactive B cells can play multifaceted roles in autoimmunity, through both Ab-dependent and Ab-independent mechanisms.

**Ab-dependent mechanisms:**

When their target is an extracellular receptor, aAb can act as either agonists or antagonists, thereby altering the function of the target cell (such as anti-acetylcholine receptor aAb in *myasthenia gravis*). Autoantibody binding to cell surface targets can also induce cytotoxicity via Complement-dependent cytotoxicity (CDC) or Antibody-dependent cellular cytotoxicity (ADCC). Additionally, autoantibodies can function as opsonins, coating cells and marking them for phagocytosis. Finally, aAb can form immune complexes with autoantigens, which can deposit in various tissues, triggering inflammation and tissue damage.

**Ab-independent mechanisms:**

Autoreactive B cells can act as APC (antigen presenting cells), presenting autoantigens to T cells and thereby amplifying the autoimmune response. They can also produce pro-inflammatory cytokines, such as IL-6 and TNF-α. On the contrary, a subset known as regulatory B cells contributes to immune tolerance by producing anti-inflammatory cytokines such as IL-10, TGF-β, and IL-35. These cells help suppress excessive immune responses, limit autoimmunity, and promote tolerance to autoantigens.
<figure><img src="image_6.png" /><figcaption>**Fig. 1.** Available techniques to identify and characterize autoreactive B cells.</figcaption></figure>

Abbreviations: aAb: autoantibody; aAg: autoantigen; CITEseq: Cellular Indexing of Transcriptomes and Epitopes by sequencing; LIBRAseq: Linking B cell Receptor to Antigen Specificity through Sequencing; scRNAseq: single cell RNA sequencing; NGS: next generation sequencing.

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* Most frequently, biotinylated autoantigens are incubated with fluorochrome-conjugated streptavidin, generating **labelled auto-antigen tetramers** [14]. Antigen biotinylation can be made by a chemical method, at multiple and non-specific sites of the protein. The risk of this approach is to generate aggregates when adding streptavidin, if the ratio biotin/antigen is >1:1. Another way is an

enzymatic method, using the *E. coli* biotin ligase enzyme (BirA), that covalently attaches one molecule of biotin to an AviTag peptide added genetically at a specific site of the antigen [15]. A derivative of this tetramer technique is to increase the number of autoantigens attached to the fluorochrome, thus maximizing the avidity of the autoreactive B cells to their target. The **Klickmer®** (Immudex®)
<table><caption>**Table 1**

Characteristics of the different techniques permitting to isolate autoreactive B cells.</caption><thead><tr><th>Technique</th><th>Technical principle</th><th>Advantages</th><th>Limitations</th></tr></thead><tbody><tr><td>ELISpot</td><td>Culture plates pre-coated with a specific autoantigen PBMCs incubated for a defined period (24 to 48 h) → aAg-specific ASCs secrete aAbs that bind to the autoantigens coated at the bottom of the wells. Washing to remove the cells and debris Adding secondary anti-Ig antibody conjugated to an enzyme and chromogenic substrate Formation of a colored precipitate, creating visible spots. Analysis using an ELISpot reader or a microscope: each spot corresponds to a single ASC [12,13].</td><td>Quantitative High sensitivity No need for specific equipment or specific technical skills No need for a labelled aAg</td><td>Cells not available for downstream analysis Limited to aAb production analysis, does not provide a full profile of autoreactive B cells. Only detect ASCs, unless B cells are pre-activated Subjectivity in counting spots Time-consuming</td></tr><tr><td>Flow cytometry</td><td>Recognition of a fluorescently labelled autoantigen by the autoreactive BCR at the surface of the B cells</td><td>Quantitative Provide information about the characteristics of B cells (phenotype, activation status ...)<br>Possibility to sort autoreactive B cells<br>Possibility to improve sensitivity by B-cell enrichment</td><td>Limited number of fluorochromes in the same panel<br>Labelled and well-conformed aAg needed<br>Possible lack of specificity: possible false positive due to B cells binding fluorochromes or streptavidin</td></tr><tr><td>DNAbarcoding (LIBRAseq)</td><td>Oligonucleotid-labelled autoantigens recognized by autoreactive B cells, followed by NGS sequencing</td><td>Theoretical unlimited number of markers and thus of aAg studied at one time Comprehensive characterization of the cells of coupled to scRNAseq and CITEseq (antigenic specificity, surface markers, BCR characteristics, transcriptomic program)</td><td>Cost<br>Labelled and well-conformed aAg needed<br>Require advanced skills and infrastructure for analysis (sequencing, bioinformatics)</td></tr></tbody></table>

aAg: autoantigen; ASCs: antibody-secreting cells; CITEseq: Cellular Indexing of Transcriptomes and Epitopes by sequencing; LIBRAseq: Linking B cell Receptor to Antigen Specificity through Sequencing; PBMCs: Peripheral Blood Mononuclear Cells; scRNAseq: single cell RNA sequencing.

technology consists of a fluorochrome-labelled dextran backbone, carrying a defined number of acceptor sites for mono-biotinylated ligands, such as autoantigens [16]. Kristyanko and colleagues also proposed an alternative technique by using multivalent polymeric

scaffolds called **polyisocyanopeptides**, coupled to around 50 bio-tinylated antigens [17].

* Beside the biotinylation of antigens, the use of the **histidine tag** is another widely used method to detect a protein. It is a short sequence of 6 to 10 histidine residues, typically placed at either the N-terminus or C-terminus of a target protein. Autoreactive B cells that express a specific BCR for the tagged autoantigen can be identified using fluorescent antibodies against the histidine tag [5].

* The **sortase tag** is a method used for the specific conjugation of antigens with fluorochromes or other labeling molecules. The antigen of interest is genetically engineered to include a specific aminoacid sequence that is recognized by the sortase enzyme. A fluorochrome (or other labeling molecule, such as a biotin) is prepared with a reactive group that can form a covalent bond with the sortase tag. The sortase enzyme (usually sortase A) is used to catalyze the ligation reaction. This enzyme recognizes the sortase tag sequence and facilitates the covalent attachment of the fluorochrome-labelled molecule to the antigen [18]. The main advantage of this technique is that it permits to attach a single fluorochrome, at one defined site of the antigen, and with a very good yield.

Besides classical cytometric techniques, recent studies use oligonucleotide-labelled antigens. This technology is sometimes called « LIBRAseq » for Linking B cell Receptor to Antigen Specificity through Sequencing, when coupled to the sequencing of the BCR variable region [19]. This is a Next Generation Sequencing (NGS)-based readout for BCR-antigen binding interactions that utilizes oligonucleotides conjugated to recombinant antigens. It allows to multiplex the study of several autoantigens at one time, and at the single-cell level.

Still, identifying autoreactive B cells by their recognition of an autoantigen poses several **technical challenges**.

First, autoreactive B cells are often present at very low frequencies (for example, as low as 0.01 % in [20]) within the total B cell population. This requires highly sensitive and specific techniques to isolate and study these cells effectively. Some methods can be used to improve the sensitivity of the technique for the detection of these rare populations, based on the enrichment of the autoreactive B cell prior to flow cytometry, for example with magnetic nanoparticles conjugated to antibodies targeting the fluorochrome on the antigen of interest [21].

