The use of Tregs to facilitate transplantation tolerance is an emerging area in clinical allogeneic stem cell transplantation. Based on preclinical murine studies that demonstrated that the adoptive transfer of Tregs could mitigate GVHD, several groups of investigators have begun clinical trials to determine if this approach will be efficacious in humans (39
). Obtaining sufficient numbers of these cells for transfer into individual patients as well as insuring that the acquired cell population is not contaminated by potentially alloreactive non-Treg cells have been ongoing challenges with this strategy (24
). An alternative approach to circumvent these potential obstacles is predicated on the in vitro expansion of Tregs that can be converted from conventional CD4+
T cells in the presence of IL-2 and TGF-β. Under these conditions, large numbers of in vitro-derived iTregs can be obtained for the subsequent administration into recipients. This strategy, however, is contingent upon the underlying assumption that these cells will function in a similar manner to nTregs with respect to suppression of GVH reactivity. In the current study, we examined this question to determine the functional capability of these cells to ameliorate GVHD in vivo. These studies demonstrated that in vitro-differentiated iTregs were not effective for the prevention of GVHD despite their ability to suppress alloreactive T cells responses in vitro. The failure to mitigate GVHD was attributable to limited in vivo survival, instability of Foxp3 expression and the subsequent reversion of these cells to a TH
1 cytokine phenotype.
Our results, along with those of other investigators, suggest that the ability of in vitro-differentiated iTregs to suppress disease may be a function of the magnitude of the inflammatory milieu. For example, Haribhai et al (26
) showed that these cells could protect mice from colitis in a T cell transfer model. Similarly, Zheng et al (41
) demonstrated that in vitro-differentiated iTregs could prolong survival in an autoimmune model characterized by a lupus-like syndrome. Both models, however, are characterized by less inflammation than is observed in GVHD where the administration of a lethal TBI conditioning regimen exacerbates the inflammatory milieu induced by a T cell-mediated alloresponse. In that regard, our results are consistent with those of Koenecke et al (42
) who recently showed that alloantigen-specific iTregs generated in vitro after co-culture with activated dendritic cells had a negligible effect on protection from lethal GVHD. The current study, however, extends these findings by demonstrating that the loss of Foxp3 expression is associated with the acquisition of an inflammatory phenotype characterized by IFN-γ secretion with no evidence of IL-17 production. Thus, in the presence of an inflammatory environment induced by GVHD, there is a reversion of a suppressive T cell population to one with a TH
1 cytokine phenotype.
One of the primary observations of these studies was the limited in vivo survival of iTregs which were essentially undetectable after two weeks, even after accounting for revertant cells. This was observed in all GVHD target organs examined indicating no preferential localization of these cells at sites of pathological damage. This finding is consistent with an earlier report by Selvaraj and colleagues (25
) who demonstrated that only a small percentage of iTregs were able to survive in vivo after the adoptive transfer into immunocompetent animals. In that study, iTregs disappeared from nearly all organs within the first two weeks with the exception of the bone marrow and lymph nodes. This was distinctly different from what we observed when nTregs were transferred into recipient animals where Foxp3+
cells were observed for up to eight weeks post-BMT. In the latter case, the stability of Foxp3 expression correlated with protection from lethal GVHD.
Prior studies have shown that RA is able to induce differentiation of Tregs from conventional T cells through both direct and indirect effects. Hill and colleagues reported that RA promoted iTreg generation by relieving the inhibition that was mediated indirectly by CD4+
T cells (41
). Conversely, others have noted that RA can directly enhance TGF-β-mediated Foxp3 induction while inhibiting the generation of TH
17 cells (34
). Based on these studies, we examined whether RA could enhance the stability of Foxp3-expressing iTregs in vivo and prevent phenotypic reversion in lethally irradiated transplant recipients. Although treatment with RA increased iTreg conversion, the in vitro exposure to this agent did not prevent phenotypic reversion in vivo after adoptive transfer. These results differ from what was observed in a murine model where animals immunized with ovalbumin had less reversion if they received RA-iTregs (44
). The degree of inflammation and the extent of reversion in control animals, however, were notably less in that model and suggest that other cytokines and or environmental factors are responsible for the phenotypic reversion which occurs after adoptive transfer of iTregs during GVHD.
