Generation of T_reg-Like Cells from CD4+CD25- T Cells Via Epigenetic Modification Using a Demethylating Agent Decitabine.

Regulatory T cells (T_reg) 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 T_reg found in the peripheral blood in the resting state (5–10% of CD4+ T cells), low yields and efficiencies of purification and severe limitations in ex vivo expansion without downregulating Foxp3 expression. The master gene responsible for the normal development and suppressor function in vitro and in vivo of T_reg is Foxp3 which is exclusively expressed in T_reg. It remains unclear what regulates the expression of Foxp3 in T_reg. Recently, selective CpG motifs within the Foxp3 locus have been reported to be demethylated in T_reg and heavily methylated in CD4+CD25-T cells. Thus, we sought to determine if an FDA-approved demethylating agent, Decitabine, could be used to enhance expression of Foxp3 via an epigenetic effect and convert CD4+CD25- T cells into T_reg. We incubated human CD4+CD25-FOXP3- T cells with CD3/CD28 beads (1:1 bead to cell) and hIL-2 (50u/ml) for 2–3 days and treated them with or without 0.1–10 uM Decitabine for an additional 3–4 days. 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 T_reg (10–12 fold increase above baseline). This resulted in increased expression at the protein level in 60% of treated cells (5% of PBS-treated cells). Decitabine also induced Foxp3 expression in 80% of murine CD4+CD25- T cells stimulated two days with CD3/CD28 beads and hIL-2 (10 u/ml), followed by two days of Decitabine treatment. The CD4+CD25-T cells expanded up to two fold during the stimulation and Decitabine treatment. One mouse spleen generated more than 5 x 10⁶ T_reg-like cells which is ten fold more than the number of naïve natural T_reg. In addition, these Decitabine-treated CD4+CD25- T cells (dcT) showed suppressor function in a mixed lymphocyte reaction. We next tested whether these cells were able to suppress GvHD in an allogeneic BMT model. Mice that were transplanted with dcT showed significantly higher survival rate and maintained their weight better. The T_reg surface markers, such as GITR, CTLA4, CD27, CD69 and CD101, were upregulated in the dcT. Activation of T_reg has been known to upregulate GzmB but not GzmA or Prf 1. We found that four fold more of dcT expressed GzmB than PBS-treated cells did (95.4% vs. 24.5%). Interestingly, Decitabine treatment induced GzmA expression in over 50% of the cells while about 5% of PBS-treated cells and natural Treg expressed the gene. Using Prf 1 KO and GzmB KO mice, we found that the suppressor function of the dcT is partially dependent on Prf 1, but not on GzmB. To identify cytokine(s) responsible for the suppressor function of these dcT, we analyzed supernatant from these cells and found that the level of IL-10, which has been known to be upregulated in T_reg and involved in suppressor function, was upregulated in dcT. The gene expression profiles were also compared between dcT, naïve natural T_reg, and bead-activated natural T_reg. We found that dcT expressed high level of CTLA-4, GITR, CD25 and Prf 1 which have been known to be upregulated in natural T_reg. In summary, Decitabine-treatment of CD4+CD25- T cells enhanced Foxp3 expression. These dcT showed suppressor function in both in vitro and in vivo and shared part of gene signature of natural T_reg.