The data presented here provide new insight into the biology of regulatory T cells within the context of a human autoimmune disease. CD4+
T cells isolated from patients with active RA, although still anergic, show compromised function as demonstrated by their inability to regulate proinflammatory cytokines released by effector T cells and monocytes. After Infliximab treatment, regulatory T cell–mediated suppression was restored to the level found in healthy individuals, whereas only a partial restoration was seen in regulatory T cells isolated from patients responding to methotrexate. Although it is well documented that Infliximab blocks both soluble and transmembrane TNFα, resulting in a strong inhibition of other proinflammatory cytokines, there is no unanimity about the effects of methotrexate on cytokine production in RA (21
). If proinflammatory cytokines are not efficiently suppressed in methotrexate-treated patients, this could have a deleterious effect on the function of regulatory T cells, thus explaining the different level of regulatory T cell–mediated inhibition in patients treated with these two therapies.
It is unclear why regulatory T cells isolated from RA patients are unable to suppress lymphocyte and monocyte cytokine production. One possibility is that the presence of TNFα as well as other proinflammatory cytokines may hinder the ability of regulatory T cells to prevent autoimmune disease. In a recent paper that examined the role of this cytokine with respect to regulatory T cells, TNFα impaired the ability of regulatory T cells to suppress disease in the NOD mouse model of diabetes (4
). These authors suggested that endogenous levels of TNFα might act centrally in the thymus to mediate these effects on regulatory T cells. Pertinent to their hypothesis is the recent observation that CD4+
T cells derived from the thymus of healthy donors have an increased expression of TNFRII compared with CD4+
T cells (12
), which might result in an increased susceptibility of regulatory T cells to the actions of TNFα. However, our in vitro data do not lend support to a direct effect of TNFα on regulatory T cells because, after exposure to a gradient of TNFα concentration alone, regulatory T cells were still viable and functionally active. These results indicate that more complex mechanisms are likely to operate in vivo. That regulatory T cells isolated from patients affected by chronic inflammatory disorders show altered function is not only peculiar to RA. Indeed, it has been reported recently that regulatory T cells isolated from the PB of patients with multiple sclerosis are functionally defective in terms of their ability to suppress proliferation and cytokines production by activated T cells (23
). This finding, together with our data, might shed some light on the role that regulatory T cells play in controlling the maintenance of peripheral tolerance and in the prevention of human autoimmunity.
It has been speculated previously that the spreading of suppression from CD4+
T cells to responder T cells is an important mechanism by which regulatory T cells exert their effect in the maintenance of peripheral tolerance (19
). This is an attractive explanation for how such small numbers of cells could prevent autoimmune disease. Our in vitro data suggest that, unlike regulatory T cells from healthy individuals, those derived from active RA patients cannot convey a suppressive phenotype to activated T cells. To our knowledge, this is the first time that this phenomenon has been shown to be impaired in an autoimmune disease. This defect could have a significant impact on the regulation of RA pathology because it is likely that the generation of newly formed (and fully functional) suppressor T cells is critical to control autoimmunity.
In searching for the mechanism by which these regulatory T cells act, we found that the suppression of cytokine production by regulatory T cells after treatment was contact dependent, and soluble factor independent as neutralization of IL-10 and TGFβ did not alter their suppressive function (unpublished data). However, depletion of CD4+
T cells from treated PB led to a reduction in the number of IL-10–secreting cells. This suggests that the production of IL-10 may be dependent on the presence of regulatory T cells, but that its release is by another population such as a subset of suppressor cells that do not express CD25. It has been suggested that CD4+
T cells may actively promote the differentiation of Tr1 cells that mediate their suppression through production of IL-10 (24
). As IL-10 is widely known for its antiinflammatory properties, it is tantalizing to hypothesize that the increase in this cytokine, previously reported in anti-TNFα–treated patients (20
), is orchestrated by regulatory T cells whose function has been restored. Interestingly, we were unable to detect IL-10 in any of the T cell populations analyzed, suggesting that the production of IL-10 depends on the interaction of a variety of cell types (and/or their soluble products) present in whole PBMCs.
The other major finding described in our paper is the increased regulatory T cell number in RA patients treated with anti-TNFα, which significantly correlated with a reduction in CRP value. It is tempting to speculate that the increase in regulatory T cells in responding patients is important in the amelioration of disease and may help to differentiate between responder and nonresponder patients because the changes occur within 6 wk of initiation of therapy. It is conceivable that TNFα has a direct effect on regulatory T cell viability, such as the induction of apoptosis (26
), which would explain the increased number of regulatory T cells after TNFα neutralization. As aforementioned, our in vitro data do not support this hypothesis. Moreover, we found no reduction in regulatory T cell numbers in patients with active RA compared with healthy donors, suggesting that neither apoptosis, nor any other direct effect on regulatory T cell numbers, are the primary mechanisms by which TNFα is acting. The lack of difference in CD25high
regulatory T cell numbers found in active patients, compared with healthy individuals, needs more careful study, as the CD25high
regulatory T cells derived from active RA could be “contaminated” by recently activated CD4+
T cells, which would mask the total number of regulatory T cells. Indeed, human CD4+
T cells are not a homogenous population and contain nonsuppressive fractions (10
). Of equal importance to the issues already raised is the possibility that an increase in regulatory T cells may have deleterious consequences, in particular an increased susceptibility to infections, which is a major issue in patients receiving anti-TNFα therapy (28
Further studies are required to elucidate the mechanisms that underlie these observations in patients with RA, and to relate the clinical and immunological responses in patients receiving anti-TNFα therapy. Restoration of the function of regulatory T cells or transfer of fully competent regulatory T cells could be a useful therapeutic tool in the treatment of RA (29