Secondly, ensuring that these methods can specifically identify autoreactive B cells without cross-reacting with non-autoreactive cells is challenging. It is important to note that some B cells may bind to fluorochromes or to streptavidin, leading to false-positive [22]. To alleviate this bias, dual labelling of the same antigen with 2 different fluorochromes, and with “decoy tetramers” is necessary. The antigen used to identify autoreactive B cells can also bind to other surface molecules besides the BCR of autoreactive B cells, leading to non-specific staining (for example, topoisomerase 1 [topo] could bind CCR7 [23] or toll-like receptors [24] on other cells besides anti-topo B cells). Moreover, rigorous negative and positive controls and validation steps (e.g. sort the B cells identified as autoreactive and verify that their specific aAb bind to their respective autoantigen) are necessary to ensure the accuracy of autoreactive B cell identification.

Thirdly, autoreactive B cells are identified by their ability to bind to autoantigens. However, presenting native, conformational epitopes of autoantigens in an experimental setting can be difficult. Misfolded or denatured antigens may not be recognized by autoreactive B cell receptors, leading to false negatives. The label used to detect autoantigen (e.g. histidine tag) can also interfere with the structure of the target protein.

Fourthly, BCR expression at the surface of PB and PC is weak, making it difficult to identify this B cell population via their recognition of an autoantigen. Permeabilization and intracellular staining could improve their detection [1]. Pinder et al. also described a method used to assess antigen-specific ASCs by capturing Ab on the cell surface, combining the
advantages of both flow cytometry and ELISpot. Streptavidin-conjugated anti-CD45 is used as the base for the scaffold complex, and biotinylated anti-IgG Ab is added to capture the IgG Ab secreted by the ASCs. Then, a GFP-conjugated antigen is added to detect antigen-specific IgG [25].

Finally, the diversity of BCRs complicates both the identification process of autoreactive B cells and the interpretation of its clinical relevance. Autoreactive BCRs identified in vitro can vary greatly in their affinity and specificity for self-antigens, requiring a broad range of detection assays to capture the full spectrum of autoreactivity. With techniques that use multimerized autoantigens, it is likely that the identified autoreactive B cells are both of low- and high-affinity. It is important to note that the ability of a B cell to recognize a self-antigen in vitro does not necessarily mean that it plays a pathogenic role in vivo. It is possible that the low-affinity autoreactive B cells do not have the same relevance and/or functions as their high-affinity counterpart (i.e. in vitro-identified autoreactive B cells could be ignorant or have a regulatory function in vivo) [26]. In the future, findings about in vitro-identified autoreactive B cells should be validated by in vivo studies (e.g. by transferring these cells into mice) to fully assess their pathological relevance.

## 2.2. Characterizing autoreactive B cells

Once the autoreactive B cells have been identified, several techniques can be used to further characterize them.

Fluorochrome-based flow cytometry is the most commonly used technique to characterize autoreactive B cells based on their **surface markers**. Other techniques deriving from flow cytometry are also useful to gain further insight on their membrane phenotype.

- Mass cytometry (or CyTOF) is a technology that combines flow cytometry and mass spectrometry, in which antibodies used to label cells are conjugated to heavy metal isotopes that are identified using time-of-flight mass spectrometry. It enables the design of a high dimensional panel up to 50 parameters without spillover. Given that currently there are no antigens coupled to heavy metals, it is not possible to directly use mass spectrometry to identify autoreactive B cells. However, *Stensland* and *Smith* have described a method to enrich a sample in autoreactive B cells based on their antigenic specificity, using magnetic beads that recognize biotinylated antigens. After cleaving the magnetic beads, the cells can then be conventionally analyzed by mass cytometry [27].

- CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by sequencing) is a technique that combines single-cell RNA sequencing (scRNA-seq) with surface protein detection. Cells are labelled with antibodies targeting CD markers and are conjugated to unique DNA oligonucleotides. These oligonucleotides serve as barcodes for the proteins they target. After the oligonucleotide-conjugated antibodies bind to their specific targets on the cell surface, the cells are subjected to scRNAsEq. [28]. As the number of different oligonucleotides is unlimited, this technique can allow the study of more than a hundred surface markers at one time.

Another way to characterize autoreactive B cells is to study their **gene expression program**. Once the cells of interest are isolated, all the different transcriptomic approaches, such as micro-array, qPCR, RNAseq and scRNAseq [29,30] are available. The best method is determined depending on the specific research goals (pre-defined target genes or unbiased approach, high throughput requirement or not), available resources (both in terms of cost and analysis complexity), and the level of detail required (single cell or not).

Autoreactive B cells can also be defined by the **characteristics of their BCR**, such as their repertoire diversity, their level of somatic hypermutations (SHM) and their isotype. From autoreactive B cells DNA or RNA, the sequence of the V(D)J genes can be amplified respectively by multiplex PCR or 5' RACE, then the library is sequenced by a NGS technique. These processes and their applications in the field of AIDs have been recently well-reviewed by Wang et al. [31]. When cells are

analyzed at single cell level, the sequences of the light and heavy chains can be paired, to access to the full BCR CDR3 sequence. By comparison to the germline sequence, it permits access to the SHM level, and, by analyzing the CDR3 sequence, it allows to quantify the repertoire diversity (i.e. rather oligo- versus polyclonal). The same technique can be applied for the constant region of the BCR, to access the isotype of the cells. Currently, these studies concerning the B cell repertoire in AIDs have mainly been conducted on total B cells, but very few studies have specifically focused on autoreactive B cells [31,32].

Finally, recent technological developments allow the combination of all these approaches at once ("**multiomics**"), providing a comprehensive view of cellular functions and features at an unprecedented depth. The use of scRNAseq permits an integrative analysis of the whole transcriptome, along with the cell surface markers (CITEseq, with oligonucleotid-labelled Ab), the (auto)antigenic specificity (LIBRAseq, with oligonucleotide-labelled autoantigens), and paired full-length BCR sequencing, in individual cells [28]. It can also be coupled to ATACseq (Assay for Transposase-Accessible Chromatin using sequencing), providing insights into gene regulation and epigenetic modifications. The contributions and limitations of multiomics in the field of AIDs are well-reviewed in [30].

## 2.3. Studying tissue autoreactive B cells

Blood remains the most accessible source for studying autoreactive B cells. However, numerous studies show that circulating blood does not accurately reflect the immunological anomalies present within tissues, and the characteristics and roles of autoreactive B cells could differ between these 2 settings [20,33,34]. Moreover, tissue B cells are increasingly recognized as playing a key role in the pathophysiology of AID [35]. Thus, the study of autoreactive tissue-infiltrating B cells is a crucial area of research in the field of AIDs.

To this end, it is possible to use the same techniques as in the blood, described above, after tissue dissociation. However, this no longer allows access to the spatial organization of the tissue, nor to visualize the localization of autoreactive B cells and their potential interactions with other cells. Moreover, tissue dissociation can alter the cell viability and membrane markers, making their reliable characterization more difficult.

To overcome these limitations, it is possible to use classical techniques of **immunohistochemistry** or **immunofluorescence**, which were done as early as 2004 for the detection of anti-SS-A and anti-SS-B B cells in Sjogren's syndrome [36]. *Wahren-Herlenius* and *Salomonsson* provide a detailed protocol for the use of immunohistochemistry in the detection of autoreactive B cells [37]. Double staining with other membrane markers or for the presence of intracellular immunoglobulins enables further characterization of the cell or determination of Ig isotype.

