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

项目摘要/摘要。在移植物抗宿主病(GVHD)中，一种有害的同种免疫 造血细胞移植(HCT)后发生的应答，供者的CD4T辅助(Th)细胞向 多个器官及其在肠道中的存在与临床预后恶化相关。尽管他们 尽管已知Th细胞在GVHD中的作用，但Th细胞如何参与肠道疾病仍不清楚。我的实验室最近 鉴定了一种新的肠道Th细胞群，它产生丝氨酸蛋白酶颗粒酶A(Gra)，该酶 是肠道损伤和移植物抗宿主病相关死亡率的关键因素。然而，Gra Th细胞是不必要的 对于HCT的有益抗癌作用，因此是GVHD的一个有前途的治疗靶点 病人。由于目前还没有有效的Gra抑制剂，因此定义了导致 Gra Th细胞分化将发现治疗GVHD的新药物靶点，并为了解GVHD的致病机理提供线索 TH细胞在肠道内分化。在这里，我们展示了Gra Th细胞独立于 其他Th血统，需要STAT3依赖和非依赖细胞因子信号的整合 它们在疾病过程中最佳分化的途径。Gra Th细胞高水平表达STAT3- 诱导转录因子(TF)、BATF和Aiolos也是它们发育所必需的。然而， 启动Gra Th细胞分化的STAT激活细胞因子以及STAT3诱导的转录因子如何引导这一过程 GVHD期间的流程仍未定义。由于STAT3是Gra Th细胞最佳分化所必需的，我们 假设激活STAT3的细胞因子IL-6和IL-21与其他细胞因子/STAT并行发挥作用 启动Gra Th细胞分化的途径以及STAT3/BATF/Aiolos网络协同促进 移植物抗宿主病期间的Gra-Th细胞谱系。为了验证这一假设，我们将1)定义细胞因子/STAT信号 GVHD期间调节致病性Gra Th细胞分化的要求和2)我们将确定如何 STAT3/BATF/Aiolos转移因子网络及其下游效应基因调控Gra-Th细胞分化 结合基因表达、染色质占有率分析和新的全基因组CRISPR的程序 屏幕上。在这些研究完成后，我们将确定炎症和转录因子 GVHD中Gra Th细胞发育所需的要求，并验证了这些因素 疾病临床前模型中的治疗靶点。这项工作意义重大，因为Gra Th细胞是主要的 肠道病理和致命性疾病的驱动力。因此，信号转导途径和基因参与了 我们发现的Gra Th细胞转录信号可能是GVHD患者的潜在药物靶点。 这些实验也很有创新性，因为我们将利用一种新型的Gzma-GFP报告鼠标 尖端测序技术(RNA-Seq、Cut&Run)，揭示STAT3驱动的TF网络如何 推动致病性Th细胞在传统的Th17/T滤泡辅助细胞范式之外的分化。

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.