Generation of Treg-Like Cells from CD4+CD25- T Cells Occurs Via Both Foxp3 Dependent and Independent Pathways
Authors: Choi Jaebok , Ritchey Julie , DiPersio John F. 
Published: October 2019

Abstract
Regulatory T cells (Treg) contribute to the maintenance of self-tolerance and have been demonstrated to both suppress autoimmune diseases and mitigate GvHD in mouse models. Major obstacles for their routine use in human clinical trials to reduce autoimmunity or GvHD include the low number of Treg found in the peripheral blood in the resting state (5–10% of CD4+ T cells), low yields and efficiencies of purification and severe limitations maintaining suppressor function after ex vivo expansion. The master gene responsible for the normal development and suppressor function of Treg is Foxp3 which is exclusively expressed in Treg. Recent identification of demethylated CpG islands within the Foxp3 locus only in Treg lead us to investigate whether an FDA-approved demethylating agent, decitabine, could be used to enhance expression of Foxp3 via an epigenetic effect and functionally convert CD4+CD25- T cells into Treg. Treatment of human CD4+CD25-FOXP3- T cells with decitabine in the presence of anti-CD3/CD28 beads and hIL-2 (50u/ml) induced FOXP3 expression. Optimal expression of FOXP3 was seen in those cells incubated with 1–5 uM decitabine. Real time RT-PCR demonstrated levels of mRNA for FOXP3 that were comparable to that seen in bead-activated natural Treg (10–12 fold increase above baseline). This resulted in increased expression at the protein level in 60% of decitabine-treated cells (dcT) (5% of PBS-treated cells (pbsT)). Decitabine also induced Foxp3 expression in 80% of anti-CD3/CD28 bead-activated murine CD4+CD25- T cells. To determine the duration of Foxp3 expression in dcT, we performed intracellular staining for Foxp3 each day for seven days and found that about 50% of dcT were Foxp3+ at day 7. Consistent with these results, decitabine also induced marked increase GFP expression (from undetectable levels) in anti-CD3/CD28 bead-activated CD4+CD25- T cells from Foxp3-ires-GFP KI mice (kindly provided by Tim Ley). In order to identify regulatory elements which mediate decitabine induced expression of Foxp3, we cloned both the 6kb 5′ promoter and the 6kb intron 1 of the human FOXP3 locus upstream of luciferase and tested expression of luciferase in stable Jurkat transfectants +/− decitabine by BLI. Decitabine induced 5–7 fold increase luciferase only in those 5′ transfectants. Of note is that dcT could potently suppress proliferation of CD4+CD25- T cells in vitro (ratio 1:1) in response to both anti-CD3/CD28 bead activation and to allo-APC in mixed lymphocyte cultures. Transwell experiments demonstrated that the suppressor function of dcT is cell-contact dependent. Surprisingly, we found that their suppressive properties are independent of Foxp3 expression in that dcT from Foxp3 KO mice were equally suppressive in bead-based proliferation assays as WT dcT. These data strongly suggest that decitabine may allow for the reexpression of genes such as Foxp3 and critical genes downstream of Foxp3 which mediate the suppressor phenotype in vitro. In murine T-cell depleted BMT model (B6→Balb/c), conventional T-cells (Tconv) (10×106) incubated with anti-CD3/CD28 beads and decitabine were found to promote enhanced engraftment with reduced GvHD, compared to mice receiving PBS-treated Tconv. In addition, addition of dcT with Tconv (1:1 ratio) resulted in decreased GvHD and improved survival, compared to Balb/c recipients receiving B6 Tconv and pbsT. In summary, decitabine-treatment enhanced Foxp3 expression in CD4+CD25- non-Treg cells. These dcT efficiently suppressed the proliferation of allo-reactive T cells in vitro and mitigated GvHD in vivo. The suppressor function of dcT was cell-contact dependent but unexpectedly Foxp3-independent.