We reasoned that IL-6 might be one cytokine that contributed to instability of Foxp3 expression given prior studies which have shown that IL-6 deleteriously affects the generation of iTregs induced by TGF-β by blocking Foxp3 expression (45
). This has been shown to occur by up regulation of the TGF-β inhibitor SMAD7 (8
). IL-6 has also been shown to play a pivotal role in driving the differentiation of naïve T cells to become TH
17 cells and in inhibiting the generation of CD4+
T cells (46
). Moreover, Horwitz and colleagues (47
) have reported that iTregs are more resistant to the inhibitory effects of IL-6 compared to nTregs. Thus, we considered that transfer of these cells might be more effective if the effects of IL-6 which is significantly increased during GVHD (28
) could be blunted. Blockade of IL-6 signaling, however, had no effect on stabilization of Foxp3 expression or preventing phenotypic reversion. Further analysis revealed that in vitro-differentiated iTregs did not express the IL-6R after either in vitro conversion or in vivo isolation of this same population after transfer. Down regulation of the IL-6R on iTregs has been shown to inhibit signaling though IL-6 (41
), providing an explanation for why these cells were not affected by antibody administration. Down regulation of the receptor is also a potential explanation for the absence of IL-17 secretion in revertant iTregs as the inability to respond to IL-6 would prevent TH
17 differentiation (41
One of the main findings was the observation that in vitro and in vivo-generated iTregs appear to be differentially regulated by IL-6. Specifically, blockade of IL-6 signaling was able to augment the in vivo induction of Tregs from the conventional CD4+ T cell pool. Under these conditions, mice treated with antibody blockade had an increased number of Foxp3+ cells in all tissue sites due to enhanced in vivo conversion which correlated with augmented GVHD protection. Since naive T cells express the IL-6R, we surmise that, in contrast to in vitro-differentiated iTregs, antibody administration was able to direct the differentiation of these cells towards a regulatory pathway due to their susceptibility to IL-6 signaling blockade. These results have direct clinical relevance given the availability of a tocilizumab which is a humanized version of this antibody and has been FDA-approved for the treatment of patients with rheumatoid arthritis. Thus, administration of this antibody may be a means to augment Treg development in vivo without the need for more time consuming and costly cellular therapy approaches.
In summary, these studies demonstrate that in vitro-differentiated iTregs are ineffective at attenuating the severity of GVHD. This lack of protection is characterized by instability of Foxp3 expression which results in reversion to an inflammatory TH1 cytokine phenotype. Reversion is not due to increased production of IL-6 nor is in vitro exposure to retinoic acid able to stabilize iTregs, indicating that other mechanisms are responsible for this unstable phenotype. These results have implications for the potential use of in vitro-differentiated iTregs for the abrogation of GVHD and suggest that additional studies that are focused on augmenting the stability of Foxp3 expression in these cells is warranted before their use is contemplated in the clinical setting. Finally, blockade of IL-6 signaling is an effective approach to augment the generation of iTregs in vivo which may be a clinically feasible strategy to enhance Treg-mediated suppression and reduce GVHD severity.
Allogeneic bone marrow transplantation (BMT) is the most potent form of immunotherapy directed against hematological malignancies. However, the success of this therapeutic modality is limited by the development of graft versus host disease (GVHD) which is the major complication of allogeneic BMT. The adoptive transfer of regulatory T cells is an emerging clinical strategy to prevent GVHD, but the relative role of natural (nTregs) versus induced Tregs (iTregs) is not defined. This has implications for the use of these cells as cellular therapy for GVHD prevention. In this study, we demonstrate that in vitro-expanded iTregs are much less potent than nTregs at preventing GVHD. This is due to the loss of Foxp3 expression and reversion to a proinflammatory phenotype after in vivo transfer. These cells, however, can be induced in vivo after blockade of IL-6 signaling and potently suppress GVHD. These results indicate that the conditions under which iTregs are generated as well as the corresponding proinflammatory cytokine milieu in which they function are critical factors in their ability to mitigate GVHD.