Rheumatoid arthritis (RA) patients who have never received treatment for RA have been found to have defective early B cell tolerance checkpoints, resulting in impaired removal of developing autoreactive B cells. However, it is unclear whether these defects in B cell tolerance checkpoints are a primary aspect of the disease or are the result of ongoing inflammatory processes in these patients. The aim of this study was to assess the impact of standard immunosuppressive treatments, methotrexate and anti–tumor necrosis factor α (anti-TNFα) agents, on early B cell tolerance checkpoints in RA patients.
Blood samples were obtained from RA patients before and after treatment with methotrexate and/or anti-TNFα agents. B cells were tested pre- and posttherapy for reactivity of recombinant antibodies cloned from single B cells, which allowed us to determine the evolution of the frequency of autoreactive clones in the mature naive B cell compartment in RA patients before and after treatment. B cells from healthy donors were used as controls.
Posttreatment frequencies of autoreactive mature naive B cells were elevated in the blood of RA patients. Nevertheless, the frequencies after treatment remained similar to those observed in the same patients before treatment.
Despite the achievement of clinical improvement in RA patients following treatment with methotrexate and/or anti-TNFα agents, these therapies did not correct the accumulation of peripheral autoreactive mature naive B cells in these patients, suggesting that inflammation is not responsible for the defective early B cell tolerance checkpoints in RA.
The efficacy of B cell depletion with anti-CD20 therapy in the treatment of rheumatoid arthritis (RA) suggests a critical role for B cells in the pathology of RA (1), although the pathogenic mechanisms are unknown. We previously described abnormalities in early B cell tolerance checkpoints in patients with active RA who had not received treatment for RA, which deviated from the evolution of autoreactive B cell frequencies during B cell development in healthy donors (2). Random V–D–J recombination generates a large number of autoreactive B cells that are counterselected in healthy donors at 2 major checkpoints. A first checkpoint occurs in the bone marrow between the early immature and immature B cell stages, and silences most polyreactive and antinuclear antibody (ANA)–expressing B cells (3, 4). A second counterselection step takes place in the periphery, and further removes autoreactive B cells that recently emigrated from the bone marrow before they enter the long-lived mature naive B cell pool (3, 4). In clinically active RA, we found that both central and peripheral B cell tolerance checkpoints were defective, resulting in the accumulation of a large number of autoreactive B cells in the mature naive B cell compartment of these patients (2).
It remains to be determined whether defective B cell tolerance checkpoints, resulting in altered antibody reactivity selection, is a primary aspect of the disease or merely represents a byproduct of the disease induced by cytokine imbalances. Methotrexate and anti–tumor necrosis factor α (anti-TNFα) agents remain the standard of care in RA (5), although little is known about their effect on the loss of B cell tolerance and the persistence of autoreactive B cells. In the present study, we analyzed patients with RA who showed clinical improvement after either methotrexate or anti-TNFα therapy, and demonstrated that B cell tolerance checkpoints remain defective posttherapy, as indicated by the increased frequency of self-reactive mature naive B cells in these patients. This suggests that the early B cell tolerance defects in RA do not result from the production of proinflammatory cytokines, but rather are more likely due to intrinsic genetic predisposition.
PATIENTS AND METHODS
Patients and controls.
Patients with RA treated with either methotrexate (patients RA01, RA02, and RA03) or anti-TNFα agents (patients RA05, RA11, and RA24) (see Supplementary Table 1, available on the Arthritis & Rheumatism Web site at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131) were enrolled at the Hospital for Special Surgery in New York. Patients RA01 and RA03 were further studied after anti-TNFα was either added or introduced as a substitute to their initial regimen several years later. All of the patients had discontinued treatment with corticosteroids for at least 3 months prior to study start. All patients, except patients RA11 and RA24, and 1 healthy donor (a 53-year-old Hispanic woman) had been studied before any treatment (2). RA patients were compared to 6 healthy adult blood donors as controls (3, 6–8). All blood samples were collected after individuals signed informed consent forms, in accordance with Institutional Review Board–reviewed protocols.
