The relative contribution of the different elements of the immune system to the pathogenesis of rheumatoid arthritis (RA) remains controversial (1, 2). The genetic link with HLA–DR4 (3) implies a T cell–dependent process. It is now evident that T cell–B cell interaction is a complex 2-way process, in which the function of each cell type is dependent on the other (4). Moreover, this interaction may, in special cases, be atypical, as for example, with rheumatoid factor (RF) B cells, which can potentially present any T cell with its cognate antigen and in return receive a positive survival signal (5).
A specific subset of autoreactive B lymphocyte clones, capable of self-perpetuation, has recently been proposed to be involved in disease persistence in RA (2). In this model, a dual role for B lymphocytes, and in particular, those committed to producing RF, has been suggested. First, they may differentiate into plasma cells that produce autoantibodies capable of forming small immune complexes. Interaction of such small immune complexes with the immunoglobulin receptor Fcγ receptor type IIIa (FcγRIIIa) on macrophages, in joints, and in other tissues may be responsible for the production of proinflammatory cytokines (6, 7). The FcγRIII-dependent arthritis of the K/BxN transgenic mouse may be a useful model for this mechanism (8). Second, daughter plasma cells may perpetuate the survival of parent RF B lymphocytes by providing a constant supply of self-complexed IgG (2).
Therapeutic B lymphocyte depletion provides a new opportunity to assess the roles of B lymphocytes in the pathogenesis of RA and other autoimmune diseases. B lymphocyte depletion has recently been introduced as a therapy for a range of autoantibody-associated disorders, including RA, IgM-associated neuropathies, immune thrombocytopenic purpura, autoimmune hemolytic anemia, systemic lupus erythematosus, and dermatomyositis (9–15). Encouraging results have been reported, and a randomized controlled trial of this therapy in RA is currently in progress.
Selective B lymphocyte depletion has been made possible by the availability of the chimeric anti-CD20 monoclonal antibody rituximab (16–21). CD20 is a B lymphocyte–restricted antigen that is expressed on B lymphocyte precursors and mature B lymphocytes. It is lost during differentiation into plasma cells. Rituximab has been proven to be very effective in depleting normal and malignant B lymphocytes in vivo. In humans, peripheral B cell depletion occurs within days, and studies in primates have shown that up to 70% of B cells in lymphoid organs are also rapidly cleared (16, 17, 21). In autoimmune diseases, rituximab has been used in many cases as monotherapy (10–12). However, B lymphocyte depletion with rituximab alone is probably incomplete, and in patients with lymphoma, long-term benefit from rituximab is increased by combination with other drugs (20). For these reasons, in the treatment of RA, it has initially been used in combination with cyclophosphamide and corticosteroid (9).
We have treated 23 patients with RA with rituximab, alone or in combination with other agents. The detailed clinical responses in these patients have been reported previously (9, 15). The majority of patients showed substantial clinical improvement, which was sustained for up to 33 months and up to 17 months after the return of circulating B lymphocytes. However, in all but 2 patients, the disease subsequently relapsed. The key objective of the present study is to understand the mechanism of this relapse. Critically, this requires an understanding of whether disease persistence depends on the existence of specific T cell clones, specific B cell clones, or the continued production of antibodies by long-lived plasma cells.
Circulating antibody levels are, at present, the only practical indices for monitoring the autoimmune response in RA. The most prevalent autoantibody reactivity in RA is RF, with IgG-RF being particularly implicated in the formation of small immune complexes (2, 22), but a significant proportion of patients also have raised levels of antibodies to cyclic citrullinated peptides (anti-CCP) (23) and to proteins such as the immunoglobulin heavy-chain binding protein (anti-BiP) (24). In the absence of T cell responses to IgG, there is particular interest in the possible roles of T cell responses to these other antigens and their interrelationships with RF B cells. For 22 of our 23 patients with RA (described in refs.9 and15), we have serial immunologic data covering the period of B lymphocyte depletion, during which no other therapy was introduced. We report herein serial observations of the total serum immunoglobulin and specific autoantibody levels together with the levels of protective antibodies to pathogens in these patients, and we report the relationships of those changes to regression and relapse of inflammation following B lymphocyte depletion.
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- PATIENTS AND METHODS
We found that B lymphocyte depletion had a selective effect on different circulating antibody populations in patients with RA. Sera from patients with a positive clinical response to treatment, in which an ACR response of ≥20% was achieved at 6 months, showed a significant fall in autoantibody and CRP levels, and this was not observed in nonresponders. Although total serum immunoglobulin levels also fell, the percentage decrease in IgA-RF, IgG-RF, and IgG anti-CCP antibodies was significantly greater in all patients. This was not the case for antimicrobial antibodies. Our findings extend the preliminary observations in patients with other autoimmune conditions, in whom a positive clinical response was associated with a significant fall in autoantibody levels following B lymphocyte depletion (9–11, 28).
