Differentiation of pathogenic granzyme A-producing CD4+ T cells in graft-versus-host-disease

项目总结/摘要。在移植物抗宿主病（GVHD）中， 在造血细胞移植（HCT）后发生的免疫应答中，供体CD 4+辅助性T（Th）细胞运输至 多个器官及其在肠中的存在与恶化的临床预后相关。尽管他们 尽管Th细胞在GVHD中的作用是已知的，但Th细胞如何促进肠道疾病仍不清楚。我的实验室最近 鉴定了一种新的肠Th细胞群，其产生丝氨酸蛋白酶颗粒酶A（GrA）， 对肠道损伤和GVHD相关死亡率至关重要。而GrA+ Th细胞则明显低于对照组， HCT的有益抗癌作用，因此代表了GVHD中有希望的治疗靶点 患者由于有效的GrA抑制剂目前不可用，因此定义了导致 GrA+ Th细胞分化将揭示GVHD的新药物靶点， Th细胞在肠内分化。在这里，我们表明，GrA+ Th细胞分化独立于 需要整合STAT 3依赖和非依赖细胞因子信号 在疾病期间进行最佳分化的途径。GrA+ Th细胞表达高水平的STAT 3- 诱导转录因子（TF）BATF和Aiolos，这也是它们发育所必需的。然而，在这方面， 启动GrA+ Th细胞分化的STAT激活细胞因子以及STAT 3诱导的TF如何引导这一分化 GVHD期间的过程仍然不确定。由于STAT 3是最佳GrA+ Th细胞分化所必需的，我们 假设STAT 3激活细胞因子IL-6和IL-21与其他细胞因子/STAT 启动GrA+ Th细胞分化的途径，STAT 3/BATF/Aiolos网络协同促进 aGVHD期间GrA+ Th细胞谱系。为了检验这一假设，我们将1）定义细胞因子/STAT信号传导 在GVHD过程中调节致病性GrA+ Th细胞分化的要求和2）我们将确定如何 STAT 3/BATF/Aiolos TF网络及其下游效应基因调控GrA+ Th细胞分化 使用组合基因表达、染色质占有率分析和新型全基因组CRISPR的程序 屏幕完成这些研究后，我们将确定炎症和转录因子 GVHD中GrA+ Th细胞发育所需的条件，并验证了这些因素， 临床前疾病模型中的治疗靶点。这项工作是非常重要的，因为GrA+ Th细胞是一种主要的 肠道病理和致命疾病的驱动因素。因此，参与细胞凋亡的信号通路和基因 我们鉴定的GrA+ Th细胞转录特征可能是GVHD患者的潜在药物靶点。 这些实验也是高度创新的，因为我们将利用一种新的Gzma-GFP报告小鼠， 尖端的测序技术（RNA-Seq，CUT&RUN），以揭示STAT 3驱动的TF网络 驱动传统Th 17/T滤泡辅助细胞范例之外的致病性Th细胞分化。

PROJECT SUMMARY/ABSTRACT. In graft-versus-host disease (GVHD), a detrimental allo-immune response that occurs after hematopoietic cell transplant (HCT), donor CD4+ T helper (Th) cells traffic to multiple organs and their presence in the intestines correlates with worsened clinical prognosis. Despite their known role in GVHD, how Th cells contribute to intestinal disease remains unclear. My laboratory has recently identified a novel population of intestinal Th cells that produced the serine protease granzyme A (GrA) which were critical for intestinal damage and GVHD-associated mortality. However, GrA+ Th cells were dispensable for the beneficial anti-cancer effects of HCT and therefore represent a promising therapeutic target in GVHD patients. As effective GrA inhibitors are currently unavailable, defining the molecular mechanisms that lead to GrA+ Th cell differentiation will uncover novel drug targets for GVHD and provide insight into how pathogenic Th cells differentiate within the intestines. Here we show that GrA+ Th cells differentiate independently from other Th lineages and require the integration of both STAT3-dependent and -independent cytokine signaling pathways for their optimal differentiation during disease. GrA+ Th cells expressed high levels of the STAT3- induced transcription factors (TFs) BATF and Aiolos that were also required for their development. However, the STAT-activating cytokines that initiate GrA+ Th cell differentiation and how STAT3-induced TFs guide this process during GVHD remain undefined. As STAT3 is required for optimal GrA+ Th cell differentiation, we hypothesize that the STAT3-activating cytokines IL-6 and IL-21 function in parallel with other cytokine/STAT pathways to initiate GrA+ Th cell differentiation and that a STAT3/BATF/Aiolos network cooperatively promotes the GrA+ Th cell lineage during aGVHD. To test this hypothesis we will 1) Define the cytokine/STAT signaling requirements that regulate pathogenic GrA+ Th cell differentiation during GVHD and 2) We will determine how STAT3/BATF/Aiolos TF network and its downstream effector genes regulate the GrA+ Th cell differentiation program using combined gene expression, chromatin occupancy analysis and a novel genome-wide CRISPR screen. Upon completion of these studies we will have defined the inflammatory and transcription factor requirements that are required for the development of GrA+ Th cells in GVHD and validated these factors as therapeutic targets in pre-clinical models of disease. This work is highly significant as GrA+ Th cells are a major driver of intestinal pathology and lethal disease. Therefore, the signaling pathways and genes involved in the GrA+ Th cell transcriptional signature that we identify may be potential druggable targets for GVHD patients. These experiments are also highly innovative as we will leverage a novel Gzma-GFP reporter mouse with cutting-edge sequencing technologies (RNA-Seq, CUT&RUN) to uncover how a STAT3-driven TF network drives pathogenic Th cell differentiation outside of the traditional Th17/T follicular helper cell paradigm.