Rheumatoid arthritis (RA) is an autoimmune disease that results in joint inflammation and destruction. Autoreactive T cells and B cells are crucial to its pathogenesis. Autoreactive T cells are predominantly deleted in the thymus, but this process is not stringent. Thus, autoreactive T cells can and do escape into the peripheral T cell pool; subsequent activation can result in autoimmune pathologic conditions. CD4+CD25+ FoxP3+ regulatory T (Treg) cells, which comprise 5–10% of CD4+ T cells, are crucial for the maintenance of peripheral tolerance (1, 2).
In adult RA and juvenile idiopathic arthritis, Treg cells were found to be enriched in the synovial fluid of inflamed joints as compared with their levels in peripheral blood, suggesting active homing or expansion at sites of inflammation (3–8). These Treg cells expressed forkhead box P3 (FoxP3) and suppressed both proliferation and cytokine production by CD4+CD25– effector T (Teff) cells. Moreover, increased numbers of Treg cells in the synovial fluid directly correlated with limited disease (3), suggesting that Treg cells aid in disease remission.
However, it is not known how Treg cells modulate immunity, because the number of Treg cells is paradoxically increased during disease, and since there is also variability in Treg cell function among different studies. Ehrenstein et al (7), for example, demonstrated that Treg cells from RA patients showed compromised function as compared with those from healthy controls, whereas other investigators (8) showed that Treg cells obtained during active RA were equally suppressive or were more suppressive than those from healthy controls. In this case, the increased suppressive function was offset by the responder Teff cells themselves being more refractory to suppression and by the presence of inflammatory cytokines (9). Because these studies examined heterogeneous patient populations, disease stages, and therapeutic regimens, these variables likely contributed to the differences between the studies. In addition, investigators have differed in the criteria they use to identify Treg cells, with some studies focusing only on CD25bright populations while others used all CD25+ T cells (8, 10). Because expression of CD25 is also elevated in activated Teff cells, the number of which is increased during disease, varying degrees of Teff cell contamination can render interpretation difficult. It is also unclear if arthritis resulted from a primary Treg cell dysfunction or a secondary defect due to persistent inflammation.
To eliminate these confounding processes, we examined Treg cell development and function in a well-characterized murine model of RA. K/BxN mice were generated by crossing KRN T cell receptor (TCR)–transgenic mice with NOD mice (11). The disease is fully penetrant in all progeny and follows a predictable course of progressive symmetric distal polyarthritis that resembles RA in humans. Arthritis results from autoreactive KRN T cells recognizing peptide 281–293 of the glycolytic enzyme glucose-6-phosphate isomerase (GPI) that is bound to I-Ag7 (the NOD-specific class II major histocompatibility complex allele) (12, 13). Incomplete thymic deletion allows autoreactive KRN CD4+ T cells to persist in the periphery and to become activated by endogenously presented GPI. KRN T cells then provide help to GPI-specific B cells, giving rise to arthritogenic antibodies. Treg cells are enriched in arthritic K/BxN mice (14, 15), and the loss of Treg cells results in earlier and more extended disease, suggesting that although Treg cells do not prevent arthritis, they may nevertheless mitigate it (15). Similar findings have been reported for collagen-induced arthritis, in which depletion of CD25+ T cells exacerbated arthritis and adoptive transfer of CD4+CD25+ Treg cells or FoxP3-transduced T cells ameliorated the disease (16–18).
To understand how antigen-specific Treg cells develop during the course of arthritis, we crossed K/BxN mice with FoxP3gfp reporter mice to unequivocally identify Treg cells. We then analyzed Treg cell selection in the thymus and followed their expansion and function during the progression of arthritis.
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There is considerable interest in harnessing CD4+CD25+ Treg cells to control autoimmune diseases (25, 26). In RA patients, the number of CD4+CD25+ Treg cells is increased in inflamed joints, and these cells show variable activity against their Teff cell counterparts. Results have differed among studies partly due to differences in the criteria used for Treg cell identification, as well as the disease states and patient populations studied. It is also not clear what factors control their tropism, function, growth, and death during disease.
In the present study, we used K/BxNgfp mice to model the development and function of arthritogenic Teff cells and Treg cells in a well-characterized murine model of RA. The K/BxN mouse model has many advantages with regard to RA in humans. The progression of arthritis has been extensively characterized and follows a predictable course: K/BxN mice develop overt joint inflammation at 4–5 weeks, which peaks at 7–8 weeks, and is followed by a chronic phase in which joint destruction and deformity predominate (27). In addition, the arthritis shows regional involvement, with the distal joints being more affected than the proximal and axial joints. This feature allowed us to determine if there is regional expansion of Treg cells at sites of disease. Moreover, by examining GPI reactivity, we focused specifically on the arthritogenic KRN Teff cells and Treg cells in this system. Last, we used FoxP3gfp to unequivocally identify Treg cells.
