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Impaired early B cell tolerance in patients with rheumatoid arthritis

Autoantibody production is a characteristic of most autoimmune diseases including rheumatoid arthritis (RA). The role of these autoantibodies in the pathogenesis of RA remains elusive, but they appear in the serum many years before the onset of clinical disease suggesting an early break in B cell tolerance. The stage of B cell development at which B cell tolerance is broken in RA remains unknown. We previously established in healthy donors that most polyreactive developing B cells are silenced in the bone marrow, and additional autoreactive B cells are removed in the periphery. B cell tolerance in untreated active RA patients was analyzed by testing the specificity of recombinant antibodies cloned from single B cells. We find that autoreactive B cells fail to be removed in all six RA patients and represent 35–52% of the mature naive B cell compartment compared with 20% in healthy donors. In some patients, RA B cells express an increased proportion of polyreactive antibodies that can recognize immunoglobulins and cyclic citrullinated peptides, suggesting early defects in central B cell tolerance. Thus, RA patients exhibit defective B cell tolerance checkpoints that may favor the development of autoimmunity.

Rheumatoid arthritis (RA) is a common chronic inflammatory disease that affects ‫ف‬ 1% of the population (1). An important characteristic that distinguishes RA from other inflammatory and degenerative joint diseases is the production of autoantibodies directed against self-antigens including antibody Fc regions (rheumatoid factors), type II collagen, and cyclic citrullinated peptides (CCP; [2][3][4][5]. These antibodies appear in the serum of RA patients many years before the onset of clinical disease suggesting an early break in B cell tolerance (6). However, the underlying mechanisms that account for autoantibody production in RA have not been defined. Mouse models reveal an important role for T and B cells in the development of inflammatory arthritis (7,8). In humans, an important role for B cells in RA was recently demonstrated by successful treatment of RA patients with anti-CD20 monoclonal antibodies that eliminate B cells (9,10). Although little is known about the characteristics of RA B cells, some express unusual B cell receptors (BCRs) with 11-amino acid-long CDR3 Ig chains that accumulate in the joints of RA patients (11,12). It is un-clear if those unusual BCRs result from altered B cell development and reflect impaired B cell tolerance.
We previously analyzed how B cell tolerance was established in humans by following the evolution of autoantibody-producing B cells during B cell development (13). Using an RT-PCR method that allowed us to clone and express in vitro recombinant antibodies amplified from single B cells, we found that 55-75% of early B cell precursors expressed self-reactive antibodies and that autoantibodyproducing B cells in healthy donors were removed from the population at two discrete checkpoints (13). The first checkpoint occurs in the bone marrow between the early immature and immature B cell stage. The second counterselection step of autoantibody-expressing B cells takes place in the periphery, at the transition from new emigrant to mature naive B cells (13). We applied the same method to characterize how B cell tolerance was established in RA patients. We found that central and peripheral B cell tolerance checkpoints were defective in RA patients and allowed the accumulation of peripheral mature naive autoreactive B cells that may contribute to RA pathogenesis.
The online version of this article contains supplemental material.

RESULTS
To determine whether B cell tolerance is established properly in RA patients, we enrolled nine active RA patients who met the Revised Criteria of the American College of Rheumatology and examined the reactivity of antibodies from six of them (Table I). Eight patients were either naive to steroids, disease-modifying antirheumatic drugs, and biologics, or off these medications for at least 4 mo (see Materials and methods and Table I).
Only patient RA07 had been taking hydroxychloroquine for 2 mo before the day when blood was drawn. In total, we cloned and expressed in vitro 176 and 177 antibodies from single new emigrant (CD19 ϩ CD10 ϩ IgM ϩ CD27 Ϫ ) and mature naive (CD19 ϩ CD10 Ϫ IgM ϩ CD27 Ϫ ) RA B cells, respectively, and compared them to control antibodies from four previously reported and three additional healthy donors (Table S1 available at http://www.jem.org/cgi/content/full/jem.20042321/DC1; 13-15).

