Further characterization of Foxp3+ cells was performed by flow cytometry of cells isolated from blood, spleen, and liver graft seven days after transplantation. (A) Gated CD3+ and CD4+ or CD8+ double positive cells were further analyzed for CD25 and Foxp3 expression. (B) CD4+CD25+Foxp3+ regulatory T cells. Dual treatment resulted in greater numbers of CD4+CD25+Foxp3+ regulatory T cells in the liver than single drug or no treatment. Interestingly, low dose tacrolimus treatment alone increased levels of CD4+CD25+Foxp3+ regulatory T cells in peripheral blood. (C) CD8+Foxp3+ regulatory T cells. Plerixafor treatment alone significantly increased the percentage of CD8+Foxp3+ T cells in spleens and liver allografts at 7 days. However the highest concentration of CD8+Foxp3+ T cells in peripheral blood, spleen, and in liver allografts was found in animals receiving dual treatment. Quantitative data are represented as group means (bars) (n=3). *P < 0.05 and **P < 0.01.