Several recent studies have shown that the frequency of Th17 cells and Treg cells in peripheral blood is altered by treatment with ABT or TCZ in patients with RA [9–14, 19–21]. Generally Th17 cells promote autoimmunity and inflammation, including RA [6, 22], whereas Treg cells play a role in maintaining immune responses and preventing autoimmunity [8, 23]. In addition these two cell types are closely related. Initial studies reported that these two cell subsets are reciprocally regulated and mutually exclusively differentiated [24]. However, recent studies reported the existence of Foxp3+ Treg cells that secrete IL-17 [25] and of Ror-?t?+?Th17 cells that differentiated from Foxp3+ Treg cells [26]. These data suggest that the development of these two subsets is less stringent than previously thought and that transition from Treg cells to Th17 cells may occur under some circumstances.
Unexpectedly we did not found any difference in the Foxp3/Ror-?t ratio between RA patients at baseline and healthy controls. Previous studies have shown that Th17 cells were increased and Treg cells were decreased in active RA patients (9, 10). However some studies showed no significant differences in the percentages of Th17 cells (11) or Treg cells (13). It may be helpful to directly compare the expression levels of master regulators and the percentages of the T cell subsets and examine whether both analyses correspond. This still remains to be addressed.
Regarding ABT treatment of RA patients, it has been shown that Treg cells are decreased after 3 or 6 months’ treatment [13, 14, 19]. On the other hand, Picchianti Diamanti A et al. reported that Treg frequency was not reduced but that function was recovered in patients with previous TNF?-blocking agent failure [20]. As for Th17 cells, a modest decrease was reported only in anti-CCP antibody-positive patients [14], or only after 12 months but not 6 months of treatment [19]. The combined data indicate that the main effect of ABT on the peripheral helper T cell population is to reduce Treg cells. Our result, that the Foxp3/Ror-?t ratio was decreased after ABT treatment is compatible with the results of those previous studies. The mechanism of the decrease in the Treg cell population is not clear; however, our results suggest that the down-regulation of Foxp3 expression by ABT leads to a reduction in Treg cells. It is of note that Foxp3/GATA3 and Foxp3/T-bet ratios showed no significant changes with treatment. These data suggested that ABT has different effects on the expression of Ror-?t, T-bet and GATA3, and that only Ror-?t expression is up-regulated. This effect may be due to reciprocal regulation of Ror-?t and Foxp3 and/or conversion of Foxp3+ Treg cells to Ror-?t+ Th17 cells [27]. ABT therapy induces a paradoxical phenomenon, i.e. Treg cells are decreased but inflammation is alleviated, which is in contrast to the effect of anti-TNF? and anti-IL-6 therapies [9, 12]. This phenomenon may be due to the critical role of the CD28-CD80/86 axis in the induction of Treg cells, and the functional substitution of Treg cells by ABT, a fusion protein of cytotoxic T lymphocyte-associated antigen 4 (CTLA4) and immunoglobulin G Fc chain, because the main machinery by which Treg cell exerts an inhibitory function is CTLA4 [28].
On the other hand, the effects of TCZ on T cell populations include an increase in Treg cells [9, 11, 12] and a decrease in Th17 cells [9, 10], although some investigators have reported that one or both of these populations were unchanged [11, 12, 21]. In our study, Foxp3 expression and the Foxp3/Ror-?t expression ratio was increased after TCZ therapy. This result is basically in line with previous cell population analyses. IL-6, together with TGF-?, induces the expression of Ror-?t and the generation of Th17 cells; at the same time, IL-6 inhibits TGF-?-induced Treg differentiation [29, 30]. It is therefore conceivable that a blockade of IL-6 signaling by TCZ attenuated the expression of Ror-?t and augmented the expression of Foxp3. Another interesting finding of our study is that there was a decrease in the Ror-?t/GATA3 ratio after TCZ therapy. Although a reciprocal relationship between Th2 cells and Th17 cells has not been established, TCZ therapy may up-regulate GATA3 expression and the Th2 population. Consistent with our result, Guggino et al showed an increase in Th2 (CD4+ IFN-?– IL-17– IL-4+) cells after 3 months of therapy with TCZ [10].
In our study we did not find any relationship between clinical response to TCZ or ABT and change in the expression of master regulators. This finding may be due to the small sample size, and the small variance in clinical responses especially in patients treated with TCZ, who all showed a similar response. Alternatively, it is possible that the clinical response occurs at a time point after the alteration of master regulator gene expression. In other words, up-regulation or down-regulation of genes is induced by treatment with ABT or TCZ, but differences in subsequent events may modulate their effect. These subsequent events may include for example: modification of transcription factors, efficacy of induction of Th17 cells and Treg cells, or activation of these cells. Impaired function of Treg cells in RA, and its recovery after therapies with ABT or anti-TNF agents has been reported [13, 20, 30–32]. To address these issues, a further experiment using a larger population may be necessary.
Our study has some limitations. First, the expression of master regulator genes is not limited to CD4?+?T cells. For example, it has been reported that Foxp3 is expressed in CD8+ T cells or B cells with regulatory properties and that these cells play a role in RA [33, 34]. Therefore the Foxp3 expression that we measured in our study may reflect not only Foxp3 expression in CD4+ T cells but also that in CD8+ T cells and B cells. Second, Th17 cells and Treg cells do not always strictly develop as discussed above, and the expression of the master regulator genes may not always correlate with the population and function of T cells. Treg cells that produce IL-17 have been identified in patients with RA [35]. Third, because of the limited numbers of patients, it was unable to compare the expression ratios in subpopulations. In addition to the response to the treatment as discussed, sexes, status of autoantibodies, and previous biologics may make differences.