RA new emigrant B cells express remarkable antibody repertoires
The B cells from our nine patients revealed at least three distinct patterns of antibody sequences. Consistent with previous reports on RA, the proportion of Ig s with unusual Ն 11-amino acid-long CDR3s in new emigrant B cells was significantly increased from 0 to 4.1% in controls to 5.7 to 17.9% in RA patients (P ϭ 0.0008, Fig. 1 and Tables S2-S16 available at http://www.jem.org/cgi/content/full/ jem.20042321/DC1; 11,12). The Ig antibody repertoires of RA01, RA02, and RA08 new emigrant B cells were also remarkable in that they displayed significantly increased upstream J 1 usage (RA01: 53.1%, P ϭ 0.025; RA02: 51.4%, P ϭ 0.02; and RA08: 47.9%, P ϭ 0.027; Fig. 2 A). In contrast, J 1 usage frequency in new emigrant B cells from seven control healthy donors and from the other RA patients ranged from 21 to 36% and 21 to 41%, respectively ( Fig. 2 A; 13,14). J 1 Ig segment is eliminated when upstream V genes delete preexisting V J 1 genes by recombining with downstream J genes (16). We found that upstream V gene usage in new emigrant B cells was decreased in RA01 (9.5%), RA02 (8.5%, P ϭ 0.044), and RA08 (14.3%) when compared with controls (21-36%; Fig. 2

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Tables S9 and S10 reported in reference 13). In contrast, new emigrant B cells from another RA patient, RA06, showed increased downstream J 3-4-5 combined with upstream V gene usage (Fig. 2 C). In addition, increased J 1 usage was found in RA patient new emigrant B cells that displayed the lowest frequency of unusual Ն 11-amino acidlong Ig CDR3s (Fig. 2 D). We conclude that RA new emigrant B cells express at least three distinct patterns of Ig antibody repertoires: unusually long Ig CDR3s, an Ig repertoire biased toward either upstream J 1 and downstream V genes or downstream J 3-4-5 and upstream V genes consistent with decreased or increased secondary V(D)J recombination, respectively.

Defective peripheral B cell tolerance checkpoint in RA patients
The self-reactivity of the recombinant antibodies from six RA patients was first tested against HEp-2 cells by ELISA and indirect immunofluorescence assay (IFA; 13,17). We found that the proportion of HEp-2-reactive antibodies in new emigrant B cells from RA patients ranged from 31.8 to 55.8% (Fig. 3 A) and was not significantly different from that of control new emigrant antibodies previously reported (31-40.7%; 13, 14). In contrast, all six RA patients revealed an increase of HEp-2-reactive mature naive B cells that reached statistical significance in five of them (Fig. 3

Defective central B cell tolerance checkpoint in RA patients
Most polyreactive B cells in healthy donors are removed at a first checkpoint in the bone marrow between the early immature and immature B cell stage (13). Few low-polyreac-  The frequency of polyreactive antibodies was also increased in the mature naive B cell compartment of five out of six RA patients and reached significance for three of them (RA01: 25%, P ϭ 0.006; RA05: 30%, P ϭ 0.004; RA06: 27.6%, P ϭ 0.0023; Fig. 4 B). Thus, most RA patients fail to efficiently remove polyreactive B cells and reveal defects at the early B cell tolerance checkpoint in the bone marrow.

Presence of naive B cells reactive to Ig and CCP in RA patients
Igs and CCP are the most common autoantigens in RA (2,4,5). Five out of six of our RA patients showed elevated anti-CCP antibody levels in their serum and only RA01 was rheumatoid factor negative (  (Fig. 5 B). Most of the antibodies that showed rheumatoid factor reactivity in either controls or patients were not specific for IgG but also recognized single-stranded DNA, double-stranded DNA, insulin, and LPS and were therefore polyreactive (Table S2 available (13,17). Percentages represent frequency of polyreactive antibodies in each fraction. *, The controls for new emigrant and mature naive B cells isolated from HD01, HD02, HD03, and HD04 were previously reported (13,14).

