Figure 4. Depletion of AzaC generated B6.Foxp3^DTR/GFP Tregs by DT reduces the protective effect of AzaC in an allotransplant model depleted of natural Tregs.

(a) Balb/c mice were lethally irradiated and infused with 1×10⁷ B6.Foxp3^DTR/GFP pan T cells, depleted of nTregs (using anti-CD25 antibodies), 11 days after TCD-BM transplant (CD45.1). Mice were subsequently treated with AzaC alone (AzaC+PBS), DT alone (PBS+DT) neither drug (PBS+PBS), or both drugs (AzaC+DT). Some mice received irradiation and only TCD-BM (BM only) (b) AzaC prolongs the survival of mice transplanted with 1×10⁷ pan T cells depleted of nTregs when compared to mice treated with both AzaC and DT. ^ǂ The effect of AzaC generated Tregs. (p=0.02) ^ǂǂ The direct antiproliferative effects of AzaC upon Teffs (p<0.0001). (c) nTregs are required in the donor graft for optimal mitigation of GvHD by AzaC. Lethally irradiated Balb/c were infused with either 1×10⁷ nTreg depleted pan T (TRD) or 1×10⁷ nTreg replete pan T prior to treatment with PBS or AzaC on days +15, +17, +19 and +21. Mice receiving nTreg depleted pan T cells had significantly reduced survival when compared to mice receiving nTreg replete pan T following treatment with AzaC (pan T AzaC vs. TRD AzaC, p=0.0004). Data combined from multiple independent experiments.