Here we show that Rapa enhances TGFβ-induced Foxp3 expression in human naïve (CD4+25-45RA+) T-cells and, more importantly, resulted in potent in vitro and in vivo suppressive function. Unlike previous studies, Rapa plus TGFβ also induced Foxp3 expression and suppressive function in CD4+25- cultures which contained naïve and memory T-cells. In as short as 2 weeks of culture, an average of 240×109 or 200×109 Rapa(d0)/TGFβ iTregs could be generated from CD4+25- or CD4+25-45RA+ T-cells, respectively, in a single apheresis unit, which would provide a high ratio of iTregs to Teffs at the time of initial transplant for recipients of UCB, bone marrow or PB hematopoietic stem/progenitor cell grafts. Alternatively, iTreg yield from a buffy coat may be sufficient, which would be favorable over donating an apheresis product 14 days prior to the stem/progenitor cells. The availability of the needed cGMP reagents makes this a practical approach for multiple iTreg infusions for GVHD prevention or therapy as well as opens the possibility for generating a master cell bank for frozen iTregs that could be used as partially HLA class II matched products. Cultured iTreg maintained Foxp3 expression and in vitro suppressive function after cryopreservation and thawing (data not shown).
A concern in the field has been whether nTregs and especially iTregs would be unstable and reprogrammed to become Teffs. We do not have evidence for this occurrence in our xenogeneic GVHD model since both nTregs and iTregs comparably suppressed GVHD lethality after a long-term observation period (82 days) even at suboptimal Treg:PBMNC ratios. Both Treg populations comparably suppressed human CD4+ and CD8+ T-cells derived from PBMNCs to low levels on day 18 post-transplant, indicating that neither Treg population was substantially contributing to the peripheral CD4+ T-cell pool. Although it is possible that iTregs experienced reprogramming(27
), an initial experiment in which PBMNC and Treg can be distinguished based upon HLA-A2 allotype demonstrated that, despite being easily detectable in blood on day 6 (~100 cells/µl) neither nTreg or iTreg were found in the blood or spleen of animals examined at death, even as early as day 18 (data not shown). These data agree with our published data that nTregs typically persist in the circulation only 1–2 weeks in vivo(19
) and are not found in GVHD target organs, although persistence in this xenogeneic system may not be predicative for persistence in humans. However, while nTregs and iTregs were comparable in significantly reducing GVHD-associated weight loss in the first 1 month post-transplant, nTregs were superior at later times post-transplant (data not shown). One potential explanation for the superior weight curves with nTregs vs. iTregs is the relative higher frequency of IL-4 secreting cells in Treg cultures, since increased IL-4 production has been shown to ameliorate disease in a murine model of GVHD(30
Multiple types of murine and human iTregs have been defined, although significant differences exist between species. For example, while TGFβ or ATRA individually induce Foxp3 expression and suppressive function in murine T-cells(9
), human T-cells do not acquire suppressive function until ATRA (which enhances TGFβ function by increasing SMAD3 expression(11
)) is combined with TGFβ(16
). Rapa, in addition to inhibiting mTOR, enhances TGFβ signaling by inhibiting Smad7, a negative regulator that targets TGFβ receptors for degradation(31
). In contrast, IFNγ suppresses TGFβ signaling(33
) and peripheral induction of Tregs in mice(34
) by inducing Smad7. We show >50% of naïve human CD4+25- T-cells expanded in TGFβ secrete IFNγ (). Therefore, Rapa is likely able to induce Treg from cultures containing naïve and memory cells because, unlike ATRA or aryl hydrocarbon receptor ligands, it inhibits both IFNγ secretion and Smad7 downregulation of TGFβ signaling. In addition to inducing Treg in cultures containing a mixture of naïve and memory T-cells, Rapa/TGFβ induced Foxp3 expression and suppressive function in purified CD4+25-45RA- (data not shown). Similarly, Zhang, et. al. found Rapa enhanced TGFβ induction murine Treg and could improve allograft survival in a mouse islet transplant model(17
). Stable Foxp3 expression in nTreg is controlled through the Treg specific demethylated region (TSDR) in the Foxp3 gene(35
). To date, no method to induce Treg in murine or human cells has resulted in TSDR methylation, and we found no evidence for Rapa/TGFβ iTreg induced from either naïve or unfractionated CD4+25- T-cells (Figure S2
). However, the enhanced TGFβ signaling found in these cells might drive stable expression through another mechanism. For example, naïve CD4+25- T-cells treated with TGFβ and aryl hydrocarbon receptor agonists induce Smad1 expression, which then binds the Foxp3
enhancer and regulates Foxp3 expression(36
). Decitabine, a DNA methyltransferase inhibitor previously shown to increase Foxp3
gene methylation and expression, did not enhance Foxp3 expression or suppressive function in Rapa/TGFβ iTreg, providing further evidence that Foxp3
methylation status does not control Treg induction (Figure S3).
In summary, we have shown that that Rapa enhances TGFβ-dependent Foxp3 expression and generates iTregs with potent in vivo suppressor function. Our protocol generates an average of 240×109 iTregs from CD4+25- or 200×109 iTregs from CD4+25-RA+ T-cells capable of suppressing third party responses in vivo. Because all the necessary clinical reagents are available and iTreg were cultured on a clinically relevant scale (≥500×106 cells), these data set the stage for clinical trials testing the feasibility and safety of iTreg cellular therapy for human diseases.