Use of FoxP3 Diptheria Toxin Receptor (Foxp3^DTR) Donor T Cells to Define the Mechanism Involved in the Inhibition of GvHD by Azacytidine in Vivo

Abstract 4113

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the primary treatment for high risk and relapsed hematological malignancies. However, the desired graft versus leukemia (GvL) effect is frequently accompanied by Graft versus 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 or,¹ 2) the direct anti-proliferative and pro-apoptotic effects of azaC on allogeneic T cells.²

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).³ To induce GvHD, 1×10⁷ panT cells obtained from Foxp3 DTR 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 injected with PBS (n=5) or DT (n=5) (without azaC) developed severe GvHD and died by day 26 post HSCT. In sharp contrast, 100% of mice (n=5) 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=5) resulted in significantly prolonged survival compared to the PBS treated mice (p=0.003) but in contrast to the azaC group, failed to survive beyond day 60 (p=0.003). Furthermore, the azaC alone group had higher percentages of Tregs (CD4+/CD25+/FoxP3+, p=0.001), CD3+ T cells (p=0.002), and B220+ B cells (p=0.02) than the azaC +DT group (consistent with less clinical GvHD) and less gastrointestinal GvHD when graded by an independent pathologist. These results suggest that BOTH pharmacologic conversion of alloreactive FoxP3- donor T cells to FoxP3-expressing Tregs and direct toxicity to allogeneic T cells by azaC represent important mechanisms involved in the amelioration of GvHD by azaC in vivo.