Epigenetic Control Of GvHD And GvL Using The Hypomethylating Agent Azacitidine

Abstract

Allogeneic bone marrow transplantation (BMT) represents the most effective treatment for patients with high risk and relapsed hematologic malignancies because allogeneic donor T cells provide a graft-versus-leukemia (GvL) effect. However, the same cells can cause graft-versus-host disease (GvHD), one of the major complications. Regulatory T cells (Tregs) have been shown to suppress GvHD while preserving GvL, their use provides a promising strategy in the allogeneic transplant setting if three major obstacles will be overcome: 1) the low numbers of Tregs, 2) loss of suppressor activity following ex vivo expansion and 3) the lack of Treg-specific markers to purify ex vivo expanded Tregs. The FOXP3 transcription factor, which is exclusively expressed in Tregs, can convert effector T cells (Teff) into Tregs when ectopically overexpressed. The Foxp3 locus is unmethylated in Tregs while highly methylated and silenced in all other T cells. The hypomethylating agent azacitidine (AzaC) can modulate this methylation status and induce stable FOXP3 expression (>7 days) in Teff. Furthermore, we have shown that these AzaC-induced FOXP3+T cells are suppressive phenotype in vitro. Thus, we hypothesize that AzaC treatment of mice after allogeneic BMT will dramatically mitigate GvHD while preserving GvL via upregulation of Foxp3 in alloreactive Teff. In murine T cell depleted (TCD) BMT model (B6→Balb/c) with delayed infusion of conventional T cells (Tconv) (2X106) at day 11 post BMT, followed by subcutaneous treatment of AzaC (2 mg/kg at days 15, 17, 19, and 21 post BMT), we found that AzaC dramatically suppressed GvHD caused by allogeneic donor T cells while maintaining donor engraftment of all lineages. The AzaC group had significantly higher FOXP3+Tregs than in PBS control group and that these Tregs were derived from donor T cells, suggesting that the suppression of GvHD was mediated by AzaC-induced Tregs. We further tested whether AzaC treatment of mice transplanted with allogeneic T cells preserve GvL while mitigating GvHD. Using the same murine allogeneic BMT model, Click Beetle Red luciferase-expressing A20 leukemia cells (Balb/c derived; 1X104) were injected with TCD BM and 10×106 Tconv and in vivo bioluminescence imaging was performed to assess tumor burden. We found that AzaC treatment mitigated GvHD without abrogating GvL or donor engraftment. Thus, the adminstration of hypomethylating agents like AzaC might be a promising strategy to treat GvHD.