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Define the role indoleamine-2,3-dioxygenase (IDO) plays in driving pathogenic B cells responses leading to arthritis and determine if inhibitors of the IDO pathway can be used in conjunction with B cell depletion therapy to prevent the re-emergence of autoantibodies and arthritis following reconstitution of the B cell repertoire.
Immunoglobulin transgenic mice were treated with the IDO inhibitor 1-methyl-tryptophan (1MT) and followed for the extent of autoreactive B cell activation. Arthritic mice (K/BxN) were treated with B cell depletion therapy alone or in combination with 1MT. Mice were followed for the presence of autoantibody secreting cells, inflammatory cytokines, and joint inflammation.
1MT did not affect the initial activation or survival of autoreactive B cells, but did inhibit their ability to differentiate into autoantibody secreting cells. Treatment with anti-CD20 depleted the B cell repertoire and attenuated arthritis symptoms; however, arthritis symptoms rapidly returned as B cells repopulated the repertoire. Administration of 1MT prior to B cell repopulation prevented the production of autoantibodies, inflammatory cytokines, and flare in arthritis symptoms.
IDO activity is essential for the differentiation of autoreactive B cells into antibody secreting cells, but is not necessary for their initial stages of activation. Addition of 1MT to B cell depletion therapy prevents the differentiation of autoantibody secreting cells and recurrence of autoimmune arthritis following reconstitution of the B cell repertoire. These data suggest that IDO inhibitors could be used in conjunction with B cell depletion as an effective co-therapeutic strategy in the treatment of rheumatoid arthritis.
The inflammatory autoimmune disease rheumatoid arthritis (RA) has classically been thought to be mediated by T cells, either by direct infiltration of tissues or indirectly through release of inflammatory cytokines (1, 2). Increasingly, it is becoming apparent that B cells also play a critical role in driving inflammatory autoimmunity in RA (3). In addition to producing pathogenic autoantibodies, B cells can trigger autoimmune responses through the presentation of self-reactive antigens to T cells and the production of inflammatory cytokines. The most convincing evidence supporting the role for B cells in RA is the recent success of B cell-mediated therapies (4). However, the factors important in initiating and maintaining autoreactive B cell responses remain unknown.
An exciting new strategy to treat RA relies on B cell depletion using a chimeric monoclonal Ab directed against the B cell-specific cell surface marker CD20 (Rituximab) (4). The addition of Rituximab to the treatment regimen led to reduced autoantibody levels and clinical improvement in the majority of patients, with some showing a complete resolution of inflammation (5). Similarly, B cell depletion has been shown to be effective in several mouse models of arthritis (6, 7). Unfortunately, the primary limitation of B cell depletion therapy in both humans and mice is that eventually the B cells return and the repopulation of the B cell repertoire correlates with the return of arthritis symptoms in many individuals (8, 9). A co-therapeutic strategy to inhibit the activation of autoreactive B cells upon repopulation would help to lengthen the effectiveness of the therapeutic window and could improve clinical outcomes in RA patients.
Recently, our laboratory has identified indoleamine-2,3-dioxygenase (IDO) as an important factor in driving the initial stages of B cell-mediated autoimmune responses (10). IDO is an IFN-γ inducible enzyme that catalyzes the initial and rate-limiting step in tryptophan degradation (11). In humans, elevated tryptophan degradation has been shown to correlate with disease activity in RA patients (12). Likewise, we have shown that IDO activity is highest during the acute phase of disease in the K/BxN mouse model of inflammatory joint disease (10). Inhibition of IDO activity in K/BxN mice with the pharmacological inhibitor, 1-methyl-tryptophan (1MT) led to reduced levels of inflammatory cytokines, diminished autoantibody titers, and an attenuated course of disease. This alleviation of arthritis was not due to a reduction in regulatory T cells or an altered T helper cell phenotype, but rather resulted from a diminished autoreactive B cell response (10). This work demonstrated a previously unappreciated role for IDO in stimulating B cell responses; however the role that IDO played in B cell activation remained unknown.