In theory, it would also be possible to use **spatial transcriptomics techniques**, although, to our knowledge, this has not yet been done in the study of autoreactive B cells.

Overcoming these challenges involves continuous advancements in immunological techniques, bioinformatics, and a deeper understanding of B cell biology. Enhancing the sensitivity, specificity, and accuracy of detection methods is crucial for advancing our knowledge of autoreactive B cells and their role in autoimmune diseases.

# 3. Lessons learned from studies on autoreactive B cells in AIDs

The development of the techniques described above, some of which are very recent, has led to an increasing number of studies on autoreactive B cells in various AIDs. In this section, we will describe the results of these different studies, particularly the insights they provide into the pathophysiology of these AIDs and the role that autoreactive B cells play in them. In Table S1 and in Fig. 2, we recapitulate the key findings from these studies.
<figure><img src="image_7.png" /><figcaption>**Fig. 2.** Autoreactive B cells characteristics Tolerance breakdown leading to the autoreactive B cells could result from defects intrinsic to the immune system (central or peripheral tolerance default) or extrinsic (caused by abnormal exposition or modification of an aAg). Autoreactive B cells could activate through different activation pathways (EF versus GC) depending on several factors such as the aAg or the AID; resulting in different types of terminally differentiated cells (LLPC, SLPC, MBCs). Homing: autoreactive B cells traffic through the different lymphoid and non-lymphoid organs in reaction to different signals such as chemokines. Autoractive B cells can play a role in AID pathophysiology through different functional mechanisms, either dependent (i.e. complement activation, opsonisation, ADCC, direct target neutralization) or independent of aAb production (cytokine or growth factor production, antigen presentation to T cells). Abbreviations: aAg: autoantigen; ADCC: antibody-dependent cytotoxicity; BCR: B cell receptor; BM: bone marrow; EF: extrafollicular; ER: endoplasmic reticulum; GC: germinal center; LLPC: long lived plasma cell; MBC: memory B cell; PTM: post translational modifications; SHM: somatic hypermutation; SLPC: short lived plasma cell; Tfh: T follicular helper cell; TLO: tertiary lymphoid organs.

Created with **Biorender.com**</figcaption></figure>

### 3.1. Presence and proportion of autoreactive among total B cells

Autoreactive B cells can be identified in healthy controls (HCs) but have been found at higher frequencies in patients with AIDs such as SLE [38–40], rheumatoid arthritis (RA) [32,40,41], PV [8,42], vasculitis [43,44], and Sjögren's syndrome (SS) [39]. Their proportion among total B cells in the peripheral blood varies a lot (from 1/12500 [45] to 17% [7]), both depending on the disease and on the methodology employed. In the studies that analyzed tissue autoreactive B cells, their percentage was higher than in peripheral blood (up to 60% of desmoglein 3 [Dsg3] + B cells in the skin of PV patients [46] and 17% of transglutaminase 2 [TG2] B cells in the intestine of coeliac disease patients [47]). In SS,

autoreactive B cells were also found at a higher frequency in salivary glands than in circulating blood [33]. Interestingly, in PV, Dsg3+ B cell presence in blood was found to precede the autoimmune condition diagnosis [5].

### 3.2. Differentiation state and isotype of autoreactive B cells

These characteristics may vary upon the underlying disease. In SLE, most of the ENA+ B cells were naïve [39,40], whereas in PV and in SS, Dsg3+ and SS-A+ B cells were predominantly composed of MBCs [9]. Some circulating PB could also be found among autoreactive B cells in RA [20,48,32,41] and in coeliac disease [49], but not in PV [5,42]. In
systemic sclerosis (SSc), topo+ B cells can be found into the transitional and naive B cell compartments [50].

The membrane phenotype can also vary depending on the target of the autoreactive B cells within the same AID. For example, in SS, rheumatoid factor (RF)-positive B cells were compared to anti-nuclear antibodies (ANA)-positive B cells, the former having mostly an unswitched memory phenotype, and the latter a switched memory phenotype [33]. The same was true in RA, in which RF+ B cells were mostly of IgM isotype, and CCP+ B cells of switched isotypes [48,32] with a MBC phenotype [20,41,51], arguing for different loss-of-tolerance mechanisms in RF+ B cells (extrafollicular differentiation pathway) versus in CCP+ (follicular differentiation pathway). RF+ B cells also over-expressed genes associated with IgM response and downregulated genes associated with germinal center (GC)-response while CCP+ B cells upregulated genes associated with class-switching [32]. These 2 differentiation pathways could even coexist for the same type of autoantigen. In SLE, the literature presents conflicting views on whether SLE autoreactivity arises from the direct activation of naive cells into PCs through an extrafollicular pathway [52] or from a GC response that generates both MBCs and PCs. *Suumonds* et al. identified two clusters of SLE patients. One was characterized by a higher frequency of ANA+ MBCs with reduced ANA+ PCs, and the other with a higher frequency of PCs with reduced MBCs. This suggests that both follicular and extrafollicular activation pathways could be implicated in SLE pathogenesis [53].

Concerning isotype, in PV, Dsg3+ B cells were isotype switched [5]. In vasculitis, PR3+ B cells were mainly of IgG1 isotype but also enriched in IgG4 isotype [6]. In coeliac disease, TG2+ B cells were found to have a higher rate of IgA isotype than non-autoreactive B cells in the intestine [47] and to be mostly of IgA isotype also in the peripheral blood [49]. In SS in salivary gland biopsies, SS-A + B cells also were predominantly of IgA isotype [36]. Autoreactive B cells were also identified into the double negative (DN) subset of B cells [36,37,54,52].

### 3.3. Repertoire and SHM levels of autoreactive B cells

In addition to the heterogeneity in their BCR constant region (isotype), autoreactive B cells are also characterized by the degree of diversity of their BCR variable region (repertoire). The diversity of the repertoire varied depending on the AID, with a polyclonal repertoire of PR3+ B cells [6], but a low diversity and presence of clonal expansion in TG2+ B cells [34] and in Dsg3+ B cells [5]. Moreover, many studies have found that autoreactive B cells were polyreactive, such as insulin+ B cells [55], topo+ B cells [50] and SS-A+ B cells [33]. Concerning RA, autoreactive B cells were found to be cross-reactive between different post-translational modifications (PTMs), such as citrullination, carbamylation and acetylation [48]. This could be explained either by “real” polyreactivity or by the existence of shared epitopes between these different PTMs.

Concerning the V(D)J gene usage by autoreactive B cells, Zhang et al. identified no specific gene usage and no CDR3 length difference between DNA+ and DNA- B cells in SLE [38]. On the contrary, insulin+ B cells had a preferential usage of the JH6 gene segments [55]; BP180+ B cells a more frequent usage of VH5 gene family [56]; and TG2+ B cells an overrepresentation of IGHV5-10-1 and IGHV5-51 usage [34]. In vasculitis, PR3+ B cells have a high proportion of lambda light chain [6].

