text: Runx3 and T-box factors control a complex program of transcriptional regulation during CTL differentiation
  Collectively, these data provide evidence that Runx3, together with T-box factors, orchestrates a complex program of transcriptional regulation in differentiating CTL (Fig. 4 C). Runx3 is present in naive CD8+ T cells before activation (12). It represses Runx1 and has a positive role in the induction of Eomes, granzyme B, perforin, and IFN-gamma. Runx3 binds to promoters and putative regulatory regions of the latter three genes, suggesting a direct effect on gene expression. Additional experiments are needed to determine whether Eomes and Runx1 are also direct target genes of Runx3. 
  Surprisingly, Runx3 contributed to the optimal expression of TNF, IL-2, and IFN-gamma at day 4 (Fig. S2). For TNF and IL-2, the requirement for Runx3 subsides by day 6 (Fig. 3 C), possibly because of compensation by Runx1, which is derepressed in Runx3-/- cells (Fig. 4 A). Runx3 continues to be required for IFN-gamma expression even at day 6, perhaps because of its role in the induction of Eomes expression (Fig. 4 A). 
  An unexpected finding was that the two T-box transcription factors, T-bet and Eomes, are up-regulated with very different kinetics in CD8+ T cells under our culture conditions and have nonredundant roles in the subsequent expression of key effector proteins (Fig. 4 C). T-bet is needed early to confer on activated CD8+ T cells the competence to produce IFN-gamma upon restimulation, but its function is less important at later times. Eomes, which is induced late and functions downstream of Runx3, may substitute for T-bet in promoting the acute expression of IFN-gamma in restimulated CTLs (8). Indeed, T-bet and Eomes both contribute to perforin expression in NK cells (8, 26), and Eomes induces granzyme B as effectively as T-bet in developing Th2 cells (7); thus, the relative roles of these T-box transcription factors vary depending on cell type. 
  Surprisingly, however, Eomes and T-bet appeared nonredundant in their ability to induce two other markers of CTL function, Prf1 and Gzmb (Fig. 4 C). Rather, T-bet and Eomes were involved in regulating granzyme B and perforin expression, respectively: up-regulation of T-bet and Eomes mRNA and protein closely preceded up-regulation of Gzmb and Prf1 mRNA and protein, respectively. T-bet had no role in perforin expression under our culture conditions, and Eo-VP16 did not affect granzyme B expression when expressed in T-bet-/- or Runx3-/- cells. Because conventional Eomes-deficient mice die before precursor cells can be isolated for bone marrow transfers (27) and because T cells conditionally deficient in Eomes have only recently been described (9), we were unable to introduce Runx3 into Eomes-deficient CD8+ T cells to test formally whether Eomes cooperated with Runx3 to induce perforin expression. 
  Collectively, our data are consistent with a transcriptional network in which preexisting Runx3 cooperates with the induced T-box factors T-bet and Eomes and IL-2Rbeta signals (unpublished data) to orchestrate CTL differentiation (Fig. 4 C). Our data recall the "feed-forward" interaction between T-bet and Runx3 that we previously described in CD4+ (Th1) T cells (15) but are distinct in two respects: in differentiating Th1 cells, T-bet is induced by TCR signals and IFN-gamma, and in turn induces Runx3 (15), whereas in differentiating CD8+ T cells, preexisting Runx3 is required to induce the T-box transcription factor Eomes. Whole-genome experiments in these and other systems will be required to establish whether cooperation between T-box and Runx family transcription factors is a general feature of cellular differentiation programs. 
  