In patients with lymphoma who were treated with rituximab, either alone or in combination with other drugs, the mean total immunoglobulin levels had been found to remain within the normal range (18, 19, 29, 30). This was taken as an indication that B lymphocyte depletion therapy would not produce significant benefit to autoimmunity if its effect were to be through reduction of autoantibody levels. Rituximab treatment in other autoimmune diseases has also not been found to result in a major drop in circulating immunoglobulin levels, with the possible exception of hemolytic anemia in children (31). In our group of patients, we did see a significant decrease in serum immunoglobulin levels, particularly in IgM, following B lymphocyte depletion, even though the mean values for all classes remained within the normal range. It is possible that in the patients studied herein, the greater drop in serum total immunoglobulins might reflect a large contribution of autoantibodies to circulating immunoglobulin levels.
Treon and Anderson (32) have suggested that only autoimmune conditions associated with pathogenic antibodies of the IgM class, e.g., polyneuropathies, might respond to rituximab treatment. In such conditions and in lymphoma, they suggested that IgM-class antibodies may be derived from CD20-positive plasmablasts. In our studies of patients with RA, we observed that the levels of all autoantibodies measured decreased, and for IgA-RF, IgG-RF, and IgG anti-CCP, the decrease was proportionately greater than the decrease in their respective total immunoglobulin classes. In addition, despite the half-life of IgG being longer than that of other immunoglobulin classes, IgG-RF and IgG anti-CCP antibodies decreased more rapidly than did IgA-RF or IgM-RF. In contrast to what was observed with autoantibodies, the levels of anti-PCP antibodies did not change significantly following treatment, and the anti-TT response closely corresponded to the changes in total serum IgG levels.
Such a selective effect on autoantibody production suggests that their production may be more dependent on the constant generation of new plasma cells from CD20-positive B lymphocytes. It is now known that some plasma cells have short lifespans, but other plasma cells may be able to live for extended periods of time (33). It is also possible that the clones responsible for antimicrobial antibodies are resident in the spleen, where they may be slowly turning over into plasma cells. Autoreactive clones are possibly in a more dynamic situation because of their constant generation and, consequently, many more may be entering the circulation. Their location in tissues other than secondary lymphoid organs may also render autoantibody-committed clones more susceptible to B lymphocyte depletion.
After rituximab administration, B lymphocyte depletion in patients occurs rapidly (within days) in the peripheral blood (17). Animal studies have shown that depletion of B cells in lymphoid tissue is rapid and unlikely to continue beyond 2 weeks (16). In this study, although patients showed differences in the timing and degree of their clinical response as measured by the ACR (15), the drop in CRP, which is an indicator of cytokine production, followed similar patterns in responding patients. This consisted of a gradual decrease in the levels of CRP over weeks to months that then reached a plateau until relapse, which was usually preceded by a rise in autoantibody levels. The reduction in autoantibody levels also occurred gradually and was reflected in the median times taken for them to decrease to 50% and 80% of pretreatment levels. These results are consistent with the effect of B lymphocyte depletion on reducing the progenitors of daughter plasma cells and thereby reducing autoantibody production. This contrasts with the rapid clinical and serologic (CRP) response of patients with RA to anti–tumor necrosis factor α treatment, which reflects the swift removal of a key effector in the inflammatory process (34). In our study, when responders were compared with nonresponders, only the former showed significant drops in autoantibody levels (as well as in CRP) in response to B lymphocyte depletion. Whether this simply reflected differences in the relative degree of B lymphocyte depletion in lymphoid organs in nonresponder patients when compared with responders could not be determined, since no tissue samples were obtained.
As shown in the representative serial studies and by the cumulative data from responding patients, the kinetics of the serologic (CRP) response to treatment paralleled the decline in circulating autoantibody levels. However, B lymphocyte return always preceded relapse. Relapse was often preceded by or coincided with a rise in 1 or more autoantibodies. On only 4 occasions were rises in autoantibodies (usually, small, transient rises in IgM-RF) detected without associated clinical manifestations of more active disease. The often long gap (up to 17 months) between B lymphocyte repopulation and relapse also suggested that relapse was not solely related to the presence of B cells.
The long period between B lymphocyte return and relapse seen in some patients treated with B lymphocyte depletion suggests that generation of new B cell clones capable of engaging in a vicious cycle of expansion may be a rate-limiting step in the recrudescence of disease. This may involve the generation of sufficient pathogenic B cell clones able to either restimulate autoreactive T cells or be precursors of autoantibody-producing plasma cells. It remains uncertain whether the reappearance of autoantibodies following B lymphocyte depletion represents the re-expansion of preexisting B lymphocyte clones or whether new clones are generated. The exact degree of B lymphocyte depletion achieved in solid lymphoid tissues in our patients remains unknown. There is also no information on what might happen to surviving B lymphocytes and to plasma cells during a period of B lymphocyte depletion. Therapeutic B lymphocyte depletion provides an excellent opportunity to learn more about these and other aspects of B lymphocyte and plasma cell biology and their roles in disease pathogenesis.