Determination of immune effectors following stem cell therapy. A) Infiltration of immune cells into the melanoma lung tumors. The lung tumors stained with monoclonal antibodies specific for mouse CD4, FOXP3, IL2 and CD8. Only respective images of CD4+, FOXP3+ and IL2+ cells from control samples are shown as there was no statistically significant difference between groups. Black arrows denote FOXP3+ and Il2+ cells. Results show significant infiltration of CD8+ cell into the melanoma lung tumors after injection of IFNγ-expressing ADSCs. Scale bars =50 μm. B) As shown in the bar graphs, mean number of CD8+ cells significantly increased in metastatic melanoma tumors of mice treated with IFNγ-ADSCs in a statistically meaningful manner. N =6; ***P < .001. C and D) Representative FACS plots for analysis of systemic CD4+ and regulatory T cells in peripheral blood obtained from the sub-ocular region of mice with established melanoma metastases. In addition, blood samples were taken from normal mice to measure normal number of CD4+ and Tregs. C) Left plot is representative FACS plot showing two distinct populations of PBMCs: lymphocytes and monocytes. Right plot is representative FACS plot indicating CD4 positive PBMCs. D) CD4+ lymphocytes were analyzed for expression of CD25 and FOXP3 by flow cytometry. Representative plots indicating analysis of Tregs in Normal, Control, EGFP-ADSCs and IFNγ-ADSCs. E) The percentage of CD4+ cells indicated a statistically significant decreased systemic level of CD4+ cells in ADSC-injected groups. The plot also shows a statistically significant decrease in the percentage of Tregs in the peripheral blood of mice with melanoma metastases treated with IFNγ-producing ADSCs. This percentage is comparable with systemic regulatory T cells of normal mice (P = .109; IFNγ-ADSCs compared to normal mice). N =6; ***P < .001, **P < .01, *P < .05.