text: Runx3 controls Eomes, perforin, granzyme B, and IFN-gamma expression in effector CTLs. Runx3+/+ or Runx3-/- CD8+ T cells were activated and transduced with retroviruses bearing an empty IRES-GFP cassette (GFP) or also encoding Eomes-VP16 (Eo-VP16) or Myc-Runx3 (Runx3). The frequency of transduced cells in the cultures was equivalent for all constructs (approximately75-90% GFP+ cells; not depicted). (A) Protein expression in whole-cell extracts (day 6) was analyzed by immunoblotting. Overexpression of Eomes-VP16 cannot be detected with the Eomes antibody, as the C-terminal epitope is within the region that has been replaced with the VP16 transactivation domain. (B) Expression of granzyme B and IFN-gamma after culture for 6 d and restimulation for 4 h with PMA and ionomycin was determined by intracellular staining. The percentage of positively stained cells is shown above the gate; the mean fluorescence intensity (MFI) of granzyme B staining for the total population is shown below the gate. The vertical gray lines indicate the MFI for WT GFP+ cells. Results are representative of at least two independent experiments. (C) Schematic diagram of the transcriptional network involving Runx3 and T-box factors. T-bet is induced by TCR signals and is essential for early IFN-gamma expression. Runx3 is present in naive CD8+ T cells and represses Runx1 and induces Eomes, perforin, granzyme B, and IFN-gamma expression. Eomes may participate in sustaining late IFN-gamma expression, whereas Runx3 and Eomes (but not T-bet) may cooperate to activate perforin expression. The dotted line indicates the partial effect of T-bet deficiency on Gzmb mRNA but not granzyme B protein expression. 
  