iTreg cells were generated as previously described by Vaeth et al. [26]. The DNA was isolated and analysed for methylation of the TSDR region. No differences in the methylation rate were detectable in Foxp3+ aTreg cells generated under the different experimental conditions (Fig. 3E). Foxp3+ Treg cells, isolated from all four cultures, revealed almost 100%

demethylation Sotrastaurin order of the TSDR region whereas the TSDR of the Teff cells was completely methylated. As expected, the TSDR of GFP+iTreg cells was still up to 60% methylated as indicated by the colour-coded matrix. We therefore assume again that the Foxp3+ cells detectable in our cultures are expanded nTreg cells. Next, we rechallenged isolated CD4+CD25+ cells with CD19+ allogeneic B cells. The Foxp3 frequency was determined on day 0 and 4 of restimulation culture. Restimulation

cultures of aTreg cells generated with aCD4, aCD4+Rapa or untreated cultures resulted in reduced frequencies GSK2118436 of Foxp3+ Treg cells (Fig. 3F). Only aCD4+TGF-β+RA aTreg cells displayed a stable Foxp3 frequency and even slightly increased in numbers upon restimulation. We tested the suppressive capacity of our in vitro generated aTreg cells in an acute GvHD (aGvHD) model. In summary, aTreg cells were generated as described above under aCD4- mAb mono-therapy or addition of TGF-β+RA or Rapa. On day 7 of primary culture, either aTreg cells or freshly isolated nTreg cells were enriched. A total of 2 × 105 C57BL/6 Treg cells were injected into myeloablatively irradiated BALB/c recipients together with 5 × 106 C57BL/6 BM cells. Two days after Niclosamide Treg-cell transfer, the mice were challenged with 1 × 106 CD4+/CD8+ C57BL/6 T cells from LUC

transgenic animals as previously described [27]. To visualise the distribution of the allogeneic effector T cells (Teff) and progress of aGvHD, the mice were monitored with bioluminescence imaging and for weight changes as a parameter of disease manifestation (Fig. 4A). Using this stringent model with a very low Treg to Teff ratio (1:5), transferred nTreg cells were unable to ameliorate aGvHD and to prolong survival (Fig. 4C and D). aTreg cells generated by aCD4 monotherapy or by addition of Rapa or TGF-β+RA prevented expansion of LUC transgenic effector T cells quantified with BLI, in contrast to controls (only transplantation of BM cells and effector T cells), mice that received aTreg cells from untreated culture conditions, or mice that had received nTreg cells in which LUC transgenic effector T cells massively infiltrated lymph nodes (LNs) and the intestinal tract (Fig. 4B and C). Improved survival after allogeneic BM transplantation further corroborated the in vivo effectiveness of the generated aTreg cells (Fig. 4D).