These experiments are the first to demonstrate the expansion and function of antigen-specific Treg cells during the initiation and progression of spontaneous arthritis. In these experiments, we confirmed our previous findings that incomplete deletion of KRN thymocytes by endogenous GPI resulted in an increased frequency of GPI-specific Treg cells in K/BxNgfp mice through selective deletion of Vβ6bright FoxP3– Teff cells and induction of FoxP3+ Treg cells in the thymus (14). In contrast to other models of autoimmune disease in which Treg cell deficits resulted in pathologic changes, K/BxN mice displayed enhanced populations of GPI-specific Treg cells (14, 15, 28–30). These Treg cells proliferated quite well, both in vitro and in vivo, in response to endogenously presented GPI, they were functional, and they suppressed Teff cell proliferation in vitro. Treg cell numbers expanded in parallel with Teff cell numbers in the spleens and LNs during arthritis. In K/BxN mice with a ubiquitous self antigen, both Teff cell and Treg cell responses were initiated in the spleen, with subsequent extension to the LNs and with a preference for the draining LNs, indicating selective tropism to inflamed joints from the increased inflammation and/or GPI presentation during disease. However, this enhanced activity was offset by an increased resistance of GPI-specific Teff cells and antigen-presenting cells to suppression.
A recent study described similar dynamic changes in the spatiotemporal expansion of Teff cells and Treg cells in the draining LNs during the induction and progression of adjuvant-induced arthritis that presumably reflected changes in the concentration of the inciting antigen (31). Those findings complement the findings of our studies, with the exception that their waxing/waning Treg cell course likely reflected the monophasic course in adjuvant-induced arthritis compared with the progressive expansion of Teff cells and Treg cells in the K/BxN model.
Despite the enrichment in Treg cell activity, K/BxN mice nevertheless developed arthritis. Our in vitro studies demonstrate effective Teff cell suppression at ratios that approximate the physiologic ratio found in vivo. Why, then, are Treg cells ineffective in vivo? There are several possible explanations. First, Treg cells and Teff cells are differentially regulated during ontogeny. CD4+FoxP3– single-positive thymocytes reached mature levels during the first week of life. In contrast, CD4+FoxP3+ single-positive thymocyte development was substantially delayed and did not achieve mature levels until 21 days of age (32). The delayed appearance of Treg cells in K/BxN mice would result in unopposed Teff cell reactivity during the initiation of arthritis. Second, the precursor frequency of GPI-specific T cells is supraphysiologic in K/BxN mice. The self antigen is also ubiquitously expressed and can be presented by all antigen-presenting cells, including B cells. Under such conditions, autoreactive B cells can be driven into memory B cells despite abundant Treg cells (33). Thus, for a humorally driven arthritis, Treg cells may be less effective than for a T cell–driven disease such as colitis. We propose that the developmental regulation and numeric superiority of KRN Teff cells overcome Treg cell suppression at the initiation of disease. Treg cells might simply arrive too late and in too few numbers.
During the ensuing arthritis, Treg cells are highly activated, with >30% of Treg cells proliferating in a 12-hour period. However, this vigorous Treg cell activity is attenuated by 3 factors: Treg cells undergo shorter proliferative bursts and higher apoptosis rates than Teff cells upon antigen stimulation, Teff cells become more refractory to Treg cell suppression as they differentiate into memory T cells, and antigen-presenting cells from arthritic mice abrogate Treg cell suppression. This inhibition of Treg cell suppression required close contact, but it is unknown whether this is due to increased resistance of antigen-presenting cells to Treg cells or to enhanced antigen presentation to Teff cells. It is also unclear whether these features are due to changes in the costimulatory molecules or to secretion of paracrine cytokines in activated antigen-presenting cells from arthritic mice. Studies to dissect these mechanisms are ongoing.
Our findings parallel observations in human RA and provide an explanation for the paradoxical occurrence of disease in the presence of abundant Treg cells. By elucidating the factors and mechanisms that augment Treg cell activity, it may be possible to achieve control of disease. K/BxNgfp mice thereby provide a valuable model in which to examine Teff cell/Treg cell homeostasis during disease and therapy.