Adjuvant-induced arthritis (AIA), an experimental model of human rheumatoid arthritis (RA), can be induced in the Lewis (RT.1l) rat by subcutaneous (SC) challenge with heat-killed Mycobacterium tuberculosis (Mtb) H37Ra (1). The 65-kd mycobacterial heat-shock protein (Hsp65) is one of the targets of the T cell response in arthritic Lewis rats (2, 3). Interestingly, patients with RA also show T cell reactivity to mycobacterial Hsp65 (4). Induction of antigen-specific T cell tolerance has been used extensively for the immunomodulation of autoimmune diseases in various animal models (5). However, most of these tolerogenic approaches are successful in the prevention of autoimmunity but generally fail to control ongoing, established disease. Gene therapy has emerged as a potent method for the modulation of autoimmune diseases (6–8).
In this study we tested an alternative and effective tolerogenic approach for modulation of AIA using B cells that express a defined antigen (9–11). We introduced a gene fusion construct of mycobacterial Hsp65 and IgG heavy-chain retrovirally ex vivo into B cells of Lewis rats, and then injected these cells intraperitoneally (IP) into syngeneic Lewis rat recipients, followed by induction of AIA by Mtb H37Ra injection. In parallel, we induced tolerance by injecting soluble mycobacterial Hsp65 IP in Lewis rats. We then tested the relative efficacy of these 2 approaches for both the prevention and the treatment of AIA. We also examined the immune response to mycobacterial Hsp65 in Lewis rats that were tolerized with mycobacterial Hsp65-IgG–expressing B cells.
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- MATERIALS AND METHODS
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Systemic or mucosal administration of an antigen can readily inactivate potentially pathogenic antigen-specific T cells and prevent the development of autoimmunity in animal models (5, 14). However, some of these tolerogenic approaches may cause untoward effects such as anaphylaxis (15) or exacerbation of disease (16, 17). Moreover, the effectiveness of these tolerogenic regimens in modulating ongoing disease in the clinical setting has not yet been fully established (14).
In this study, we observed that the injection of soluble mycobacterial Hsp65 in Lewis rats significantly reduced the severity of the subsequently induced AIA but failed to ameliorate established AIA. In contrast, the administration of mycobacterial Hsp65-IgG–expressing tolerogenic B cells not only was effective in reducing the severity of subsequent AIA, but also ameliorated established AIA in Lewis rats. Our findings corroborate the results from earlier reports of the success of antigen/epitope-IgG–expressing B cell–based therapy for the prevention and treatment of autoimmunity in animal models of experimental autoimmune encephalomyelitis, experimental autoimmune uveitis (EAU), and type I diabetes (9–11). In the EAU model, the B cell–mediated antigen-specific tolerance has been reported to last for more than 6 months (9).
Attempts have been made in the past to build protective immunity against Hsp65 by administration of human Hsp60–containing vaccinia virus (18) or injection of naked DNA encoding mycobacterial Hsp65 (19) or human Hsp60 (20). These attempts were successful in preventing subsequent induction of AIA. However, the effectiveness of such approaches in established disease was not demonstrated. In this regard, our results show that the B cell–mediated treatment was effective in suppressing ongoing AIA.
At present, the precise mechanisms involved in the modulation of AIA by mycobacterial Hsp65-IgG–expressing B cells are not fully understood. However, the results of the above-mentioned studies in other models of autoimmunity have shown that the presence of B cells is essential, and that presentation of class II major histocompatibility complex, engagement of CTLA-4, and B7 signaling are involved in B cell–mediated tolerization of the disease-related T cells (21). It has also been reported that the number of CD4+,CD25+ regulatory T cells is increased in NOD mice after application of this regimen (22). However, no evidence of a deviation in the cytokine response has been observed (10).
Mauri and coworkers demonstrated that B cells producing interleukin-10 (IL-10) can ameliorate collagen-induced arthritis (23). These B cells were nonspecifically activated in vivo with anti-CD40. Although production of IL-10 by LPS-activated B cells, as used in the present study and in our previous investigations (Scott DW, et al: unpublished observations), may be involved in the mechanisms of suppression, it should be pointed out that our approach involves induction of antigen-specific tolerance by the administration of genetically modified B cells, and therefore it is not a generalized immunosuppressive regimen. With regard to the T cell tolerance induced by soluble antigen administration, other investigators have shown that it involves the induction of anergy that is attributed to a lack of adjuvant-mediated costimulation, immune deviation (Th1 to Th2 type), and CD4+,CD25+ regulatory T cells (14).
We observed that the pretreatment of Lewis rats with mycobacterial Hsp65-IgG–expressing B cells induced an anti–mycobacterial Hsp65 antibody response but a decrease in the mycobacterial Hsp65–directed T cell proliferative response during the early phase of AIA. Furthermore, this modulation of the humoral and cellular immune responses to mycobacterial Hsp65 corresponded to the protection against AIA observed with this pretreatment. We (12) and others (13) have previously shown that antibodies produced during the course of AIA are protective rather than being pathogenic. Therefore, our results suggest that tolerogenic mycobacterial Hsp65-IgG–expressing B cell treatment offers protection from AIA, in part through suppression of the T cell response along with an enhancement of the protective antibody response to mycobacterial Hsp65.
At this time, we do not know the precise mechanistic differences that might account for the differing effects on established AIA of B cell–mediated compared with soluble antigen–induced tolerogenic approaches. However, a major difference in immune activation exists in this context, in that there is a continuing in vivo antigen presentation by mycobacterial Hsp65–expressing B cells, as compared with a single antigen exposure in the case of soluble antigen administration. The former regimen might facilitate the induction of disease-regulating T cells at a regular pace, which in turn might successfully down-regulate the activity of continuously emerging arthritogenic T cells following Mtb H37Ra challenge. In contrast, the latter regimen, involving the inactivation and/or deletion of pathogenic T cells by soluble antigen–induced tolerance, might be ineffective in suppressing the activity of the emerging pathogenic T cells. Mtb is a lipid-rich particulate antigen that tends to provide an antigen depot for sustained release of arthritogenic stimulus, and therefore a disease-modulating regimen that can provide a similarly sustained protective stimulus (e.g., mycobacterial Hsp65-Ig–expressing B cells) might have a better chance to succeed, compared with a one-time protective challenge (e.g., soluble antigen injection), in suppressing ongoing AIA.
Detailed mechanistic studies comparing the antiarthritis activities primed by these 2 tolerogenic regimens are currently in progress in our laboratory. Since T cell responses to mycobacterial Hsp65 have also been reported in patients with RA as well as in patients with juvenile RA (4), the use of mycobacterial Hsp65-IgG–expressing tolerogenic B cells might be an effective approach for the treatment of these debilitating autoimmune disorders.
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- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
Dr. Moudgil 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 design. Satpute, Scott, Moudgil.
Acquisition of data. Satpute, Soukhareva.
Analysis and interpretation of data. Satpute, Scott, Moudgil.
Manuscript preparation. Satpute, Scott, Moudgil.
Statistical analysis. Satpute.
Principal investigator. Moudgil.