Studies that analyzed the level of SHM also found different results depending on the AID and the autoantigen that is recognized. ENA+ B cells in SLE, RF+ B cells in RA and TG2+ B cells in coeliac disease had low SHM levels [32,34,40,47,49], suggesting an extrafollicular response, whereas CCP+ B cells in RA [32,57] had a high percentage of SHM, in favor of a GC response [58]. Interestingly, Vergroesen et al. found that CCP+ B cells have higher levels of SHM than tetanus-toxoid (TT) + B cells. In SS, data are conflicting, with one study finding low levels of SHM in SS-A+ B cells, and another high level of SHM [33]. In

vasculitis, PR3+ B cells had low levels of SHM, but a reduced PR3 binding when reverted to germline [6], proving that SHM is an essential component of developing autoreactivity. In PV, Cho et al. found that Dsg3+ B cells had high level of SHM and did not longer recognize their autoantigen when reverted to their germline sequence [5]. Compared to Dsg3+ B cells identified before PV diagnosis, Dsg3+ B cells after diagnosis circulated at an increased frequency, with a higher level of SHM and a higher affinity [5], which suggest that Dsg3+ clones derive from the maturation of pre-existing clones generated before the disease onset with a continuous affinity maturation. In SSc, one clone of transitional B cells was found to bind topo without SHM [50]. In autoimmune hepatitis, soluble liver antigen (SLA) + B cells could also bind SLA when reversed to their germline configuration, albeit with a lower affinity than in their mutated version [59].

### 3.4. Signaling pathways, mechanisms of emergence and defective tolerance

Multiple studies have identified defects in tolerance mechanisms that usually eliminate or inactivate autoreactive B cells, and specific pathways implicated in the emergence of autoreactive B cells.

The individual genetic background plays a role in the emergence of autoreactive B cells: in RA, the percentage of citrullinated fibrinogen+ B cells was higher in patients of a PTPN22 risk allele (which is known to be associated with several AIDs) than in patients with the non-risk allele [60].

Some studies underlined the importance of T cell help for the secretion of aAb by autoreactive B cells. In SLE and in RA, autoreactive B cells secreted higher levels of ENA or ACPA after stimulation with an anti-CD40 mAb, mimicking T cell help [40]. In PV, production of Dsg3 Ab by Dsg3+ B cells was abolished when CD4+ T cells were depleted or when interaction with class II HLA was blocked [61,62].

Autoreactive B cells were found to express several signaling molecules. In RA, CCP+ B cells expressed higher levels of cytokines receptors, such as IL-7R, and of the anaphylatoxin receptor C5aR1, than in CCP- B cells [45]. In PV, Dsg + B cells upregulated the IRF5 transcription factor, implicated in the interferon pathway [8].

It was also shown that autoreactive B cells express less inhibitory receptors: in RA, CCP+ B cells downregulated the CD72 gene [32], and have a reduced expression of FcγRIIb [20], permitting them to escape to inhibitory control. In a transgenic mice model with Dsg3+ B cells, mice did not develop cutaneous lesions unless they had also a FcγRIIb deficiency [63]. On the contrary, *Bonasia* et al. showed that PR3+ B cells had a higher expression of the inhibitory marker CD22 than their non-autoreactive counterparts [64]. Their hypothesis to explain this result is that it is a regulatory feedback mechanism aimed at dampening the autoimmune response.

Many studies also found that autoreactive B cells expressed high levels of activation markers, such as CD69 in SLE and RA [40]. Interestingly, *Mahendra* et al. found that CCP+ B cells upregulated genes associated with B cell activation when compared to hemagglutinin (HA) + B cells from influenza-vaccinated HCs [51]. In a recent study, CCP+ and TT+ B cells clustered into different subsets of switched MBCs, with resting TT+ B cells and recently activated CCP+ B cells [48]. In these 2 aforementioned studies, the delay between the vaccination and the sample was not specified. On the contrary, *Kristyanko* et al. compared CCP+ B cells to TT+ B cells before and just after a booster vaccination. Before the injection, the levels of activation and proliferation were higher in CCP+ B cells than in TT+ B cells. However, after the boost, these levels became comparable [20]. This could plead in favor of a continuous and antigen driven activation of CCP+ B cells, mimicking what happens during a booster vaccination [20].

The BCR glycosylation profile was found to play a role in the survival of autoreactive B cells. CCP+ B cells were found to have higher levels of N-glycosylation sites in their BCR than TT+ B cells. These glycosylation sites could allow CCP+ B cells to escape negative selection
mechanisms in GC by interaction with glycan receptors [57]. Kissel et al. showed an increase BCR signaling linked to the presence of glycans at the surface of CCP+ B cells, which were also able to delay BCR down-modulation after antigenic stimulation, lowering the threshold for B cell activation [65]. Thus, in contrast to the selection of B cells against infectious recall antigens that are primarily driven by affinity for cognate antigens, activation of autoreactive B cells could also be mediated by their BCR glycosylation.

Volkov et al. proposed another explanation to this phenomenon: the glycosylation of the V domains could impair the autoantigen recognition, reducing the affinity of the BCR, and thus allowing the CCP+ B cells to escape from the negative selection mechanisms into the GC [66]. In mice, introduction of V domains glycosylation induced a decrease of affinity for an autoantigen and provided a selection advantage [67].

Regarding the **loss of tolerance mechanisms**, different theories are discussed, suggesting either an antigen-specific tolerance breakdown (central and/or peripheral), or conversely a global loss of tolerance mechanisms. In vasculitis, PR3+ B cells percentage rise with the maturation status of B cells, suggesting an antigen-specific peripheral tolerance default [43,44]. Moreover, percentages of PR3+ B cells were higher in each B cell subset (transitional, naïve, unswitched and switched MBCs) in vasculitis patients compared to HCs, indicating a possible central tolerance defect associated with the peripheral tolerance defect [44]. Berti et al. also analyzed PR3+ B cells in the bone marrow of HCs. Their percentage was higher in the bone marrow than in peripheral blood of HCs, and similar to that of the peripheral blood of vasculitis patients. Thus, results suggest a tolerance defect into or upon leaving the bone marrow in vasculitis patients [68]. On the contrary, in SLE, two studies found a reduction in the percentage of ANA+ and DNA+ B cells from transitional to the naïve B-cells compartment, and from the naïve to the antigen-experienced B-cells compartment [7,39], in favor of at least in part preserved peripheral tolerance mechanisms. In favor of a general (non-antigen specific) loss of tolerance, Suurmond et al. identified that the increased number of ANA+ PCs in SLE patients was due to a general expansion of total PCs, rather than to a lack of negative selection against ANA+ PCs. They conclude that SLE results from a global aberrant B-cell differentiation (i.e. PCs expansion regardless of their antigenic specificity) rather than a defect in antigen-specific B-cell tolerance [69].

Another tolerance mechanism, **B cell anergy**, was found to be globally defective in several AIDs. In SLE patients, the percentage of ANA+ B cells in the anergic compartment (unresponsive to BCR stimulation) was lower than in HCs [7]. In HC, high affinity insulin+ B cells were only found in the anergic compartment, whereas in type-1 diabetes (T1D) patients, insulin+ B cells were absent of this anergic subset. This finding coincides with a loss of the entire anergic B cell compartment, irrespective to their antigenic specificity, at the time of T1D diagnosis. The authors suggest that it could be explained by a breach of anery due to an environmental factor such as infection, on a predisposing genetic background (PTPN22 polymorphisms). This global loss of anery could also explain the association of T1D with other AIDs in the same patient [55].