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for half of the patients (RA01: 19.2%, P ϭ 0.016; RA03: 14.8%, P ϭ 0.041; and RA04: 14.0%, P ϭ 0.037; Fig. 5 C). The percentage of anti-CCP antibodies remained elevated and ranged from 10 to 17.9% in the mature naive B cell compartments of RA patients (Fig. 5 D). Anti-CCP antibodies showed low levels of reactivity when compared with serum anti-CCP IgG antibodies from positive controls (Fig. 5, C and D). Most of the antibodies that reacted to CCP were in fact polyreactive antibodies (Tables S2-S13 available at http: //www.jem.org/cgi/content/full/jem.20042321/DC1). However, none of the polyreactive antibodies from healthy individuals recognized CCP (Fig. 5 C). We conclude that a fraction of new emigrant and mature naive B cells from RA patients express polyreactive antibodies that can recognize RA-specific antigens IgG and CCP with low affinity.
RA antibodies with unusual Ն11-amino acid-long Ig CDR3s are frequently self-reactive The proportion of B cells expressing antibodies with unusual Ն11-amino acid-long Ig CDR3s is increased in most RA patients (11,12). We analyzed the reactivity of those unusual clones and found that most antibodies containing Ն11-amino acid-long CDR3 Ig chains were either HEp-2reactive and/or polyreactive (77.8%, 14 out of 18; Table II (13). Most polyreactive antibodies enriched in RA peripheral B cells are also reactive with RA-specific antigens and show low rheumatoid factor and anti-CCP reactivity. This early break in B cell tolerance may explain why RA patients relapse after successful treatment with anti-CD20 monoclonal antibodies that target immature and mature B cells but not their precursors (10). The second peripheral checkpoint that accounts for additional removal of autoreactive B cells in the periphery between the new emigrant and the mature naive B cell stage is also defective, and allows the accumulation of a large number of self-reactive B cells in the mature naive B cell compartment of all RA patients. What are the defective mechanisms that may be responsible for the early break in B cell tolerance in RA patients? The biased repertoires observed in our RA patients were found in new emigrant B cells and may reflect abnormal bone marrow B cell development. In the mouse, three central mechanisms are responsible for self-reactive antibody silencing: deletion, anergy, and receptor editing (18)(19)(20)(21)(22). Light chain antibody sequences from RA01, RA02, and RA08 patients showed an increase in downstream V genes associated with the most upstream J, J1, suggesting inefficient secondary recombination and therefore a potential defect in receptor editing. Consistent with our data, defects in secondary recombination have also been reported in other rheumatic diseases such as systemic lupus erythematosus (SLE) and may therefore account for the increased frequency of polyreactive and autoreactive B cells in the periphery (23,24). In contrast, Ig chains from RA06 new emigrant B cells showed an increase in upstream V genes associated with downstream Js suggesting extensive secondary recombination in those cells. Interestingly, extensive secondary recombination has also been reported in B cells from an untreated SLE patient and suggests that it can be an abnormal mechanism shared by different rheumatic diseases (25). BCR signaling plays an important role in controlling receptor editing by regulating recombination activating genes in mice and in humans (14,(26)(27)(28). Defects in BCR signaling may result either in a failure to induce receptor editing or in a premature termination of secondary recombination at the immature B cell stage. Alternatively, other BCR signaling defects may fail to terminate secondary recombination. X-linked agammaglobulinemia (XLA) patients suffer from defective BTK genes, which encode a cytoplasmic tyrosine kinase that plays an essential role in mediating BCR signaling (29,30). We recently reported that XLA B cells showed extensive secondary recombination events and expressed autoreactive BCRs thereby demonstrating that proper BCR signaling is essential in regulating thresholds for human B cell tolerance (14). Little is known about BCR signaling defects in RA B cells, but abnormal BCR signaling has been reported in SLE B cells that also suffer from defective B cell tolerance (31,32). More recently, a missense polymorphism in the protein tyrosine phosphatase PTPN22 gene have been shown to segregate with RA and SLE patients (33,34). This missense polymorphism in PTPN22 disrupts its interaction with Csk tyrosine kinase and potentially alters these proteins' normal function as regulators of BCR signaling (33).