Here, we use Ig transgenic (tg) mice to define the stage at which B cell activation is influenced by IDO. We demonstrate that IDO activity is involved in the differentiation of autoreacitve B cells into antibody secreting cells, but is not required for the initial stages of B cell activation or germinal center formation. This suggests that IDO plays a role in establishing the autoreactive B cell profile at the onset of the autoimmune response. As such, inhibitors of IDO activity should be most useful therapeutically at the initiation of autoreactive B cell responses. We propose that inhibition of IDO activity at this critical stage will prevent the establishment of the autoreactive B cell profile, thereby reducing subsequent joint inflammation and damage. To test this hypothesis, we combine 1MT with B cell depletion therapy using antibodies to CD20. We demonstrate that the addition of 1MT inhibits the differentiation of autoantibody secreting cells following B cell depletion therapy and prevents the recurrence of autoimmune arthritis. These data suggest that inhibition of the IDO pathway could be an effective strategy to use in conjunction with B cell depletion therapy in the treatment of RA.
KRN TCR tg and VH147 Ig tg mice on a C57BL/6 background have been described (13, 14). NOD mice were purchased from Jackson Laboratories. To obtain arthritic mice, KRN and VH147/KRN tg C57BL/6 mice were crossed with NOD mice yielding KRN and VH147/KRN (C57BL/6 x NOD)F1 mice designated K/BxN and VH147 K/BxN, respectively. All mice were bred and housed under specific pathogen free conditions in the animal facility at the Lankenau Institute for Medical Research. Studies were performed in accordance with National Institutes of Health and Association for Assessment and Accreditation of Laboratory Animal Care guidelines with approval from the LIMR Institutional Animal Care and Use Committee.
Mice were given 400 mg/kg/dose (100μl total volume) of D/L-1MT (Sigma) diluted in Methocel/Tween (0.5% Tween 80, 0.5% methylcellulose (v/v in water) twice daily by oral gavage (p.o.), a single 200μg dose (200μl total volume) i.p. of anti-CD20 Ig (clone 5D2, Genentech) or isotype control antibody (mouse IgG, Jackson Immunoresearch) diluted in PBS, or a combination of 1MT + antibody. Control mice were given an equal volume of carrier alone (Methocel/Tween).
The two rear ankles of K/BxN mice were measured axially across the ankle joint using a Fowler Metric Pocket Thickness Gauge. Ankle thickness was rounded off to the nearest 0.05mm. At the termination of the experiment, ankles were fixed in 10% buffered formalin for 48 hrs, decalcified in 14% EDTA for 2 wks, embedded in paraffin, sectioned, and stained with H&E.
Cells from the draining lymph nodes (LNs) were plated at 4 × 105 cells per well and diluted serially 1:4 in Multiscreen HA mixed cellulose ester membrane plates (Millipore) coated with GPI-his (5μg/ml). The cells were incubated on the Ag-coated plates for 4hr at 37°C. The Ig secreted by the plated cells was detected by Alkaline Phosphatase-conjugated goat anti-mouse total Ig secondary Ab (Southern Biotechnology Associates) and visualized using NBT/BCIP substrate (Sigma).
Serum samples were plated at an initial dilution of 1:100 and diluted serially 1:5 in Immunlon II plates coated with GPI-his (5μg/ml). Donkey anti-mouse total Ig-HRP (Jackson Immunoresearch) was used as a secondary Ab. Ab was detected using ABTS substrate (Fisher). The serum titer was defined as the reciprocal of the last dilution that gave an OD>3x background.
LN cells were harvested and cultured (2×106/ml) in either media alone or PMA (50 ng/ml) + ionomycin (500 ng/ml) for 24 h. The supernatants were then harvested and analyzed for the levels of cytokines by cytometric bead array (BD Biosciences). The samples were stained according to manufacturer instructions and analyzed on a FACSCanto II flow cytometer (BD Biosciences) using FACSDiva software (BD Biosciences). Cytokine concentrations were calculated by comparing to standard curves using FACS array analysis software (BD Biosciences).
1×106 spleen or joint draining LNs cells were stained with recombinant glucose-6-phosphate isomerase (GPI)-his and Annexin-V or antibodies to B220, CD25, CD69, CD80, CD86, and MHC Class II. Peripheral blood was collected from control, 1MT, anti-CD20, and anti-CD20 + 1MT treated K/BxN mice and stained with antibodies to pan CD45 and the B cell-specific isoform B220. Samples were analyzed on a FACSCanto II flow cytometer using FACSDIVA software. Data was analyzed using Cellquest software (BD Biosciences). Gating on live lymphocytes was based on forward and side scatter, with 100,000 events collected for each LN and spleen sample and 2,000 events for each blood sample.