Finally, the **integrity of the target tissue, the abnormal autoantigen exposition or its modifications** seem to play a role in AIDs. This could explain why, even if global loss of tolerance exists, only certain tissues / aAg are targeted by autoreactive B cells. In SLE, tissue damage can trigger or sustain the autoimmune response by exposing nuclear antigens that are normally hidden from the immune system due to their intracellular location [70]. In a mice model with Dsg3+ B cells, Nomura et al. found that mice only developed cutaneous lesions if they received Dsg3 injections (mimicking an abnormal autoantigen exposition) or if skin inflammation was induced, but not in mice with intact skin [63]. In RA, autoreactive B cells recognize antigens that have been modified by PTM [48]. These PTM create neoepitopes, that could be not recognized as "self" by the immune system and trigger the autoimmune response. The creation of neoepitopes is also involved in coeliac disease

pathophysiology. TG2, an enzyme present in the intestine, modifies gliadin's glutamine into a deaminated form. Autoreactive B cells recognize complexes formed between deamidated gliadin and TG2 [71]. In T1D, endoplasmic reticulum (ER) stress has been linked to the autoimmune reaction. In response to an insult (such as viral infection or inflammation), the pancreatic response involves ER stress, that induces changes in the protein landscape with PTM and insulin misfolding, followed by a T and B cell autoimmune response that compromise the beta cell function [72]. Neoepitopes or abnormal exposition of autoantigens could also be caused by cancer. For example, mutations of RNA polymerase III in tumors were shown to be associated with the onset of SSc with anti-RNA polymerase III aAb [73].

### 3.5. Effector functions of autoreactive B cells

Autoreactive B cells had been shown to exert multiple roles that can take part in AIDs pathophysiology.

#### 3.5.1. Autoantibody production

Autoreactive B cells are responsible for the production of aAbs. Many studies have identified a correlation between the percentage of autoreactive B cells and the levels of circulating aAbs [9,48,39,43,44] [74]. In vasculitis, on the contrary, the percentage of PR3+ B cells was not associated with the levels of anti-PR3 aAb [43], but there was a correlation between anti-PR3 IgG levels in supernatant from in vitro cultures of PR3+ B cells, and PR3 aAb levels in the patients' serum [44]. Several studies also assessed the in vitro production of aAb by circulating autoreactive B cells. In SLE and RA [40], PV [62], SSc [75] and coeliac disease [34,49], ENA+, CCP+, Dsg3+, centromere+ and TG2+ B cells needed to be stimulated to secrete aAb. This suggests that, in these AIDs, autoreactive B cells circulate as MBCs, and need an additional trigger to differentiate into ASCs. On the other hand, topo+ B cells from SSc patients could produce aAb without an additional stimulation [75]. These last results imply that, in topo+ SSc, there is an expansion of circulating ASCs involving recent / repetitive / continuous B cell activation.

The capacity of production of aAb by autoreactive B cells seemed to vary depending on the individual's diseased status: Dsg3+ MBCs from PV patients produced anti-Dsg3 aAb after stimulation, while Dsg3+ MBCs from HCs did not [42]. A similar pattern was observed for PR3+ B cells in vasculitis [44] and for topo+ B cells in SSc [26].

#### 3.5.2. Cytokines and growth factors production

Autoreactive B cells have been found to produce various molecules that can have an implication in the pathophysiology of AIDs. In RA, CCP+ B cells produce amphiregulin, a protein that leads to migration and proliferation of fibroblast-like synoviocytes and differentiation of osteoclasts [51]. Autoreactive B cells also express and/or produce a wide range of cytokines, such as IL-15 with IL-15RA [51], IL-8 (which drive neutrophils recruitment), IL-6 and TNF-α [20] by CCP+ B cells, and IL-1β, IL-23 and IL-12 by Dsg3+ B cells [8]. In SSc, the role of topo+ B cells depended on their BCR affinity: low affinity topo+ B cells have been reported to produce anti-inflammatory cytokines, whereas high affinity topo+ B cells produced pro-inflammatory cytokines in topo-immunized mice [26].

#### 3.5.3. Antigen presentation and T cell costimulation

Autoreactive B cells can act as potent APCs, presenting self-antigens to autoreactive T cells and perpetuating the autoimmune response. In RA, CCP+ B cells upregulated genes associated with the B-T cooperation [32] and expressed high levels of co-stimulatory molecules (CD80/86) [20,45]. In autoimmune hepatitis, soluble liver antigen (SLA) + B cells could process and present SLA peptides to SLA+ T cells, inducing a proliferative response [59].

#### 3.5.4. Tolerogenic and protective role

Opposite to the aforementioned mechanisms, some autoreactive B
cells could also play a role in immune tolerance. In a recent study, Aoun et al. analyzed natural autoreactive B cells directed against type-II collagen (COL-II). These cells, characterized by a germline encoded BCR, were present in HC's peripheral blood, and their frequency was reduced in RA patients. When transferred to autoimmune-prone mice, COL-II+ cells prevented to development of arthritis. This tolerogenic role was exerted in an IL-10-independent manner, and induced the expansion of COL-II specific regulatory T cells in a contact-dependent way [76]. He et al. also evaluated the pathogenic role of murinized ACPA derived from RA patients. After passive transfer in mice, they showed that one of these aAb protected mice from antibody-induced arthritis. ACPA interacted by their Fc with macrophages, inhibiting their differentiation in osteoclasts, and limiting their contribution to tissue degradation. In humans, this clone of aAb was found to bind the citrulinated enzyme alpha-enolase, and to interact with the inhibiting receptor FcgRIIb, which induced a decrease in osteoclastogenesis and an increase in IL-10 levels [77]. Moreover, in SSc, co-culture of low-affinity topo+ B cells induced FoxP3 upregulation in CD4+ T cells, and their transfer into topo-immunized mice inhibited fibrosis development [26].

### 3.6. Localization and circulation

Most studies focused on peripheral blood autoreactive B cells, but a few analyzed B cells in target tissues, for evident reasons of accessibility, and technical limitations. The phenotype of autoreactive B cells in the tissues is different from that of peripheral blood. In RA, more than half of CCP+ B cells in the synovial fluid were PB/PC [20]. In line, in PV, skin Dsg3+ B cells were ASCs and produced more Dsg3 aAb than blood Dsg3+ B cells [61]. In vasculitis, Weppner et al. only identified very few PR3+ B cells in lung and kidney biopsies [78].

To explain that Dsg3+ B cells percentage was higher in skin than in blood, Zhou et al. proposed two non-exclusive hypotheses: (i) B cell clones might differentiate in the skin in tertiary lymphoid organs (skin-associated lymphoid tissue) and then migrate to the peripheral blood; or (ii) some low-abundance peripheral B cell clones previously differentiated in secondary lymphoid organs could migrate to the skin and expand locally [46]. In line with the first hypothesis, transplantation of PV lesions into SCID mice led to recirculation of Dsg + B cells from skin to lymph nodes and peripheral blood [46]. The mechanism of circulating autoreactive B cells entering the target tissue was also suggested in RA: Reijm et al. found that CCP+ MBCs and PBs expressed chemokine receptors such as CXCR3 permitting to enter inflamed tissues [48]. Moreover, in coeliac disease, circulating TG2+ B cells also expressed gut-homing molecules (integrin α487, CCR9 and CXCR3) [49].

These findings collectively enhance our understanding of the multifaceted roles of autoreactive B cells in AIDs. Their phenotypes and functional mechanisms appear diverse and vary from one AID to another. These characteristics may depend on the specific targeted aAg, the cell type presenting this aAg to autoreactive B cells, and the costimulatory signals they receive (such as TLR stimulation). This underscores the importance of autoreactive B cells not only in autoantibody production but also in sustaining the autoimmune response through multiple mechanisms, including antigen presentation and cytokine production.