Patients RA03, RA04, RA05, RA07, and RA09 do not show any significant evidence of defective secondary recom- bination, illustrating the heterogeneity of rheumatic diseases. However, those RA patients like others showed an increased proportion of new emigrant B cells expressing Ն11-amino acid-long CDR3 Ig chains (11,12). Interestingly, Ն11amino acid-long CDR3 Igs were enriched in new emigrant B cells but not in mature naive B cells. The disappearance of those unusual clones from the mature naive B compartment in the peripheral blood may reflect a counterselection step still functional in RA patients. Alternatively, B cells expressing Ն11-amino acid-long CDR3 Igs may not be counterselected but migrate to the joints of RA patients where they accumulate (12). In line with this hypothesis, antibodies with Ն11-amino acid-long CDR3 Igs were frequently found to be self-reactive and may recognize joint antigens. The production of 11-amino acid-long CDR3 Ig chains mostly requires the addition of nontemplate nucleotides to V-J junctions by the terminal deoxynucleotidyl transferase (TdT) whose expression is normally down-regulated by preBCR expression at the preB cell stage (35,36). Abnormal BCR signaling may fail to down-regulate TdT expression and may therefore result in the generation of those 11-amino acid-long CDR3 Ig chains as reported in some XLA patients (14,37). Alternatively, light chain gene rearrangements at the pro-B cell stage when TdT is expressed may generate 11-amino acid-long CDR3 Ig chains found in RA patients (38). Autoreactive B cells that escape central B cell tolerance are normally counterselected in the periphery and fail to enter the long-lived mature naive B cell pool (39,40). The appearance of autoreactive B cells in the periphery of RA patients may result from either defective negative or abnormal positive selection processes that are believed to control peripheral B cell tolerance (41)(42)(43). Interestingly, serum BAFF levels have been recently shown to regulate autoreactive B cell deletion by rescuing self-reactive B cells from cell death (44,45). Patients with rheumatic diseases including RA have been reported to display elevated BAFF serum levels that may therefore result in the survival of unusual autoreactive B cell clones in the blood of RA patients (46).
How could mature naive autoreactive B cells contribute to RA? A large number of mature naive B cells from RA patients express autoreactive and polyreactive antibodies that can recognize classical RA autoantigens with low affinity. A recent report showed that similar autoreactive/polyreactive mature naive B cells accumulated in the periphery of SLE patients (32). Genetic and environmental factors may select and activate those autoreactive/polyreactive mature naive B cells in a differential manner in RA and SLE patients. This would allow the development of B cells secreting isotypeswitched antibodies with high affinity to self-antigens specific for either RA or SLE. For instance, polyreactive B cells that recognize Igs may bind IgG-chromatin immune complexes in the joints of RA patients. Dual engagement of BCR and toll-like receptors such as TLR9 may activate such B cells and induce their development into rheumatoid fac-tor-producing B cells (47,48). Polyreactive RA B cells that recognize CCP may bind and internalize citrullinated peptides that can interact with RA-associated HLA-DRB1*0401 MHC class II molecules and activate specific autoreactive T cells (49). In return, those T cells would provide activation signals to CCP-reactive B cells and induce their proliferation and eventually the formation of ectopic germinal centers, resulting in the production of specific high affinity anti-CCP autoantibodies (50,51).
In conclusion, the antibody repertoire heterogeneity of our RA patients reflects different defective mechanisms of B cell tolerance resulting from potentially diverse RA genetic polymorphisms. Yet all six patients showed defective tolerance checkpoints and the presence of a reservoir of self-reactive mature naive B cells, likely contributing to RA pathogenesis.

