Figure 2. Decrease in CD4+Tregs in MIF−/− mice.

(A) Decrease in CD4+Tregs in MIF−/− mouse as compared to MIF+/+ mouse. Representative data of similar results in more than five mice are shown. Using flow cytometry analysis, the frequency of CD4+CD25+Foxp3+ T cells, CD4+GITR+ T cells, and CD4+CTLA4+ T cells was determined in single cell suspensions of tumor mass of MIF−/− versus MIF+/+ mouse. (B) Representative flow cytometry analysis of CD4+Tregs in the spleen of MIF−/− mouse versus MIF+/+ mouse. (C) Comparison of the percentage of CD4+Tregs in the spleen and tumor tissue between tumor-bearing MIF−/− and MIF+/+ mice. The frequency of Tregs was calculated as the percentage of CD4+CD25+Foxp3+ cells in the CD4+ T-cell population. Mice without tumor inoculation were used as a control. **, P < 0.01. (D) Tumor rejection in MIF−/− mice. Left panel represents a photograph of tumor-bearing and tumor-rejected MIF−/− mouse taken 4 weeks after tumor inoculation. Right panel shows changes in tumor volume in tumor-rejected versus tumor-bearing MIF−/− mice. Representative data are shown and the arrow indicates the injection site. (E and F) Recovery of CD4+ and CD8+ T cells in the spleen of tumor-rejected MIF−/− mice. The frequency of CD4+ or CD8+ T cells was determined by flow cytometry and calculated as the percentage of CD4+ or CD8+ cells in the splenocytes from mice. Tumor-naïve MIF−/− and MIF +/+ mice were used as a control. *, P < 0.05. (G) Decrease in CD4+ Tregs in the spleen of tumor-rejected MIF−/− mice, as compared to that of tumor-bearing MIF−/− and MIF +/+ mice. The frequency of Treg was calculated as the percentage of CD4+CD25+Foxp3+ cells in the CD4+ T-cell population. *, P < 0.05, **, P < 0.01. (H) Correlation of the frequency of CD4+Tregs with tumor size determined at 4 weeks after tumor injection, in all of MIF−/− and MIF+/+ mice tested.