Statistical significance was determined using an unpaired Student’s t test or the Mann-Whitney nonparametric test and Instat Software (GraphPad Software, Inc)
Our previous work demonstrated that the anti-arthritic effect of 1MT in K/BxN mice was due to its inhibition of the pathogenic B cell response, suggesting that IDO plays an important role in driving B cell-mediated autoimmunity (10). To define the mechanism by which IDO shapes the autoreactive B cell response leading to arthritis, it is important to determine the step(s) at which 1MT affects B cell activation and/or differentiation. There are several steps in the B cell response that could be affected by 1MT treatment that could each result in a diminished autoantibody response. These include the initial activation of the autoreactive B cells, their recruitment into germinal centers, or later in the response when they terminally differentiate into antibody secreting cells (ASCs). In the K/BxN model, the autoantigen targeted by the pathogenic B cells is the glycolytic enzyme, glucose-6-phosphate isomerase (GPI) (15). To follow anti-GPI B cells specifically, we used VH147 Ig tg mice that have an increased frequency of anti-GPI B cells in the pre-immune repertoire (14). Use of these mice allows anti-GPI B cells to be tracked prior to activation, through the initial stages of activation, as well as once the immune response is underway, an impossibility without the Ig tg (16). Previously, we showed that anti-GPI B cells in non-autoimmune mice were not rendered tolerant. Instead, the anti-GPI B cells in the recirculating follicular/ LN B cell pool remained naive, although they showed clear evidence of antigen encounter. Importantly, these anti-GPI B cells could be induced to secrete high levels of autoantibodies in response to cognate T cell help when bred onto the autoimmune K/BxN background (VH147 K/BxN) (14).
To determine if 1MT treatment affected the initial activation of anti-GPI B cells, VH147 K/BxN mice were treated with 1MT or carrier alone. At the peak of arthritis, the tg anti-GPI B cells from the spleen and joint draining LNs were analyzed by flow cytometry, gating on GPI-binding B cells. No differences were detected in the percentage of GPI-binding B cells in the spleen (Carrier: 25.4 ± 2.3%; 1MT: 25.9 ± 3.2%, p>0.9) or LNs (Carrier: 8.6 ± 1.1%; 1MT: 8.4 ± 2.1%, p=0.9) in 1MT compared to carrier-treated mice. Compared to non-tg B cells (Fig. 1) and anti-GPI B cells in non-arthritic mice, which remain naive (10), anti-GPI B cells in arthritic VH147 K/BxN mice showed signs of activation. They expressed elevated levels of the early activation markers CD25 and CD69, costimulatory molecules CD80 and CD86, and MHC Class II (Fig. 1a). Anti-GPI B cells in 1MT-treated VH147 K/BxN mice also expressed elevated levels of these activation markers that were indistinguishable from those in carrier-treated mice (Fig. 1a), indicating that 1MT did not block this initial stage of B cell activation.
It is possible that IDO activity was not necessary for the activation of anti-GPI B cells, but instead was required for B cell survival, such that blocking IDO activity with 1MT resulted in B cell apoptosis. To address this possibility, we measured the percentage of apoptotic GPI-binding B cells in carrier vs. 1MT-treated VH147 K/BxN mice using Annexin-V and flow cytometry. While a greater percentage of B cells in VH147 tg mice were Annexin-V+ compared to non-tg mice (Non-tg: 0.9 ± 1.5%; VH147 tg: 7.4 ± 4.0%) no differences were detected between those in VH147 tg mice receiving 1MT (8.8 ± 0.7%) and carrier (7.4 ± 4.0%). Therefore, neither the initial stages of anti-GPI B cell activation nor their survival are affected by 1MT treatment.
We next addressed whether 1MT inhibited the recruitment of anti-GPI B cells into germinal centers (GC) or their differentiation into ASCs. Recruitment into GCs was measured by flow cytometry using the GC marker PNA. GC B cells were readily detectable among the GPI-binding B cell population (Fig. 1b). Similar percentages of GPI-binding B cells were PNAhigh in the spleens (Carrier: 21.7 ± 4.6%; 1MT: 22.7 ± 5.8, p>0.9) and draining LNs (dLN) (Carrier: 18.3 ± 6.2%; 1MT: 15.5 ± 6.9%, p=0.3) of 1MT and carrier-treated VH147 K/BxN mice (Fig. 1b). To measure the number of anti-GPI B cells that differentiated into ASCs, joint dLN samples from 1MT and Carrier-treated VH147 K/BxN mice were quantitated by ELISpot (Fig. 1c). High numbers of GPI-reactive ASCs were present in the dLNs of carrier-treated VH147 K/BxN mice (174 ± 45). In contrast, very few anti-GPI ASCs were detectable in 1MT-treated VH147 K/BxN mice (23 ± 7). As expected, no anti-GPI ASCs were found in the dLNs of either carrier (0.3 ± 0.3) or 1MT-treated (0.2 ± 0.2) control non-arthritic VH147 BxN mice. Taken together, these data demonstrate that IDO activity is not required for the initial activation, survival, or recruitment of GPI-reactive B cells into GCs, but instead exerts its effect later in their differentiation into autoantibody secreting cells.
