Azacitidine mitigates GvHD via its differential effects on expansion of nTregs in vivo.. Mus musculus

Azacitidine (AzaC) mitigates Graft vs. Host Disease (GVHD) in both murine preclinical transplant models and in human clinical trials while maintaining a robust Graft vs. Leukemia (GVL) effect. Previous studies have failed to investigate the role of natural Tregs on the mitigation of GVHD by AzaC; instead focusing on the generation of suppressive regulatory T cells (Tregs, CD4+CD25+FOXP3+) through the in vivo conversion of alloreactive donor T effectors (Teff CD4+ CD25-, FOXP3-) and the direct anti-proliferative effects of AzaC on allogeneic T cells. Using B6.Foxp3DTR/GFP mice in which Tregs can be specifically ablated through administration of Diphtheria toxin, we demonstrate that nTregs are required in the donor graft for AzaC to optimally protect against GvHD and that nTregs, unlike T effectors (Teff, CD3+FOXP3-), are resistant to the anti-proliferative effects of AzaC. Gene expression analysis identified the potent cell cycle inhibitor, p21, was significantly upregulated in Teff but not nTregs after treatment with AzaC. Furthermore, we demonstrate that Teff cells deficient in p21 are less sensitive to the antiproliferative effects of AzaC. These results, demonstrate that nTregs are essential for AzaC to fully protect against GvHD and have important clinical implications for future clinical trials testing AzaC as a novel method of GVHD prophylaxis in man. Overall design: n=3 per group nTregs and AzaC generated Tregs, nTregs were purified from the spleens of B6.Foxp3GFP mice and Treg depleted T cells were obtained from B6. Foxp3GFP x B6.CAGDSRED. T Cells were co-cultured at a 1:10 ratio of nTreg to Teff and were activated for 2 days in the presence of anti-CD3/CD28 beads (bead:cell 1:1; Invitrogen) and Xcyte medium supplemented with L-glutamine (4mM), penicillin (100 U/mL), streptomycin (100 µg/mL), and human recombinant IL-2 (hIL-2; 500 U/mL). The activated T cells were incubated in the presence of AzaC (1µM) (Sigma-Aldrich) or PBS for an additional 2 days. Cells were sorted using FACS Aria II (BD) to isolate nTreg (CD4+DSRED-FOXP3GFP+), CD4+ Teff (CD4+DSRed+FOXP3GFP-), CD8+ Teff (CD8+DSRed+FOXP3GFP-).