TGF-β mediated conversion of CD4+
cells has been used in various models to generate Foxp3+
iTreg with suppressive capacity (5
). However, a number of studies have now demonstrated that Foxp3 expression in iTreg is unstable upon re-stimulation in the absence of exogenous TGF-β (18
). In this report, we have re-evaluated the factors that control the stability of Foxp3 expression in iTreg both in vivo and in vitro. We have mainly used “pure” iTreg generated from Foxp3 GFP-KI mice to avoid any complicating factors produced by contaminating Foxp3−
T cells. We have confirmed the observations of other groups (18
) that Foxp3 expression is rapidly lost when iTreg are re-stimulated in vitro in the absence of TGF-β. In contrast with a previous study indicating that both IL-2 and TGF-β were required to sustain expression of Foxp3 (27
), we now demonstrate that in the absence of stimulation via the TCR, Foxp3 expression is remarkably stable in vitro for at least 2 weeks of culture even when the iTreg proliferate in response to the addition of exogenous IL-2. TCR stimulated iTreg that had lost Foxp3 expression did not become cytokine producing Th1 or Th17 cells although a low levels of IFN-γ producing cells could be detected in both Foxp3+
populations. Nevertheless, as reported by Polansky et al (18
) the TSDR of the induced Foxp3+
T cells remained fully methylated.
The major focus of this report was the fate of iTreg that had been transferred to normal recipients. Some studies (21
) have reported that iTreg in vivo rapidly lost Foxp3 expression and suppressor function, but these observations run counter to the studies that have demonstrated the remarkable therapeutic efficacy of iTreg in several autoimmunity models as well as their capacity to rescue Scurfy mice when administered shortly after birth. The results of our in vivo studies parallel the in vitro experiments. In the absence of TCR stimulation, iTreg isolated from DLN or spleen maintain high levels of Foxp3 expression for long periods of time (up to one month, data not shown) and exhibit minimal proliferative responses. This observation differs from the studies of Selveraj and Geiger (21
) who demonstrated that iTreg rapidly lost Foxp3 expression in most sites in the absence of TCR stimulation and only a small number of Foxp3+
T cells could be detected in LN or bone marrow. One possible difference between the studies is that they transferred polyclonal Treg, while we transferred iTreg generated from OT-II mice on a RAG−/−
background that expressed a restricted TCR repertoire. The iTreg that had retained Foxp3 expression in the absence of stimulation retained a methylated phenotype of their TSDR. Upon stimulation with antigen by immunization with peptide in IFA or antigen-pulsed spleen cells, the OVA-specific iTreg rapidly lost Foxp3 expression. The cells that had lost Foxp3 proliferated more rapidly than those that retained Foxp3 expression, but did not differentiate to Th1 or Th17 cells. The fate of the iTreg in vivo was markedly dependent on the strength of the TCR signal, as weak stimulation with lower doses of peptide or lower numbers of DC that resulted in a decreased proliferative response induced less loss of Foxp3. We could find no evidence for a stable subpopulation of Foxp3+
cells as suggested by Selveraj and Geiger (21
), as the minor population of Foxp3+
cells that remained after stimulation with antigen in vivo, when transferred to a new host, lost Foxp3 expression upon a second exposure to antigen in vivo.