## 4. Autoreactive B cells as biomarkers and therapeutic targets

Based on the identification and characterization of autoreactive B cells presented above, some studies have assessed their role as potential AIDs biomarkers. They found that elevated levels of autoreactive B cells or specific phenotypic profiles of autoreactive B cells might be associated with disease activity and severity in several AIDs. For example, in SLE, Malkiel et al. identified an inverse correlation between the percentage of anergic ANA+ B cells and SLEDAI score [7]. A higher frequency of DNA+ B cells was also found in active than in quiescent SLE patients [39]. In vasculitis, there was a trend toward a higher proportion

of PR3+ B cells in patients with active disease compared to patients in remission [43]. At diagnosis or during a flare, levels of CRP were higher in patients with a switched memory / naive PR3+ B cells ratio > 1 [44]. In SSc, the ratio of high affinity to low affinity CD27+ topo+ B cells correlated positively with skin fibrosis score, and inversely with lung volume [26]. After in vivo PBMCs culture, Wortel et al. found a higher production of anti-topo IgG in SSc patients with interstitial lung disease (ILD), and a correlation between anti-topo IgG production and lung fibrosis score [75]. However, in SLE, the different ANA+ B cells profiles (i.e. high frequency of PCs or high frequency of IgG memory B cells) were unrelated to disease activity and remained stable over time [53].

Some studies also evaluated the **evolution of autoreactive B cells after treatment** and their **link with therapeutic response**. In coeliac disease, TG2+ B cells, as well as specific antiTG2 aAb, are undetectable in patients under a gluten-free diet [49].

Belimumab is a monoclonal antibody that specifically inhibits B-cell activating factor (BAFF/Blys), a key regulator of B cell survival and activity. By binding to BAFF, belimumab prevents it from interacting with its receptors on B cells, thereby reducing the proliferation and activity of autoreactive B cells. In SLE, belimumab increased the percentage of ANA+ B cells in the anergic compartment compared to patients without belimumab [7].

Rituximab (RTX) is a monoclonal antibody that targets CD20 on B cells, leading to their depletion, and is used to treat various autoimmune diseases. Special attention has been paid to the evolution of autoreactive B cells after RTX treatment. Cho et al. observed a drop in anti-Dsg3 aAb levels after RTX, along with unaffected anti-TT titers, suggesting that anti-Dsg3 aAb production is not mediated by bone marrow long-lived PC (that are CD20-negative), but rather by short-lived PB continuously replenished by the MBC pool (that are CD20-positive) [5]. All studies agree that autoreactive B cells reappear during B cell reconstitution after RTX [8, 33, 56, 79]. Some found that they reemerge at the same percentage as before RTX [8] or even at higher levels [79]. Even if they reappear, RTX seemed to modify autoreactive B cells characteristics. The percentage of transitional and naive PR3+ B cells was higher at B cell recurrence than at baseline, whereas the percentage of memory PR3+ B cells was lower (Berti 2023). In PV, Maho-Vaillant et al. observed that, at M36 after RTX, naive and non-switched Dsg + B cells levels were at a frequency close to baseline, whereas there was a decrease in the Dsg + switched MBC levels and a disappearance of Dsg + ASCs. They also noted that Dsg3+ T follicular helper cells and IL-21 levels dramatically decreased after RTX. These results suggests that the therapeutic effect of RTX in pemphigus is not only explained by a direct long-lasting depletion of Dsg + B cells, but also by the impact of the specific B cell depletion on the Dsg + T follicular cell survival, resulting in an inefficient maturation and class-switching process of re-emerging Dsg + B cells caused by the specific T follicular cells disappearance [80]. A normalization of the IL-1β gene expression in Dsg3+ circulating B cells was observed 36 months after RTX [8]. Moreover, in bullous pemphigoid, BP180+ B cells cytokine production was modified after RTX treatment, with a decrease in the production of inflammatory cytokines (IL-15, IL-6, TNF-α) and an emergence of anti-inflammatory cytokines production (IL-10 and IL-1RA) [56]. Some teams assessed the predictive value of autoreactive B cells percentage for treatment response. In vasculitis, a baseline switched memory / naive PR3+ B cells ratio > 1 was associated with a poor therapeutic response [44]. During follow-up, the percentage of PR3+ B cells did not differ between patients who experienced relapse and patients who remained in remission after B cell recurrence. However, PB frequency within the PR3+ B cell pool was higher in patients who experienced relapse and associated with a shorter time to relapse [79].

Daratumumab is a recent therapeutic option for AIDs [81]. It targets CD38, a molecule that is highly expressed on plasma cells. Even more recently, anti-CD19 CAR (chimeric antigen receptor)-T cells have been employed to target B cells in the field of AIDs [82]. However, to date, no study evaluated the impact of daratumumab nor of anti-CD19 CAR-T
cells specifically on autoreactive B cells.

Alongside treatments that nonspecifically target all B lymphocytes (RTX, belimumab, daratumumab, anti-CD19 CAR-T cells), some teams have focused on therapeutic options aimed at **targeting autoreactive B cells**. These approaches could permit to achieve an antigen-specific depletion of B cells, without generating a general immunosuppression and thus reducing the risks of infectious adverse events.

The general principle is to fuse an autoantigen with a toxic/inhibitory molecule or to a cytotoxic cell that will inhibit or kill the autoreactive B cell (Fig. 3). Various strategies have been described, such as CAAR (chimeric autoantibody receptor)-T cells [83–86], universal anti-fluorescein isothiocyanate CAR-T cells [87], bispecific autoantigen-T cell engagers [88], fusion molecules [89], inhibitory liposomal nanoparticles [90], inhibitory functional scaffolds of autoantigens [17,91], antigen-drug conjugates [92], and disarmament by BCR engagement [93]. Principles et applications of these strategies are presented in Table 2. These therapeutic strategies are promising but also have limits. In theory, all the therapeutic strategies using an autoantigen could be neutralized by the circulating aAb in the patient's serum, before binding their specific B cell. It will be needed to assess these caveats in future pre-clinical and clinical studies.

<figure><img src="image_8.png" /><figcaption>**Fig. 3.** Therapeutic strategy principles to target autoreactive B cells. CAAR-T cells are modified T cells engineered to express a receptor that mimics the autoantigen targeted by autoreactive B cells, allowing them to bind and destroy these harmful cells specifically. Anti-FITC CAR-T cells are modified T cells that recognize FITC, used in combination with an FITC-labelled autoantigen, which binds to autoreactive B cells, allowing their specific destruction by the CAR-T cell. BIAATEs are engineered bispecific antibodies designed to simultaneously bind autoreactive B cells and T cells. One arm of the BIAAT targets the B cell receptor (BCR) specific for an autoantigen, while the other arm binds to CD3 on T cells. Fusion molecules are composed of the Fc part of Ig, coupled to the autoantigen of interest. It allows the elimination of autoreactive B cells by Antibody-Dependent Cellular Cytotoxicity. Inhibitory nanoparticles are liposomal structures that carry at their surface the autoantigen and an inhibitory molecule, such as CD22 ligand. It induces a tolerogenic targeting of autoreactive B cells. Functional autoantigen scaffolds are multivalent polymeric structures that are associated with multiple autoantigen molecules, and with an inhibitory molecule (such as CD22 ligand), which specifically inhibit autoreactive B cells. Toxic/drug conjugated autoantigens are autoantigens that are directly coupled to a toxic molecule, to directly kill the target autoreactive B cells.</figcaption></figure>

