Fig. 4.

Lack of IFN-γ sensitivity protects activated CD8 + T cells from IFN-γ-induced apoptosis. a Adoptively transferred Thy1.1⁺ Pmel cells were sorted from cell suspensions prepared from similar size tumors (n = 3) 5 days after treatment with Pmel^Ag or Pmel^Ag+IL-12 cells, and analyzed for IFNγR1 and IFNγR2 expression by qPCR. Data are expressed as fold expression of Pmel^Ag+IL-12 over Pmel^Ag ±SD (p = 0.07). 1 × 10⁶ ex vivo expanded wild-type Pmel^Ag or Pmel^Ag+IL-12, and IFNγR^−/− Pmel^Ag cell were recultured in fresh media. b Cell viability was determined after 24, 48, and 72 h of culture using the LIVE/DEAD^® assay. c Apoptosis was measured by annexin V stain 24 h after culture. Data are presented as the average ratio of CD8⁺ annexin V⁺ over CD8⁺ annexin V⁻ cells ± SD (n = 3). d RNA isolated from cells after 24 h of culture was analyzed by qPCR for expression of Bcl-2 and Bax. These values were used to calculate Bax/Bcl-2 ratio which directly associates with apoptosis [20]. Data are presented as the average change in Bax/Bcl-2 ratio relative to their respective baseline measurement ± SD (n = 3). Two-tailed Student’s t test was used, *p < 0.05, **p < 0.005, and ***p < 0.0001. Data shown are representative of at least three independent experiments