Role of Notch signaling during the early priming and activation of alloreactive T cells

项目总结 移植物抗宿主病(GVHD)是异基因骨髓最严重的并发症。 移植(allo-BMT)。移植物中存在的同种异体反应性T细胞介导了GVHD的发病 识别同种异体抗原并损害宿主组织。大多数GVHD疗法依赖于全球免疫抑制和 与感染风险增加和移植物抗肿瘤效应降低有关。新的战略 预防移植物抗宿主病，在保护宿主组织的同时保持其对肿瘤的杀伤活性 同种异体免疫T细胞是必需的。我们的实验室发现了Notch信令和类Delta 1/4(Dll1/Dll4)Notch 配体作为致病异基因T细胞反应的关键调节因子，以及有吸引力的新治疗靶点。在……里面 在小鼠和非人类灵长类动物模型中，抑制供体T细胞的Notch可导致长期保护 来自GVHD发病率和死亡率。抗Notch单抗在体短期阻断Notch的研究 特异性Notch受体或配体钝化同种异体反应性T细胞的致病性而不诱导 广泛的免疫抑制，降低GVT活性，或引发慢性Notch抑制的毒副作用。 而在移植时一次注射抗DLL1/DLL4 Notch配体抗体就足以治疗长期的 长期GVHD控制，移植后48小时给予DLL1/DLL4阻滞剂并不能预防 GVHD。因此，在T细胞激活的初始阶段，Notch信号的早期脉冲对于 建立致病T细胞状态。然而，我们目前还不清楚短期缺口是如何 抑制起到了持久的GVHD保护作用。同种异体反应性T细胞依赖两种中枢功能进行调节 疾病：转运到外周靶器官并产生促炎细胞因子。我们的初步数据 显示Notch抑制保留了次级淋巴器官中初始T细胞IL-2的产生和扩张 同时损害多种炎性细胞因子的分泌，并归巢到胃肠道。因此， 我假设在T细胞启动和激活的早期阶段传递给异基因T细胞的Notch信号是 对于转运到肠道并启动持续的炎性细胞因子反应至关重要。我推测这些东西 两个关键的机制解释了为什么短期的缺口抑制可以预防许多严重的、病理性的 GVHD的后果。为了探索这一假设，我将使用MHC不匹配的GVHD模型来 研究Notch抑制如何影响异基因T细胞的肠道嗜性。此外，我将使用RNA的组合 测序、转座酶可及染色质测序分析(ATAC-SEQ)和裂解 靶点和释放使用核酸酶(切割和运行)染色质分析，以提供全基因组的视角 在allo-T细胞启动/激活的早期阶段Notch信号的转录和表观遗传效应。 综上所述，这一建议将阐明早期肺炎影响的细胞和分子机制 缺口信号在塑造异基因骨髓移植后的同种免疫反应中的作用。

PROJECT SUMMARY Graft-versus-host-disease (GVHD) is the most serious complication of allogeneic bone marrow transplantation (allo-BMT). GVHD pathogenesis is mediated by alloreactive T cells present in the graft that recognize alloantigens and damage host tissues. Most GVHD therapies rely on global immunosuppression and are associated with increased risks of infection and decreased graft-versus-tumor effects. New strategies for GVHD prevention that protect host tissues while simultaneously preserving the tumor-killing activity of alloimmune T cells are needed. Our laboratory identified Notch signaling and Delta-like1/4 (Dll1/Dll4) Notch ligands as critical regulators of the pathogenic allo-T cell response, and attractive new therapeutic targets. In both mouse and non-human primate models, Notch inhibition in donor T cells leads to long-term protection from GVHD morbidity and mortality. Short-term in vivo Notch blockade using monoclonal antibodies against specific Notch receptors or ligands blunts the pathogenicity of alloreactive effector T cells without eliciting broad immunosuppression, reducing GVT activity, or triggering toxic side effects of chronic Notch inhibition. While a single injection of anti-Dll1/Dll4 Notch ligand antibodies at the time of transplant was sufficient for long- term GVHD control, Dll1/Dll4 blockade administered >48 hours after transplant provided no protection against GVHD. Thus, an early pulse of Notch signaling during the initial stages of T cell activation is critical for establishing a pathogenic T cell state. However, we do not currently understand how short-term Notch inhibition exerts long-lasting GVHD protection. Alloreactive T cells rely on two central functions to mediate disease: trafficking to peripheral target organs and producing proinflammatory cytokines. Our preliminary data show that Notch inhibition preserves initial T cell IL-2 production and expansion in secondary lymphoid organs while impairing secretion of multiple inflammatory cytokines and homing to the gastrointestinal tract. Therefore, I hypothesize that Notch signals delivered to allo-T cells during early stages of T cell priming and activation are critical for trafficking to the gut and initiating a sustained inflammatory cytokine response. I speculate that these two key mechanisms explain why short-term Notch inhibition prevents many of the severe, pathological consequences of GVHD. To explore this hypothesis, I will use MHC-mismatched models of GVHD to investigate how Notch inhibition influences allo-T cell gut tropism. Moreover, I will use a combination of RNA sequencing, Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq), and Cleave Under Targets and Release using Nuclease (CUT&RUN) chromatin profiling to provide a genome-wide view of the transcriptional and epigenetic effects of Notch signaling during early stages of allo-T cell priming/activation. Altogether, this proposal will elucidate the cellular and molecular mechanisms that underlie the impact of early Notch signals in shaping the alloimmune response after allo-BMT.