Abbreviations: BIAATE: bispecific autoantigen-T cell engagers; CAAR-T cell: chimeric autoantibody receptor-T cells; CAR-T cell: chimeric antigen receptor-T cells; FITC: Fluoresceine isothiocyanate; scFv: single-chain variable fragment. Created with Biorender.com

<table><caption>**Table 2**

Therapeutic approaches allowing a specific targeting of autoreactive B cells.</caption><thead><tr><th>Therapeutic strategy</th><th>Mechanism of action</th><th>Field of application and effect</th></tr></thead><tbody><tr><td>CAAR T cells</td><td>Genetically modified T cells that express a chimeric receptor composed of:<ul><li>An autoantigen reproducing the structure recognized by the autoreactive BCR</li><li>A signaling domain that activate the T cell upon binding to the autoreactive B cell</li></ul></td><td>Dsg-3 CAAR-T cells ameliorated the skin phenotype in a mouse model of PV [83]. Phase 1/2 clinical trial in humans currently underway (NCT04422912) with promising preliminary results [84]<br>MUSK CAAR-T cells reduced anti-MUSK IgG without decreasing B cells or total IgG levels in a myasthenia gravis mice model [85]<br>TSH-receptor CAAR-T cells improved thyroid hormones levels in a Grave's disease mice model [86]<br>Anti-FITC CAR-T cells could specifically kill CCP+ hybridoma cells, and CCP+ B cells from RA patients via recognition of corresponding FITC-labelled citrullinated peptide epitopes [87].</td></tr><tr><td>Universal anti-FITC CAR-T cells</td><td>Derivative of CAAR-T cells developed to create a customized CAR-T cell permitting to target autoreactive B cells with different specificities, by using FITC-labelled antigenic peptide epitopes and anti-FITC CAR-T cells</td><td></td></tr><tr><td>BIAATEs</td><td>Engineered bispecific antibodies designed to simultaneously bind autoreactive B cells and T cells. One arm of the BIAATE targets the BCR specific for an autoantigen, while the other arm binds to CD3 on T cells.</td><td>BIAATE consisting of the immunodominant domain of PLA2R and the single-chain variable fragment of an antibody against the T cell antigen CD3, successfully induced T cell-dependent depletion of PLA2R+ B cells isolated from membranous nephropathy patients and reduced the anti-PLA2R aAb levels in a mice model [88]<br>AKS-107: human IgG1 Fc molecule fused with human insulin chains, that bound to insulin-specific BCR but not to insulin metabolic receptor. Specifically depleted insulin+ B cells and prevented spontaneous diabetes development in NOD mice [89].</td></tr><tr><td>Fusion molecules</td><td>Molecule composed of the Fc part of Ig, coupled to the autoantigen of interest. Allows the elimination of autoreactive B cells by Antibody-Dependent Cellular Cytotoxicity.</td><td>Liposome carrying both autoantigens and ligands of the inhibitory coreceptor CD22, permitting to induce a tolerogenic program that selectively causes apoptosis of specific B cells [90].<br>In vitro successful inhibition of BCR downstream signaling effects by a scaffold conjugated with 50 peptide antigens and CD22 ligand [17].</td></tr><tr><td>Inhibitory liposomal nanoparticle</td><td>Liposomal structures that carry at their surface the autoantigen and an inhibitory molecule, inducing a tolerogenic targeting of autoreactive B cells.</td><td>Polymeric backbone coupled with the autoantigen PLP and cell adhesion inhibitory peptides induced anergy of autoreactive B cells in a mice model of EAE [91].</td></tr><tr><td>Inhibitory functional scaffold of autoantigen</td><td>Multivalent polymeric scaffolds conjugated with multiple autoantigens, and associated with an inhibitory molecule.</td><td>Autoantigen PLP conjugated with dexamethasone protected mice against EAE [92].</td></tr><tr><td>Toxic/drug conjugated autoantigens</td><td>Directly conjugate a drug or a toxic molecule to an autoantigen.</td><td>Tetramer display of PLP depleted autoreactive B cells, (continued on next page)</td></tr><tr><td>Disarmament by BCR engagement</td><td>Multivalent, high avidity BCR binding "disarming"</td><td></td></tr></tbody></table>
<table><caption>**Table 2 (continued)**</caption><thead><tr><td>Therapeutic strategy</td><td>Mechanism of action</td><td>Field of application and effect</td></tr></thead><tbody><tr><td></td><td>autoreactive B cells by a sustained BCR engagement and cross-linking</td><td>decreased inflammatory cytokines levels and ameliorated symptoms in a mice model of EAE [93].</td></tr></tbody></table>

aAb: autoantibodies; BCR: B cell receptor; BiAATEs: Bispecific AutoAntigen-T cell Engagers; CAAR: chimeric autoantibody receptor; EAE: experimental autoimmune encephalomyelitis; FITC: fluorescein isothiocyanate; PV: pemphigus vulgaris; RA: rheumatoid arthritis.

## 5. Unanswered questions and perspectives

Despite all these studies on the characteristics of autoreactive B cells and the therapeutic strategies targeting them, some gray areas remain.

First, there are discrepancies concerning the range of autoreactive B cells frequency into the peripheral blood, even at the scale of one AID and one aAg specificity. For example, in vasculitis, a team found around 4–5% of PR3+ B cells [44,79], another 0.1% [64] and as low as 0.0075% [6]. These inconsistencies could derive from several technical aspects. By flow cytometry, the fluorescence threshold used to identify autoreactive B cells can be more or less conservative, the autoantigen can be associated with only one or with two different fluorochromes to improve the specificity of the autoreactive B cell detection. The origin of the autoantigen can vary (e.g. native, recombinant antigen, extracts of granules of leukocytes) and influence the quality of the staining. These technical considerations are of importance, because it influences the analysis of the characteristics of these B cells. Indeed, it may be biased if autoreactive B cells are mixed with non-autoreactive B cells; or on the contrary if all autoreactive B cells are not identified.

Second, the mechanisms by which the B cell tolerance breakdown occurs remain poorly understood. Several hypotheses have been proposed, some of which implicate defects intrinsic to the immune system, while others rely on extrinsic factors such as target organ injury, or environmental events, like infections. Among intrinsic defects, data about default in B cell central tolerance mechanisms are scarce due to difficulties in accessing bone marrow samples. Still, Berti et al. found that the percentage of PR3+ transitional B cells was higher in vasculitis patients than in HCs [44], suggesting that central tolerance can be defective. However, a global central tolerance defect could not explain why autoimmunity only targets one or few antigens or organs, why it generally appears in adults, and why the disease progresses with alternating phases of flare-ups and remission. Some data also point out the implication of peripheral tolerance defaults, for instance, the increase in PR3+ B cells percentage with the maturation status of B cells [43]. This peripheral tolerance defect could be due to several causes, such as (i) the increase in the peripheral concentrations of BAFF as described in SLE [94] (ii) the special glycosylation pattern of their BCR [57] or (iii) the low levels of inhibitory receptor expression at their surface [64], permitting them to survive in spite of the various tolerance checkpoints. Still, many studies found autoreactive B cells in HCs, suggesting that the presence of autoreactive cells is not sufficient to trigger the disease [32,38,43]. Moreover, even if global loss of anergy [57] or abnormalities in B cell differentiation [69] have been described in some studies and could explain the association of different AID in the same patient, AID symptoms remain in most cases directed toward certain restricted and specific organs. These AIDs could then also depend on abnormal exposure or post translational modification of certain aAg, exposed in an inflammatory context. Thus, the definitive tolerance breakdown could be driven by the autoantigen exposition, due to a tissue damage (trauma, infection provoking ER stress, apoptosis ...), in a predisposition immune context of tolerance loss. In RA, CCP+ B cells were found to be mostly naive, and needed to be activated by autoantigens to secrete aAb [40]. In line, the levels of activation and proliferation were comparable between CCP+ B cells and in TT+ B cells just after a boost TT vaccine injection

