The role of Runx1 in the regulation of T cell anergy

摘要：T细胞无能是一种程序化的细胞低反应状态，可阻止T细胞的传播。 对自我的免疫反应。因此，无能为力是自我容忍的关键组成部分。因此， T细胞中无能程序的发展和维持受到许多机制的严格控制，两者都 细胞内在的和细胞外在的。由于无能在自我耐受中很重要，因此理解 调节无能程序的启动和调节的复杂生物学。我们在野生型中发现 (WT)转录因子RUNX1表达降低的小鼠与无能T细胞相关，定义为 细胞表面标记CD73和FR4的共表达。此外，T细胞中RUNX1的条件性缺失 使用CD4-CRE可以产生更高频率的共表达CD73和FR4的T细胞。这支持 RUNX1抑制CD4+T细胞无能发展的假说。CD4-CRE RUNX1 CKO小鼠 T细胞成熟受阻，外周淋巴器官中仅有极少数的CD4+T细胞。绕过这一点 成熟缺陷，并测试RUNX1在外周CD4+T细胞中的作用，我们产生了新的1：1混合 应用雌激素受体(ER)-Cre RUNX1 CKO骨髓混合的骨髓嵌合体(BMC)系统 B6.SJL野生型(WT)骨髓。这通过删除外周血细胞中的RUNX1来绕过成熟缺陷 在他莫昔芬完全成熟后，以他莫昔芬可诱导的方式。此外，一半的人 细胞是RUNX1充足的，允许分析T细胞的内在效应。在这个系统中，我们发现 RUNX1调节细胞内源性无能的诱导，因为CD73和CD73共表达的频率增加 外周CD4+T细胞中FR4仅见于ER-CRE RUNX1 CKO细胞，而不存在于B6.SJL WT细胞 源自同一只鼠标。拟议研究的目标1将确定关键基因靶点RUNX1 调节以控制CD4+T细胞无能。为此，通过RNA测序确定的候选基因将是 在我们的新型ER-CRE系统中进行了分析，并将从功能上评估它们在无能中的作用。令人惊讶的是，这两个 他莫昔芬治疗动物体内ER-CRE、RUNX1、CKO和B6、SJL、CD_4~+T细胞不能增殖 体外TCR刺激，表明RUNX1调节一种控制耐受性的细胞-外在信号机制。 目的2将确定无能RUNX1缺陷的CD4+T细胞用于耐受野生型CD4+T细胞的关键机制。 为了完成这一目标，我们将研究候选基因在野生型细胞中的作用。对事件的进一步调查 RUNX1在CD4+T细胞无能中的作用将有助于深入了解免疫系统是如何启动和维持的 自我宽容。

Abstract: T cell anergy is a programmed state of cellular hyporesponsiveness that prevents the propagation of an immune response to self. Thus, anergy is a critical component of self-tolerance. Consequently, the development and maintenance of the anergy program in T cells is tightly controlled by many mechanisms, both cell-intrinsic and cell-extrinsic. Since anergy is important in self-tolerance, it is critical to understand the complex biology that regulates the initiation and regulation of the anergy program. We have found in wild-type (WT) mice that lower expression of the transcription factor Runx1 is associated with anergic T cells, defined by co-expression of the cell-surface markers CD73 and FR4. Furthermore, conditional deletion of Runx1 in T cells using a CD4-Cre produces a higher frequency of T cells that co-express CD73 and FR4. This supports the hypothesis that Runx1 suppresses the development of anergy in CD4+ T cells. CD4-Cre Runx1 cKO mice have a block in T cell maturation and have very few CD4+ T cells in peripheral lymphoid organs. To bypass this maturation defect, and test the role of Runx1 in peripheral CD4+ T cells, we have generated a novel 1:1 mixed bone marrow chimera (BMC) system using Estrogen Receptor (ER)-Cre Runx1 cKO bone marrow mixed with B6.SJL Wild Type (WT) bone marrow. This bypasses the maturation defect by deleting Runx1 in peripheral CD4+ T cells in a tamoxifen inducible manner after they have completed maturation. Furthermore, half the cells are Runx1-sufficient allowing for analysis of T cell-intrinsic effects. In this system, we have found that Runx1 regulates the cell-intrinsic induction of anergy, as increased frequency of co-expression of CD73 and FR4 in peripheral CD4+ T cells is only seen in the ER-Cre Runx1 cKO cells and not the B6.SJL WT cells derived from the same mouse. Aim 1 of the proposed studies will determine critical gene targets Runx1 regulates to control CD4+ T cell anergy. To do this, candidate genes identified by RNA-sequencing will be analyzed in our novel ER-Cre system, and their role in anergy will functionally assessed. Surprisingly, both the ER-Cre Runx1 cKO and the B6.SJL CD4+ T cells in the tamoxifen treated animals fail to proliferate upon in vitro TCR stimulus, suggesting that Runx1 regulates a cell-extrinsic signaling mechanism controlling tolerance. Aim 2 will define key mechanisms anergic Runx1-deficient CD4+ T cells use to tolerize wild type CD4+ T cells. To complete this aim we will examine the role of candidate genes in wild type cells. Further investigation of the role of Runx1 in CD4+ T cell anergy will provide insight into how the immune system initiates and maintains self-tolerance.