Fig. 2.

Tumor-derived MIF directly inhibits immune activation of T cells. (A) Purified T cells from B6 mice, 1 × 10⁵, were incubated with 50 U/ml IL-2 and 12.5 ng/ml IL-15. [³H]Thymidine was added after 72−96 h of culture and the cells harvested after overnight thymidine incorporation. Incorporated average counts per minute (cpm) from triplicate wells (error bars indicate standard deviations) treated with cytokines only (cntrl) or wells containing dilutions of day 3 culture supernatants from non-transfected AGN2a (wild type), AGN2a transfected with d-siRNA for MIF (MIF siRNA), or AGN2a transfected with d-siRNA for GFP (GFP siRNA) are shown. Dilutions of the culture supernatants are indicated on the right of the figure. GFP and wild-type differed significantly from MIF si-RNA (P < 0.02) while the 1:100 dilution of MIF siRNA treated cell culture supernatant did not significantly differ from the control. (B) The amounts of MIF in culture supernatants from AGN2a (solid bars) and from U2OS/MIF cells were normalized by ELISA and then used in the same thymidine incorporation assay. Average thymidine incorporation and standard deviation of triplicate wells are shown, representative of three experiments. MIF from either source resulted in significant loss of proliferative activity (P < 0.01) at 10 pg/ml, while differences at 1 pg/ml were not statistically significant, demonstrating dose dependency.