The experiments using VH147 Ig tg mice show that IDO plays an activating role in establishing the autoreactive B cell profile at the onset of the autoimmune response. As such, inhibitors of IDO activity will be most useful prior to the activation of the autoreactive B cell repertoire. Therapeutically, this critical stage in B cell activation can be reinstated using B cell depletion with a chimeric monoclonal Ab directed against the B cell-specific cell surface marker CD20 (17). Because anti-CD20 Ig specifically targets immature, naive, and memory B cells, the B cell repertoire will be depleted starting at the precursor B cell stage, effectively “rebooting” the immune system (17). This is an ideal situation to test whether 1MT is able to inhibit the establishment of the autoreactive B cell profile in a therapeutic setting where the initiation phase of the autoimmune response has been reinstated. We hypothesized that administration of 1MT would inhibit the reactivation of autoreactive B cells as they are regenerated, thus preventing the recurrence of autoimmune arthritis.
Before we could test the efficacy of anti-CD20 + 1MT combination therapy, we first needed to determine whether anti-CD20 could inhibit arthritis in the K/BxN model, and if so, the timeframe in which anti-CD20 depletion remained effective. To do this, K/BxN mice were treated with anti-CD20 Ig or isotype control antibody at two different timepoints, starting either after the onset of arthritis symptoms (4–5 wk of age) or prior to the initiation of arthritis (3 wk of age). In the first strategy, K/BxN mice were allowed to develop arthritis and then were treated with anti-CD20 or an isotype control antibody (Fig. 2a). Given this treatment regimen, anti-CD20 had only a minimal effect on arthritis progression. This is similar to findings using anti-human CD20 to deplete B cells in K/BxN mice expressing a human CD20 tg (18). In the second strategy, K/BxN mice were treated with anti-CD20 or isotype control antibody prior to arthritis onset (Fig. 2b). Anti-CD20 treatment did not affect the time of arthritis onset; however, within 14 days (5 wk of age), the anti-CD20 group began to show reduced arthritis symptoms. This reduction in ankle inflammation continued until 6–7 wk of age, when arthritis dramatically flared. The half-life of anti-CD20 in the serum has been reported to be approximately 4–5 days, with the B cell repertoire returning to full strength in approximately 4–6 weeks (19). Therefore, it was possible that the flare in arthritis seen 4 weeks post-treatment was due to the return of autoreactive B cells to the repertoire.
To determine the effectiveness of B cell depletion and importantly, the timing of reappearance of B cells following depletion, B cell percentages in the peripheral blood were monitored by flow cytometry. By 1 week post antibody treatment, B cell numbers were significantly decreased in anti-CD20 treated mice compared to those receiving isotype control Ig (Fig. 2c). B cells began to approach normal levels approximately 3 wks following anti-CD20 treatment and were close to levels in control mice by 5 wks post-Ab treatment. This timing of B cell repopulation did indeed coincide with the flare in arthritis seen in Fig. 2b. Therefore, the data from these initial experiments demonstrated that while anti-CD20 treatment was unable to prevent the initiation of arthritis, it was able to halt the progression of arthritis, provided treatment was begun prior to the onset of joint inflammation. Furthermore, these experiments established a timecourse of B cell depletion in this model, with both B cells and arthritis symptoms reappearing 3 weeks after anti-CD20 treatment.