Peripheral B cells were purified from the blood of RA patients by positive selection with anti-human CD20 magnetic microbeads (Miltenyi Biotec). CD20-enriched peripheral B cells from RA patients were stained with fluorescein isothiocyanate (FITC)–conjugated anti-human CD27, phycoerythrin (PE)–conjugated anti-human CD10, biotin-labeled anti-human IgM, and allophycocyanin (APC)–conjugated anti-human CD19 (BD PharMingen). Biotinylated antibodies were revealed using PE-Cy7–conjugated streptavidin (BD PharMingen). Single CD19+CD10−IgM+CD27− mature naive B cells were sorted on a FACSVantage sorter (Becton Dickinson) into 96-well polymerase chain reaction (PCR) plates containing 4 μl lysis solution (0.5× phosphate buffered saline containing 10 mM dithiothreitol, 8 units RNasin [Promega], 0.4 units 5′-3′-RNase inhibitor [Eppendorf]), and immediately frozen on dry ice. All samples were stored at −70°C. Additional flow cytometry analyses of Treg cells were performed using FITC-conjugated anti-CD62L, PE-conjugated CD25, PE-Cy7–conjugated CD4, and APC-conjugated CD127.
Reverse transcription (RT)–PCR analysis and production and purification of antibodies.
RNA from single cells was reverse-transcribed in the original 96-well plate in 12.5-μl reactions containing 100 units of Superscript II reverse transcriptase (Gibco BRL) for 45 minutes at 42°C. RT-PCR reactions, primer sequences, cloning strategy, expression vectors, and antibody expression and purification have been described previously (3). Ig sequences were analyzed using Ig BLAST comparison with GenBank sequences. The heavy-chain third complementarity-determining region was defined as the interval between the conserved cysteine at position 92 in the VH framework 3 and the conserved tryptophan at position 103 in JH segments.
Enzyme-linked immunosorbent assays (ELISAs) and immunofluorescence assays.
The antibody concentrations used and their reactivity against specific antigens have been described previously (3). A high (polyreactive ED38) HEp-2–reactive antibody was used as a positive control in the HEp-2 reactivity and polyreactivity ELISAs. This control antibody is used to establish that recombinant antibodies with absorbance values (expressed as the optical density at 405 nm) higher than 0.5 could be considered reactive with the coated antigen. Antibodies were polyreactive when they recognized 4 antigens, including single-stranded DNA, double-stranded DNA, insulin, and lipopolysaccharide. For detection of ANAs, HEp-2–coated slides (Bion Enterprises) were incubated with purified recombinant antibodies at 50–100 μg/ml. FITC-conjugated goat anti-human IgG was used as the detection reagent.
Impact of methotrexate and anti-TNFα agents on peripheral B cell subpopulations.
Methotrexate and anti-TNFα agents are the standard of care in RA (5). Recent studies analyzing B cell subpopulation changes in RA patients after anti-TNFα treatment have provided results that are conflicting (9, 10). Therefore, with the use of flow cytometry, we assessed the frequency of different B cell subpopulations in the blood of the same RA patients before and after therapy with methotrexate and/or anti-TNFα agents. We found that the frequency of CD19+CD10+IgMhighCD27− new emigrant/transitional B cells was not affected by any treatment, and this subset represented 1.5–8% of the total B cells in all RA patients (Figure 1, top panel).
Similarly, the frequencies of CD19+CD10−IgM+CD27− mature naive B cells and CD19+CD10−CD27+ memory B cells were not significantly altered by either methotrexate or anti-TNFα treatment (Figure 1, middle and lower panels). However, 3 of 5 patients receiving TNFα blockade showed decreased frequencies of naive B cells and an increased proportion of CD27+ circulating memory B cells (Figure 1, middle and lower panels), as has been previously reported by others (10).
Induction of expansion of the CD62L− regulatory T cell population by infliximab, but not by other TNFα blocking agents.
