The inflammatory autoimmune disease rheumatoid arthritis (RA) has classically been thought to be mediated by T cells, either directly, by infiltration of tissues, or indirectly, through the release of inflammatory cytokines (1, 2). It is becoming increasingly 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 of 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.
A promising strategy for treating RA relies on B cell depletion using a chimeric monoclonal antibody directed against the B cell–specific cell surface marker CD20 (rituximab) (4). The addition of rituximab to the treatment regimen was shown to reduce autoantibody levels and improve clinical signs and symptoms in the majority of RA patients, with some showing 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 cotherapeutic strategy aimed at inhibiting 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.
Researchers in our laboratory recently 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 interferon-γ (IFNγ)–inducible enzyme that catalyzes the initial and rate-limiting step in tryptophan degradation (11). 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 pharmacologic inhibitor 1-methyltryptophan (1-MT) led to reduced levels of inflammatory cytokines, diminished titers of autoantibodies, 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, it resulted from a diminished autoreactive B cell response (10). This work demonstrated a previously unappreciated role of IDO in stimulating B cell responses; however, the role of IDO in B cell activation remained unknown.
In the present study, we used Ig-transgenic mice to define the stage at which B cell activation is influenced by IDO. We demonstrated that IDO activity is involved in the differentiation of autoreactive B cells into antibody-secreting cells (ASCs) 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 initiation of the autoimmune response. Thus, inhibitors of IDO activity should be most useful therapeutically at the initiation of autoreactive B cell responses. We proposed that inhibition of IDO activity at this critical stage would prevent the establishment of the autoreactive B cell profile, thereby reducing subsequent joint inflammation and damage. To test this hypothesis, we combined 1-MT treatment with therapeutic B cell depletion using antibodies to CD20. We demonstrated that the addition of 1-MT 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 for use in conjunction with B cell depletion therapy in the treatment of RA.
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Autoantibodies are the hallmark of many autoimmune diseases, including RA (20). One of the most promising strategies for controlling 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). New strategies for inhibiting the activation of autoreactive B cells upon repopulation would clearly help to increase the therapeutic effectiveness of B cell depletion with a CD20-specific antibody. Recently, we identified the IDO pathway as a major contributor to autoantibody production in a mouse model of RA (10). Inhibition of IDO with 1-MT attenuated arthritis progression by reducing autoantibody levels, suggesting an important role of IDO in driving autoreactive B cell responses.
In the present study, we used Ig-transgenic mice to show that IDO activity is essential for the differentiation of autoreactive B cells into ASCs but is not necessary for the initial stages of B cell activation. Furthermore, the addition of 1-MT to B cell depletion therapy prevented the reemergence 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 cotherapeutic 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 1-MT does not inhibit antibody production by purified B cells in vitro (10), suggesting that 1-MT may affect B cell differentiation indirectly by affecting the microenvironment in which the B cells are being activated. In support of this, we found that the levels of IL-4, IL-6, IL-10, and IL-13, all of which are cytokines that are necessary for B cell antibody production, were decreased in 1-MT–treated mice. Treatment with 1-MT inhibited ASC formation at the late/post–germinal center stage but did not appear to affect long-lived plasma cells, since the titers of Ig and the numbers of ASCs were not diminished in mice treated with 1-MT after the onset of arthritis. This stage-specific effect could be advantageous, in that 1-MT treatment would affect only the generation of newly formed ASCs and would not inhibit memory responses to pathogens to which acquired immunity has occurred through vaccination or prior exposure.
A positive role of IDO in driving B cell–mediated autoimmune responses is in contrast to the traditional view of IDO as having a suppressive function in T cell–mediated immunity (21–24). These findings may have implications for the development of the IDO inhibitor 1-MT as a clinical agent. Currently, 1-MT is in early-stage clinical testing as an anticancer therapeutic agent (25). Based on the presumed inhibitory action of IDO against T cells, one concern has been that the use of 1-MT might induce severe autoimmune-based toxicities. The use of 1-MT did exacerbate symptoms in some models of induced autoimmunity (26–28). However, there is no evidence of spontaneous autoimmunity resulting from 1-MT treatment in nonautoimmune mouse models (29), and our findings in the K/BxN mouse model of RA actually showed reduced autoantibody levels and evidence of improvement in inflammatory autoimmune symptoms with 1-MT treatment (10). Taken together with our findings demonstrating the role of IDO 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 cotherapeutic 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 RA patients, in whom 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 RA flare (31). Even when multiple treatment cycles are possible, long-term B cell depletion therapy may not be desirable. Although the problem is not as significant as that associated with other biologic therapies such as use of TNF-blocking agents, patients receiving B cell depletion therapy exhibit a higher risk of infections (31, 32). Furthermore, as B cells provide critical immune functions outside of their ability to produce antibodies, including cytokine secretion and antigen presentation, depletion of the entire B cell repertoire will adversely affect 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. Combination therapy with anti-CD20 plus 1-MT therefore has the potential to benefit RA patients by both eliminating pathogenic B cells that are already present and preventing the generation of new ones.