text: INTRODUCTION
  Naive CD8+ T cells differentiate into effector CTLs with the ability to lyse antigen-bearing target cells by exocytosis of lytic granules containing perforin and granzymes, and to produce inflammatory cytokines such as IFN-gamma and TNF upon restimulation through the TCR (1, 2). In vivo experiments have elucidated many critical parameters governing the development and evolution of primary CTL responses (3, 4). In this study, we have used in vitro systems such as those developed to study CD4+ T cell differentiation to define the molecular basis of effector CTL differentiation (5, 6). 
  The T-box transcription factors Eomesodermin (Eomes) and T-bet are needed for important aspects of effector and memory CTL differentiation (7). In uninfected mice, compound deletion of the Tbx21 (encoding T-bet) and eomesodermin genes is associated with a selective loss of CD8+ T cells with an IL-2Rbeta-high, memory phenotype (8). Mice deficient for both T-bet and Eomes in T cells have impaired expression of cytolytic mediators, manifest poor cytolytic activity, and fail to control acute lymphocytic choriomeningitis virus infection (9). Nevertheless, the specific roles of T-bet and Eomes in clonal expansion and CTL differentiation have not yet been resolved: in particular, it is not known whether these transcription factors function redundantly to control effector CD8+ T cell differentiation, and whether they do so directly by targeting specific effector cytokine and cytolytic genes. 
  Runx proteins, a family of three DNA-binding transcription factors, control thymocyte differentiation and the CD4/CD8 lineage decision (10-13). Runx3 and perforin mRNA are expressed by double-positive (DP) thymocytes and CD8+ single-positive (SP) thymocytes but not in CD4+ SP cells (14). Although Runx3 is not expressed in naive CD4+ T cells, its expression is up-regulated during Th1 cell differentiation, and Runx3 influences Th1 cell differentiation and function through direct regulation of the Il4 and Ifng cytokine genes (15, 16). In contrast, all three Runx proteins are expressed in mature CD8+ T cells (10, 12), and Runx3-deficient CD8+ T cells show reduced cytolytic activity (12, 13). We therefore tested whether Runx3 influenced cytolytic T cell differentiation. 
  In this report, we show that Runx3 and T-box factors synergistically regulate CTL differentiation and function. T-bet is induced quickly upon TCR stimulation and is required for early programming of cytokine production (17), whereas Eomes is induced later during differentiation and sustains IFN-gamma expression. Runx3 is required for Eomes and perforin expression, and both Eomes and Runx3 bind at the Prf1 locus; in contrast, perforin expression is unaffected in T-bet-deficient cells. T cells lacking Runx3 show decreased expression of IFN-gamma and granzyme B, and Runx3 also binds the promoter regions of the Ifng and Gzmb genes. Collectively, these results provide evidence for a complex transcriptional network in which Runx3 is a primary regulator of Gzmb expression but synergizes with T-bet and Eomes, respectively, to promote transcription of the Ifng and Prf1 genes. 
  