Studies on the function of Twist2 during CD4/CD8 thymocyte lineage commitment

Abstract

CD4/CD8 T cell lineage commitment is one of the central issues in the development of the immune system. T cell receptor (TCR) signal strength and/or duration is known to be a key in determining the lineages. Several transcription factors are known to participate during CD4/CD8 lineage commitment. Gata3 and ThPOK reportedly play critical roles in CD4 single-positive (CD4 SP) differentiation. Conversely, Runx3 and Mazr reportedly play important roles in CD8 single-positive (CD8 SP) differentiation. Particularly, ThPOK seems to be both necessary and sufficient for CD4­lineage differentiation and Runx3 plays an important role in CD8 SP differentiation by repressing ThPOK expression. However, the critical factor(s) regulating this process is still missing. For example, a defined factor(s) translating the TCR signal and co-receptor complexes into the lineage differentiation is not clear yet. Also, it has been reported that the Runx complex binds persistently to the silencer region of ThPOK in thymocyte subsets, regardless of their capability to differentiate into the CD4 or CD8 SP lineage. Thus, it has been postulated that some other factor(s) would act to resolve the distinct suppressive activity of the Runx complex on ThPOK between CD4 and CD8 SP cells. Twist2 is a basic helix-loop-helix transcription factor that generally act as transcriptional repressors. It was reported that Twist2 expression was found to be regulated by TCR signaling and to adjust the survival or death of thymocytes and Twist2 controls TCR-mediated apoptosis by regulating the expression of Nur77 and Nor-1. These results suggest that Twist2 may play an important role during lymphocyte development. Here, I show that Twist2 is a critical factor for CD4/CD8 thymocyte differentiation. Twist2 expression is differentially regulated by T-cell receptor signaling leading to differentiation into the CD4 or CD8 lineage. Twist2 expression was higher in CD8 SP thymocytes than in CD4 SP thymocytes. Twist2 interacts with Runx3 and their interaction is dependent on Twist2 carboxyl-terminal Twist-box. Also, Twist2 downregulated the expression of ThPOK like Runx3. Twist2 could bind to the silencer region of the ThPOK promoter in a lineage-dependent manner. ThPOK silencer has two important regions, which are an essential 80bp core sequence (containing NFkb and E box) and a Runx binding domain. Twist2 was specifically bound to the Runx sites. These results suggest that Twist2 interacts with Runx3 to bind to the RBS1 site of ThPOK, thereby blocking its expression. Moreover, Twist2 transgenic mice perturbed CD4+ thymocyte differentiation, whereas enhanced CD8+ thymocyte differentiation. In contrast, the development of CD8 SP thymocytes was impaired in Twist2 deficient mice. Also, Twist2 expression produced mature CD8+ thymocytes in B2m-/- mice, while its deficiency significantly impaired CD8+ cells in TCR transgenic mice favoring CD8 T-cell differentiation. These gain-of-function and loss-of-function mutations mice systems showed that Twist2 has a critical role in CD4/CD8 thymocyte lineage differentiation through interaction with Runx complex to suppress the expression of ThPOK in a CD8 SP lineage-specific manner.