It has been reported that Treg cell functions may be defective in RA patients, and treatment with infliximab induces the expansion of potentially functional CD62L− Treg cells (11, 12). Consistent with these findings, we found that infliximab treatment, for a duration of 4 months in patient RA01 and 2 months in patient RA03, induced the expansion of CD4+CD25highCD127−lowCD62L− Treg cells (13, 14) (Figure 2). In contrast, we found that treatment with other anti-TNFα agents, etanercept and adalimumab, did not generate CD62L− Treg cells. Indeed, the frequency of CD62L− Treg cells remained very low and was similar to that in control healthy donors (Figure 2) and untreated RA patients. Thus, TNFα blockade in patients with RA is not responsible for the induction of CD62L− Treg cells, whose expansion seems to be specifically dependent on infliximab.
Lack of effect of methotrexate and anti-TNFα therapy on defective peripheral B cell tolerance checkpoints in RA.
Methotrexate is one of the most commonly prescribed medications in RA, and in many patients, the disease responds positively to this inexpensive agent. In addition, anti-TNFα agents have shown remarkable efficacy in the treatment of RA (15). Moreover, the success of anti–B cell therapy has demonstrated the important role of B cells in RA (1). We aimed to characterize the effects of methotrexate or anti-TNFα agents on the early defective B cell tolerance checkpoints in RA patients, to determine whether methotrexate and anti-TNFα agents can restore the removal of autoreactive B cells. We cloned and expressed in vitro 159 antibodies from single mature naive B cells from the blood of RA patients before and after treatment with either methotrexate and/or anti-TNFα agents. Antibody reactivity was tested using ELISAs and immunofluorescence assays, as previously described (3) (for full details on the repertoire and reactivity of the antibodies tested from mature naive B cells in treated and untreated patients, see Supplementary Tables 2–11, available on the Arthritis & Rheumatism Web site at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131).
We found that the frequency of HEp-2–reactive B cells from RA patients after treatment with methotrexate was 36.8–50% (mean 41%), while after treatment with anti-TNFα agents, the frequency of HEp-2–reactive B cells was 33–43.5% (mean 39%); neither of these values was significantly different from the mean frequency of 42% found in the same RA patients before treatment (Figures 3B and C and Figures 4A—C). In contrast, the frequency of HEp-2–reactive clones in healthy donors only averaged 20% (Figure 3A and Figures 4A–C).
Recombinant antibodies were also tested by ELISA for polyreactivity against 4 different antigens, as previously reported (3). We found that after treatment with methotrexate and/or anti-TNFα agents, polyreactive clones represented 20–26% of mature naive B cells. These frequencies were also similar to those of polyreactive clones in RA patients before treatment (mean frequency of 22%) (Figures 4A, B, and D and Figure 5).
In addition, the frequencies of ANA-expressing B cells in treated RA patients were similar to those in untreated patients (range 3.8–5% in both groups) and were comparable to the frequencies of ANA-expressing mature naive B cells in healthy donors (mean 3.1%) (Figure 4E), suggesting that despite the global increase in autoreactive B cells in RA patients, ANA-expressing B cells are counterselected properly in these individuals.
In summary, the proportion of autoreactive B cells in RA patients remained elevated after either methotrexate treatment or TNFα blockade. Our results demonstrate that these antiinflammatory agents do not correct the defective peripheral B cell tolerance checkpoints in patients with RA.
A pathogenic role for B cells in autoimmune diseases has been demonstrated clearly in several mouse models, and a likely mechanism of action is found in the known antibody-independent functions of B cells, such as autoreactive T cell priming through antigen-presenting cell functions and/or cytokine production (16, 17). In humans, the success of B cell–depleting anti-CD20 treatment in RA, type 1 diabetes, and multiple sclerosis also underlines an important role for B cells in the development of these autoimmune diseases (1, 18, 19).
We previously reported that untreated patients with active RA display abnormal early B cell tolerance checkpoints, resulting in the accumulation of large numbers of autoreactive B cells in the periphery. However, it remained to be determined whether defects in early B cell tolerance checkpoints in RA were a primary feature of the disease or occurred secondary to the disease. We have shown herein that methotrexate and the 3 longest-available anti-TNFα therapies, which are widely used to inhibit inflammatory disorders, do not restore early B cell tolerance in RA patients, suggesting that B cell tolerance defects do not result from an imbalance of proinflammatory cytokines in these patients. Similarly, the defects in early B cell tolerance checkpoints that have been observed in patients with active systemic lupus erythematosus (SLE) persist in patients in clinical remission (20), suggesting that disease activity is not correlated with impaired autoreactive B cell removal (21).
