Effect of the plasmids on NK, CTL and regulatory T cell (Treg) frequencies in mice. Plasmids included: control, pGFP-IRES, pGM-CSF-IRES, pIL-21-IRES, pGM-CSF-IRES- IL-21, and pGM-CSF-GFP-IRES-Rae-1-IL-21. At 10 d after treatment, mice were sacrificed and flow cytometry was used to determine splenic total T cells CD3+, Th cells (CD3+ CD4+), CTL (CD3+ CD8+), B cells (CD3-CD19+), NK cells (CD11b + CD27+) and the proportion of Treg cells (CD4+ CD25+ Foxp3+). A: flow chart and frequency of NK, and B: flow chart and frequency of CTL in a mixed population of spleen cells. C: flow chart and frequency of Treg cells from the same tissue. D: ratios of T cells (CD3+), Th cells (CD3+ CD4+), and B cells (CD3-CD19+) in the spleen after 10 d of treatment. E: NK and CTL-mediated cytotoxity as determined by MTT assays after 10 d of treatment. All data were expressed as mean ± standard deviation (n = 3). Note: *P < 0.05 and **P < 0.01, as compared with control. #P < 0.05 and ##P < 0.01, as compared with IRES/GM-SCF and IRES/IL-21. △P < 0.05 and △△P < 0.01, as compared with IRES/GM-SCF-IL-21.