Our previous studies (8
) on the treatment of Scurfy
mice with iTreg strongly suggested that factors present in the inflammatory environment of the Scurfy host might potentiate Foxp3 expression in iTreg, as iTreg that had been transferred to Scurfy mice expanded and retained expression of Foxp3, while iTreg transferred to normal neonates lost Foxp3 expression. As culture of iTreg with IL-2 resulted in stable expression of Foxp3, we first evaluated whether treatment of mice with IL-2/anti-IL-2 complexes resulted in stabilization of Foxp3 expression. Treatment of mice with the complexes alone enhanced the proliferation of the transferred iTreg that remained Foxp3+
in the absence of stimulation. Surprisingly, treatment of mice that had received iTreg and were subsequently immunized markedly enhanced Foxp3 expression. Moreover, analysis of the methylation status of the TSDR indicated that treatment of mice with both antigen stimulation and IL-2, but not IL-2 alone, resulted in significant demethylation of the TSDR. Thus, IL-2 alone is not sufficient to induce epigenetic imprinting of Foxp3 expression. Furthermore, our studies demonstrate that culture of iTreg either with IL-2 alone or with plate-bound anti-CD3 and IL-2 did not lead to significant demethylation of the TSDR (89.5±5.1%, 76.1±6.5%, methylated). It will be interesting in future studies to clarify the differences between the in vitro and in vivo effects of antigen and IL-2 treatments on the methylation status of the TSDR.
We used several additional approaches to confirm that IL-2 played an important role in the maintenance of Foxp3 expression in vivo. First, following TCR stimulation, neutralization of IL-2 potentiated the loss of Foxp3 expression. Secondly, iTreg generated from Stat5−/−
mice demonstrated a greater loss of Foxp3 expression than iTreg from WT mice following antigen stimulation in vivo. Lastly, we used a co-transfer model in which antigen-specific iTreg inhibit the expansion of naïve antigen-specific T cells following antigen stimulation. In contrast to the stimulation of iTreg alone, stimulation of iTreg in the presence of effector cells resulted in significant maintenance of Foxp3. Both the maintenance of Foxp3 expression and T suppressor function in vivo were reduced when endogenous IL-2 was neutralized with anti-IL-2. Because optimal TCR and CD28 engagement can induce IL-2-independent cell cycle progression in vivo (27
), depletion of endogenous IL-2 did not inhibit effector T cell proliferation. These results demonstrate that IL-2 is not only essential for the development and homeostasis of nTreg (28
), but that IL-2 produced by T effector cells is also critical for the function of iTreg in vivo.
It is clear from our studies and those of other groups that the conditions for induction of TSDR demethylation are lacking in TGF-β induction cultures in vitro. Both the biologic and molecular signals leading to TSDR demethylation in nTreg and iTreg remain elusive. It is unlikely that the induction of CREB/ATF by TCR stimulation or Stat5 activation by IL-2 stimulation are directly responsible for inducing demethylation as both CREB/ATF and Stat5 only bind to demethylated TSDR (13
). Foxp3 itself is also unlikely to regulate demethylation as Foxp3 in the form of Foxp3-Runx-1-Cbf-β complexes also only bind to the TSDR after demethylation (30
). It thus remains to be determined how the synergistic interaction of IL-2 signals with TCR signals promotes in the induction of TSDR demethylation.
One major issue that is not resolved by these studies is the development of an optimal protocol for the in vitro generation of iTreg that stably express Foxp3 and concomitantly have a demethylated TSDR. This issue is of greater significance for the generation of human iTreg in vitro as it has been shown that TGF-β stimulation of human cells, while inducing Foxp3 expression, fails to induce functional iTreg (31
). One criticism that has been raised about the in vitro methods used by most investigators is that the result in the rapid induction of Foxp3 expression in the presence of CD28-mediated co-stimulation and proliferation is detrimental to the generation of stable iTreg and that the addition of agents that inhibit the mTOR pathway increases the stability of Foxp3 expression in iTreg generated in vitro (25
). However, it remains to be demonstrated that inhibition of the mTOR pathway results in demethylation of the TSDR. Previous studies have strongly suggested that IL-2 is absolutely required for the generation of iTreg in vitro (32
) and the present studies demonstrate that the administration of IL-2 in vivo can stabilize Foxp3 expression and promote demethylation of the TSDR. It is likely that the success of previous studies (5
) that demonstrated the therapeutic effects of iTreg in several distinct models of autoimmunity was secondary to the production of IL-2 by the autoreactive effector cells. Future studies should address whether IL-2 can function as an adjuvant in vivo for the prevention and treatment of autoimmune disease, graft rejection, or graft versus host disease.