As a single agent therapy administered prior to arthritis onset, 1MT inhibited B cell differentiation into ASCs. We hypothesized that administration of 1MT would also inhibit the differentiation of autoreactive B cells as they were regenerated after B cell depletion therapy, thus preventing the recurrence of autoimmune arthritis. To test this hypothesis, K/BxN mice were treated with anti-CD20 prior to arthritis onset. Three weeks later, before the B cell population returned to normal levels, the mice were treated with 1MT. The administration of 1MT affected neither the depletion nor the repopulation of B cells in the anti-CD20 treated mice (Fig. 3a). After 3 weeks of 1MT treatment, GPI-specific ASCs were measured in the joint dLNs by ELISpot (Fig. 3b). As expected, large numbers of anti-GPI ASCs were present in the dLNs of isotype control treated mice (330 ± 70). In contrast to the reduction in ASC formation seen when 1MT is administered before arthritis develops (Fig. 1 and ref. (10), administration of 1MT alone after arthritis onset did not affect ASC formation (Fig. 3b, 396 ± 40; p=0.3). Treatment with anti-CD20 alone reduced the number of anti-GPI ASCs compared to control mice (99 ± 28; p=0.03). Importantly, administration of 1MT following anti-CD20 treatment led to an even greater reduction in the number of anti-GPI ASCs present in the dLNs (42 ± 9; p=0.05) compared to those treated with anti-CD20 alone. A similar trend in reduced anti-GPI titer was measured in the serum of anti-CD20+1MT compared to anti-CD20 alone treated mice, although the values did not reach statistical significance (Fig. 3d). These data demonstrate that, similar to its effect prior to arthritis onset, 1MT inhibits the differentiation of autoreactive B cells as they are regenerated following B cell depletion therapy.
The effectiveness of 1MT at inhibiting pathogenic ASC differentiation following B cell recovery after B cell depletion suggested that it might also inhibit the flare in arthritis observed in anti-CD20 treated mice following B cell repopulation. As shown in Fig. 2b, anti-CD20 treatment did not affect the onset of arthritis, but did diminish the severity of joint inflammation until the B cell population returned approximately 4 weeks later. At this time, arthritis in anti-CD20 treated mice flared to the level seen in control-treated mice. Strikingly, administration of 1MT just prior to the repopulation of peripheral B cells (3 wks following CD20-treatment) prevented the flare in arthritis observed in anti-CD20 treated mice (Fig. 3c). Administration of 1MT alone at this timepoint had no effect on arthritis. Likewise, control mice treated with anti-CD20 followed by carrier or isotype control Ab followed by 1MT did not show this same inhibition (Fig. 3c).
At the termination of the experiment (10wk of age), rear ankles were harvested and examined for histological evidence of arthritis by staining with hematoxylin and eosin (Fig. 4). Control-treated mice showed classic signs of arthritis, with a greatly expanded synovium, panus formation, and inflammatory cell infiltrates. CD20 alone treated mice also showed inflammatory cell infiltrates. In contrast, joints from CD20 + 1MT-treated mice showed a reduction in the severity of arthritis with minimal synovial expansion and few infiltrating inflammatory cells (Fig. 4).
The levels of cytokines implicated in arthritic responses were compared in the joint draining LNs of mice treated with anti-CD20 alone and anti-CD20 + 1MT (Fig. 5). Consistent with the reduction in arthritis, mice treated with the combination of 1MT and anti-CD20 had lower levels of the classic inflammatory cytokines IL-6, IL-17, IFNγ, MCP-1, RANTES, and TNFα compared to mice treated with anti-CD20 alone. However, other inflammatory cytokines either showed no difference in levels between the two treatment groups (MIP-1α, MIP-1β) or were detectable at minimal levels in both groups (IL-1α, IL-1β). Importantly, levels of several cytokines implicated in B cell differentiation (IL-4, IL-6, IL-9, IL-10, and IL-13) were all significantly lower in the presence of 1MT (Fig. 5). 1MT did not affect cytokine levels in control mice not treated with anti-CD20 (Fig. 6). Together, our data demonstrate that 1MT is able to inhibit the reactivation of autoreactive B cells and prevent the recurrence of arthritis following B cell depletion therapy.
Autoantibodies are the hallmark of many autoimmune diseases, including rheumatoid arthritis (RA) (20). One of the most promising strategies to control pathogenic autoantibodies is B cell depletion using a CD20-specific antibody (4). However, long-term B cell depletion is difficult to maintain and repopulation of the B cell repertoire is often accompanied by the return of arthritis symptoms (8, 9). Clearly, new strategies to inhibit the activation of autoreactive B cells upon repopulation would help increase its therapeutic effectiveness. Recently, we identified the IDO pathway as a major contributor to autoantibody production in a mouse model of RA (10). Inhibition of IDO with 1MT attenuated arthritis progression by reducing autoantibody levels, suggesting an important role for IDO in driving autoreactive B cell responses. Using Immunoglobulin transgenic mice, we show here that IDO activity is essential for the differentiation of autoreactive B cells into antibody secreting cells, but is not necessary for the initial stages of B cell activation. Furthermore, the addition of 1MT to B cell depletion therapy prevented the re-emergence of autoantibody secreting cells and arthritis symptoms following reconstitution of the B cell repertoire. Our data suggest that IDO inhibitors could be used in conjunction with B cell depletion as an effective co-therapeutic strategy in the treatment of RA.