TNFα plays a central role in the pathology of RA by recruiting leukocytes to the joints, inducing the secretion of proinflammatory cytokines, such as interleukin-1 (IL-1), and participating in joint damage (15). TNFα may also impact early B cell development by modifying the frequency and absolute numbers of B cell subpopulations in the peripheral blood of RA patients (9, 10). However, conflicting results on B cell subpopulation alterations after anti-TNFα therapy have been reported by different groups (9, 10). These apparent discrepancies may be attributed to the use of different anti-TNFα agents (9, 10). Nonetheless, studies in which the same RA patients were followed up before and after anti-TNFα treatment, as in the present study, have tended to reach similar conclusions, that a decrease in mature naive B cells in conjunction with an increase in memory B cells occurs after anti-TNFα therapy (10).
Anti-TNFα therapy has been reported to affect Treg cells, which may have an impact on the functions of B cells and lead to suppression of autoreactive B cells (22). Indeed, the anti-TNFα agent infliximab rescues the function of Treg cells and induces the emergence of a subset of CD62L−CD4+CD25highFoxP3+ T cells (11, 23). In agreement with these findings, we also observed an increased frequency of CD62L− Treg cells in our patients after treatment with infliximab, when compared with the pretreatment frequencies. However, other anti-TNFα agents, such as etanercept and adalimumab, did not show any effects on Treg cell frequencies. The intrinsic nature of the antibodies targeted by infliximab and adalimumab (chimeric versus humanized) or the delivery routes used (intravenous versus subcutaneous) may account for these differences. Nonetheless, anti-TNFα agents do not systematically impact Treg cell homeostasis and function, suggesting that these cells are not responsible for RA patients' improvement after anti-TNFα therapy.
How do methotrexate and anti-TNFα therapies improve the clinical condition of RA patients? We showed that the antiinflammatory effects of methotrexate and TNFα blockade do not restore the defective early B cell tolerance checkpoints in RA. However, methotrexate has been shown to reduce the release of proinflammatory cytokines, such as IL-6 or TNFα, and to inhibit cell proliferation by interfering with DNA and RNA synthesis, potentially leading activated cells to apoptosis and cell death (24). In vitro, anti-TNFα agents showed cytotoxic effects on transmembrane TNFα-expressing cells (25). TNFα blockade can also induce the disruption of synovial lymphocyte aggregates and germinal centers, as well as their follicular dendritic cell network, thereby blocking B cell activation (9, 26). Thus, the achievement of clinical improvement following treatment with methotrexate and anti-TNFα therapy is likely a result of the blockade of proinflammatory cytokines and inhibition of cell activation, downstream of early B cell tolerance checkpoints.
We have thus demonstrated that the improvement in RA after treatment with methotrexate and/or TNFα blockade is likely due to the antiinflammatory effects of these drugs and not to a reestablishment of early B cell tolerance checkpoints, which, rather, may be controlled by intrinsic genetic factors. Indeed, we previously reported that B cell receptor (BCR) signaling was essential in regulating the central B cell tolerance checkpoint in humans (6). Little is known about BCR signaling defects in RA B cells, but abnormal BCR signaling has been reported in SLE B cells, which also display defective early B cell tolerance (20, 21, 27). More recently, the R620W missense polymorphism in the protein tyrosine phosphatase gene PTPN22, which segregates with RA, SLE, and type 1 diabetes, has been shown to encode overactive PTPN22 phosphatases that alter BCR signaling (28–32). Therefore, genetic alteration of the BCR signaling threshold may result in defective early B cell tolerance checkpoints, which are likely to precede the onset of autoimmune diseases and favor the development of autoimmunity by producing large amounts of autoreactive B cells.
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Meffre had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Menard, Meffre.
Acquisition of data. Menard, Samuels, Ng.
Analysis and interpretation of data. Menard, Samuels, Ng.
We thank Dr. S. Rudchenko for assistance with the single-cell sorter, and Drs. D. Goldenberg and D. Orange for providing blood samples from the study subjects.