Defining The Mechanism Involved In The Inhibition Of GvHD By Azacytidine In Vivo Through The Use Of FoxP3 Diphtheria Toxin Receptor (Foxp3^DTR) Donor T Cells

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the primary treatment for high risk and relapsed hematological malignancies. However, the desired Graft vs. Leukemia (GvL) effect is frequently accompanied by Graft vs. Host Disease (GvHD) in which donor lymphocytes target host tissues, often resulting in significant morbidity and mortality. We and others have shown that the hypomethylating agent, 5-azacytidine (azaC), can mitigate GvHD in both murine preclinical transplant models and in human clinical trials while maintaining a robust GvL effect. Two mechanisms have been proposed to explain how azaC reduces GvHD: 1) the generation of suppressive regulatory T cells (Tregs, CD4+CD25+Foxp3+) through the in vivo conversion of alloreactive donor T effectors (FoxP3-) into suppressive regulatory T cells (Tregs, CD4+CD25+Foxp3+) via the pharmacologic hypomethylation of the Foxp3 promoter resulting in enhanced gene expression (Choi et al 2010) or, 2) the direct anti-proliferative and pro-apoptotic effects of azaC on allogeneic T cells (Sánchez-Abarca et al. 2010).

To assess the importance of azaC-generated Tregs in ameliorating GvHD we obtained Foxp3^DTR mice in which Tregs can be selectively ablated (>97%) following the administration of diphtheria toxin (DT) (Kim et al. 2007). To induce GvHD, either 1x10⁷ panT or 1x10⁷ Treg-depleted panT cells obtained from Foxp3DTR mice were injected (CD45.2+) via the lateral tail vein on day 11 post allo-HSCT (CD45.1+) into allogeneic recipients (Balb/c). Transplanted mice were treated with PBS (carrier) or azaC (2 mg/kg) (i.p.) on days +15, +17, +19, and +21, followed by injection (i.p.) of DT or PBS on days +16, +18, +20, (DT 10 μg/kg) and +22 (DT 50 μg/kg) to deplete azaC-induced Tregs. Complete depletion of Tregs by DT was confirmed by flow cytometery.

Mice transplanted with panT cells and injected with PBS (n=10) or DT (n=10) (without azaC) developed severe GvHD and died by day ∼26 post HSCT. In sharp contrast, 90% of mice (n=10) treated with azaC alone survived >100 days, displayed no visible GvHD related symptoms, and gained weight at a similar rate as T cell depleted bone marrow controls. The treatment of mice with azaC+DT (n=10) resulted in significantly prolonged survival compared to the PBS treated mice (p=<0.001) but in contrast to the azaC group, most failed to survive beyond day 60 (p=<0.001, Fig 1a). Furthermore, the azaC alone group had higher percentages of Tregs (CD4+/CD25+/FoxP3+, p=<0.001), CD3+ T cells (p=<0.001), and B220+ B cells (p=0.04) than the azaC+DT group (consistent with less clinical GvHD) and less gastrointestinal GvHD when graded by an independent pathologist. In separate studies, mice transplanted with panT cells depleted of natural Tregs upfront (using anti-CD25 antibody), lost weight, developed GvHD and failed to survive, even when treated with azaC alone (Fig. 1b). These results suggest that the GvHD sparing effect of azaC is dependent on both the conversion of CD4+ T effectors to Tregs and the presence of natural Tregs, and eliminating this population with DT results in greater GvHD and reduced survival. Further experiments are ongoing to determine the relative importance of both natural Tregs and azaC generated Tregs to prevent GvHD in this mouse model.

Table.

Figure 1a. Foxp3DTRGFP panT cells.	Figure 1b. Foxp3DTRGFP Treg depleted panT cells

Figure 1a. Foxp3DTRGFP panT cells.	Figure 1b. Foxp3DTRGFP Treg depleted panT cells

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