Our data demonstrate that IDO plays a role in driving the differentiation of B cells into ASCs in vivo. However, blocking IDO activity with 1MT does not inhibit antibody production from purified B cells in vitro (10), suggesting that 1MT may affect B cell differentiation indirectly by affecting the microenvironment in which the B cells are being activated. In support of this, levels of IL-4, IL-6, IL-10, and IL-13, cytokines necessary for B cell antibody production, were all decreased in 1MT-treated mice. 1MT inhibited ASC formation at the late/post germinal center stage, but does not appear to affect long-lived plasma cells, as Ig titers and numbers of ASCs were not diminished in mice treated with 1MT after the onset of arthritis. This stage-specific effect could be advantageous in that 1MT treatment would only affect the generation of newly formed ASCs and not inhibit memory responses to pathogens to which we have acquired immunity through vaccination or prior exposure.
A positive role for IDO in driving B cell-mediated autoimmune responses is in contrast to the traditional view of IDO having a suppressive function in T cell-mediated immunity (21–24). These findings may have implications for the development of the IDO inhibitor 1MT as a clinical agent. 1MT is currently in early stage clinical testing as an anti-cancer therapeutic (25). Based on IDO’s presumed inhibitory action on T cells, one concern has been that the use of 1MT might induce severe autoimmune-based toxicities. The use of 1MT did exacerbate symptoms in some induced models of autoimmunity (26–28). However, there is no evidence of spontaneous autoimmunity resulting from 1MT treatment in non-autoimmune mouse models (29) and our findings in the K/BxN RA model actually show reduced autoantibody levels and evidence of improvement in inflammatory autoimmune symptoms with 1MT treatment (10). Therefore, together with our findings demonstrating IDO’s role in driving autoantibody production, this suggests that the potential application of IDO inhibitors may be more far reaching than is currently appreciated.
One potential new use of IDO inhibitors that our data point to is in a co-therapuetic strategy to increase the effectiveness of B cell depletion therapy. In K/BxN mice and other mouse models of RA, treatment with anti-CD20 leads to a rapid depletion of B cells from the circulating B cell repertoire (7, 18, 19). However, as the anti-CD20 antibody is cleared from the circulation, the B cell repertoire repopulates and disease symptoms return (19). This is also seen in human RA patients where the reemergence of the B cell repertoire is often accompanied by the return of arthritis symptoms (8, 9, 30). A second treatment cycle with Rituximab will sometimes, but not always, reduce the flare in arthritis symptoms (31). Even when multiple treatment cycles are possible, maintaining patients on B cell depletion therapy may not be desirable long-term. Although not as significant a problem as with other biologic therapies such as TNF blocking agents, patients on B cell depletion therapy do exhibit a higher risk of infections (31, 32). Furthermore, as B cells provide critical immune functions outside of their ability to produce antibody, including cytokine secretion and antigen presentation, depletion of the entire B cell repertoire will adversely impact the immune system as a whole. In support of this, B cell depletion in mouse models has been shown to inhibit T cell function (6, 7).
In summary, our data suggest that adding IDO inhibitors to B cell depletion therapy is an effective way to inhibit the reemergence of autoantibody secreting cells while allowing the repopulation of the B cell repertoire. Therefore, combination therapy with anti-CD20 and 1MT has the potential to benefit RA patients by both eliminating pathogenic B cells that are already present and preventing new ones from being generated.
Grant support: This project was supported by Grant Number 5-R01 AR057847-01 from NIAMS/NIH.
The authors would like to thank Drs. Flavius Martin, Andrew Chan and Qian Gong (Genentech) for the murine anti-CD20 Ig, and Drs. Lisa Laury-Kleintop, Alexander Muller, Lauren Merlo, and Sudhir Nayak for critical reading of the manuscript and thoughtful input.
The authors did not receive any financial support or benefits from commercial sources for the work reported on in the manuscript and do not have any other financial interests that could create a potential conflict of interest or the appearance of a conflict of interest with regard to the work.