[20], as if the aAb response was triggered by the citrullinated antigen overexposure, mimicking an exoantigen response. This hypothesis could explain why some AIDs progress by flare-up and remission phases (with flare-up triggered by tissue damage / autoantigen exposure). Finally, these mechanisms could be combined as “multiple hits”: the onset of the AID symptoms need both the existence of an intrinsic immune system tolerance default, and an extrinsic tissue injury, and even be interdependent: abnormal exposition or modification of autoantigens due to organ damage could trigger the differentiation of autoreactive B cells. With this in mind, some therapeutic approaches have been developed, such as TG2 inhibitors in coeliac disease [95], or peptidylarginine deiminase inhibitors that inhibit peptide citrullination in RA [96].

Third, the pathway(s) by which autoreactive B cells activate are still debated. Several studies agree on the fact that T cell help is needed to lead to aAb secretion by autoreactive B cells, which suggests a T-dependent response [40,61,62]. Given that AIDs could have different dynamic patterns (i.e. persistent symptoms along time, or more acute evolution with flares and remission periods), and that many aAb exhibit significantly different evolutions both during the disease and in response to B cell depletion therapy, it is plausible that various activation pathways are involved depending on the AID and on the aAg. In PV, Dsg3+ B cells were found to accumulate SHM, and to be isotype switched and of recently activated MBC phenotype, in favor of a GC response [5]. In RA, many clues point to an ongoing GC differentiation of CPP+ B (high levels of SHM with accumulation of N-glycosylation sites in the variable domain, class-switched memory phenotype) [58]. Still, by contrast, RF production, found in RA and in SS seems to depend on an EF response [32]. In AIDs that evolve with more acute changes, such as SLE and vasculitis, the response pathway of autoreactive B cell is more discussed. In SLE, two patterns of B cells have been reported, with patients who had a predominant EF-derived ANA+ B cell pool, and others who have a higher percentage of GC-derived ANA+ B cells [53]. In vasculitis, PR3+ B cells were found to have low levels of SHM (in favor of an EF activation pathway), but high levels of CXCL13 were found in patients with high levels of PR3+ MBCs, suggesting a possible GC reaction [44]. Moreover, the frequency of PR3+ B cells was not associated with the levels of anti-PR3 aAb [43]. This could suggest that the production of anti-PR3 aAb depends on BM-localized long-lived PC, rather than on ASCs newly generated from circulating B cells. These considerations are of importance, because it could influence the choice of the therapeutic strategy. In GC-mediated diseases, molecules that target bone marrow long-lived PC are needed, while in EF-mediated AIDs, targeting circulating MBCs by an anti-CD20 molecule could be sufficient.

Finally, the question of the relevance of a therapeutic targeting of autoreactive B cells recognizing a specific autoantigen, which frequency is very low, arises. First, in many AIDs (such as SLE or SS), there are autoantibodies against more than one target. Moreover, the epitope spreading phenomenon, that is well-known in several AID (PV, T1D, RA and SLE), could hinder the optimal efficacy of treatments targeting autoreactive B cells. It refers to the process where the immune response initially targets a specific epitope on an autoantigen, but over time expands to target additional epitopes on the same antigen or even different antigens. Therefore, for an optimal efficacy of autoreactive B cells-targeted strategies, it could be necessary to target multiples specificities of autoreactive B cells [3]. Furthermore, the autoreactive B cells recognizing the major autoantigens of one AID (e.g. anti-PR3 in vasculitis, anti-SS-A in SS ...) may represent only the emerging part of the iceberg. Very often, other autoantigens are involved in the AID, such as anti-Trim21 that can be found in several connective tissue diseases and be a severity marker [97]. Moreover, there could be several other target autoantigens implicated in disease pathophysiology, that are not yet recognized to this day. For example, in multiple sclerosis, in which no specific B cell antigenic target has been identified, Lefranc et al. found a global modification of the IgG reactivity pattern against brain antigens, resulting in a IgG signature permitting to distinguish between patients and HCs, and even, the different clinical and evolutive forms of the
disease [98]. Additionally, patients who well-respond to B cell depletion therapies do not always show a corresponding decrease in specific aAb levels [99], such as in SLE [100] and in RA [101]. This could suggest that the specific depletion of autoreactive B cells may not be sufficient to treat AIDs. In this regard, it would be more relevant to broadly target B cells rather than the rare autoreactive B cells recognizing only a specific antigenic target.

## 6. Conclusion

The study of autoreactive B cells has provided valuable insights into their identification, characterization, and potential as therapeutic targets in AIDs. Classical and advanced techniques such as ELISpot, flow cytometry, scRNAseq, and multiomics have greatly enhanced our ability to detect and analyze these cells, revealing their roles in immune dysfunction. It underscores the complexity of autoreactive B cell behavior, including their phenotypical and transcriptomic characteristics, their emergence mechanisms (GC vs. EF pathways), and their different functional properties depending on the AID and on their target. Therapeutic strategies targeting autoreactive B cells, such as CAAR-T cells or BiAATEs, have shown promise in mitigating autoimmune responses. However, challenges remain, including improving the sensitivity and reliability of the detection of autoreactive B cells, and better understanding both their heterogeneity and their commonalities among different AIDs. Continued research into the mechanisms governing autoreactive B cells will be essential for developing more precise and effective therapies, ultimately improving outcomes for patients with AIDs.

## Funding

This project was co-founded by the European Union through the European Regional Development Fund (ERDF) within the framework of the Contrat de Plan Etat-Région (CPER) 2021-2027 for the Hauts-de-France region.

## Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Aurore Collet reports travel grant from Grifols, outside the submitted work. Sébastien Sanges reports meeting fees from Shire, Sanofi-Genzyme, SOBI, Novartis, BioCryst; consulting fees from Novartis, Takeda and Grifols; speaker fees from MSD; research grants from Novartis, BioCryst, MSD; outside the submitted work. Aurélien Chépy reports travel reimbursement from Vitalair, outside the submitted work. Vincent Sobanski reports consulting and speaking fees from Boehringer Ingelheim, EDF, Fresenius Kabi France LLC, Grifols Inc. and Ultragenyx; and research support from CSL Behring, Grifols; outside the submitted work. David Launay reports consulting and speaking fees from Takeda Pharmaceutical Company Limited, CSL Behring LLC, Biocryst Pharmaceuticals, Inc., AstraZeneca; outside the submitted work. David Launay reports funding grants with CSL Behring LLC and Octapharma AG, outside the submitted work. The other authors declare no conflicts of interest.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.autrev.2025.103851.

## Data availability

No data was used for the research described in the article.

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