MATERIALS AND METHODS
Patients. Patient 1 (RA01) is a 60-yr-old black woman who was diagnosed with RA in 1990 with symmetric polyarticular symptoms and severe morning stiffness. We first met her after 13 yr of intermittent treatment with steroids, hydroxychloroquine, and methotrexate but she had no treatments in the previous 5 mo (Table I). Initial data included an erythrocyte sedimentation rate of 127, a negative rheumatoid factor (Ͻ20), a slight elevation in anti-CCP titer (32), and multiple erosions on radiographs. Patient 2 (RA02) is a 62-yr-old Asian man who presented with 2 yr of increasing right shoulder, left elbow and bilateral wrist/hand pains, and years of increasing right knee pain. His medications at presentation were celecoxib, atenolol, and omeprazole. Data were significant for an erythrocyte sedimentation rate of 14 but elevated levels of rheumatoid factor (848) and anti-CCP antibodies (88), and radiographs with erosions and an effusion in the left elbow, and erosions in the right wrist and left IP joints. Patient 3 (RA03) is a 62-yr-old Hispanic woman who presented 3 yr prior with symmetrical polyarticular joint swelling/pain and morning stiffness, and a diagnostic work up consistent with RA. Treated intermittently with methotrexate and steroids, she stopped all of her medications 4 mo before the most recent flare because she was feeling well and presented with severe pain and swelling in both her upper and lower extremities. Medications on presentation included Prevacid and naproxen. Significant data included high titers of rheumatoid factor (1,110) and anti-CCP antibodies (240), and normal radiographs. Patient 4 (RA04) is a 36-yr-old white woman who presented with 1 yr of increasing pain and morning stiffness in her hands and elbows, but had refused all treatment. Workup included elevated levels of rheumatoid factor (55) and anti-CCP antibodies (63) but no radiographic erosions. Patient 5 (RA05) is a 30-yr-old white woman who presented with 2 yr of increasing left wrist and ankle pain and swelling, but had not taken any medication. Data included an elevated rheumatoid factor (48) but an anti-CCP antibody titer Ͻ20, with erosions (only seen with MRI) in both joints. Patient 6 (RA06) is a 64-yr-old black woman who presented with 2 yr of increasing pain and swelling in the right hand and shoulder, and polyarticular arthralgias, but had not been on any medication. Data included elevated titers of rheumatoid factor (474) and anti-CCP antibodies (Ͼ250), but no radiographic erosions. Patient 7 (RA07) is a 44-yr-old black woman who presented with 2 yr of increasing pain and swelling in her hands and knees, and had not improved while on hydroxychloroquine for 2 mo. Data included elevated titers of rheumatoid factor (267) and anti-CCP antibodies (218), but no radiographic erosions. Patient 8 (RA08) is a 28-yr-old Hispanic male who first presented 2 yr prior with morning stiffness and symmetrical swelling of the hands, wrists, knees and ankles, and responded well to steroids, methotrexate, and hydroxychloroquine. He stopped the medication 10 mo before representing with 2 wk of polyarticular arthritis more severe than on initial presentation, with elevated titers of rheumatoid factor (258) and anti-CCP antibodies (80) but no radiographic erosions. Patient 9 (RA09) is a 52-yr-old Hispanic female who presented with 1 yr of pain and swelling in her hands, wrists, knees, and ankles, but had received no medication. Data included elevated titers of rheumatoid factor (36) Table S1; 13,14). All samples were collected after patients signed informed consent in accordance with IRB-reviewed protocols.
cDNA, RT-PCR, antibody production, and purification. RNA from single cells was reverse transcribed in the original 96-well plate in 12.5 l reactions containing 100 U of Superscript II RT (GIBCO BRL) for 45 min at 37ЊC. RT-PCR reactions, primer sequences, cloning strategy, expression vectors, antibody expression, and purification were as described (13). Ig sequences were analyzed by Ig BLAST comparison with GenBank/ EMBL/DDBJ. Heavy chain CDR3 was defined as the interval between the conserved cysteine at position 92 in the V H framework 3 and the conserved tryptophan at position 103 in J H segments.
ELISAs and IFAs. Antibody concentration, reactivity against specific antigens, and IFAs were as described previously (13). High (polyreactive ED38) and weak (mGO186) HEp2-reactive and -nonreactive mGO53 and iGO13 were used as positive and negative controls in HEp2-reactivity and polyreactivity ELISAs (13,17). Those control antibodies established that recombinant antibodies with absorbance values above 0.4 could be considered reactive with the coated antigen. Antibodies were polyreactive when they recognized at least two and usually all of the four analyzed antigens that include single-stranded DNA, double-stranded DNA, insulin, and LPS. Rheumatoid factor reactivity was determined by ELISA using rabbit whole IgG as antigens and recombinant antibody binding was revealed using peroxidase-conjugated rabbit anti-human IgG (Jackson ImmunoResearch Laboratories, Inc.). Anti-CCP reactivity was determined using QUANTA Lite TM CCP ELISA plates (INOVA Diagnostics, Inc.).

Statistical analysis.
Fisher exact tests were used to analyze dichotomized data. Js were dichotomized into J1, the most upstream of all Js used in primary recombination events, versus all the other Js whose usage may involve secondary recombination events. Student's t tests were used to analyze the differences in proportions of Ն11-amino acid-long Ig CDR3s between healthy donors and RA patients. All statistical tests were considered two-tailed and probability values of P Ͻ 0.05 were considered to be statistically significant.
Online supplemental material. Healthy donor characteristics are shown in Table S1. Antibody characteristics from RA patients and control B cells are presented in Tables S2-S20. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20042321/DC1.