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

项目摘要 移植物抗宿主病（GVHD）是异基因骨髓移植最严重的并发症 移植（allo-BMT）。GVHD发病机制是由移植物中存在的同种异体反应性T细胞介导的， 识别同种异体抗原并损伤宿主组织。大多数GVHD疗法依赖于全身免疫抑制， 与感染风险增加和移植物抗肿瘤效应降低相关。新战略 GVHD预防，保护宿主组织，同时保留肿瘤杀伤活性， 需要同种免疫T细胞。我们的实验室鉴定了Notch信号和Delta-like 1/4（Dll 1/Dll 4）Notch 配体作为致病性同种异体T细胞应答的关键调节剂，以及有吸引力的新治疗靶点。在 在小鼠和非人灵长类动物模型中，供体T细胞中的Notch抑制导致长期保护 GVHD的发病率和死亡率。使用抗Notch单克隆抗体的短期体内Notch阻断 特异性Notch受体或配体减弱同种异体反应性效应T细胞的致病性， 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。 虽然在移植时单次注射抗Dll 1/Dll 4 Notch配体抗体足以长时间维持移植， 移植后>48小时给予长期GVHD控制、Dll 1/Dll 4阻断剂没有提供保护作用 GVHD。因此，在T细胞活化的初始阶段期间的Notch信号传导的早期脉冲对于T细胞活化是至关重要的。 建立致病性T细胞状态。然而，我们目前并不了解短期缺口如何 抑制发挥持久的GVHD保护作用。同种异体反应性T细胞依赖于两个中心功能来介导 疾病：运输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产生和扩增 同时损害多种炎性细胞因子的分泌和归巢到胃肠道。因此，我们认为， 我推测，在T细胞引发和活化的早期阶段，Notch信号被传递到同种异体T细胞， 对于运输到肠道和启动持续的炎性细胞因子反应至关重要。我推测这些 两个关键机制解释了为什么短期Notch抑制可以防止许多严重的病理性 GVHD的后果为了探索这一假设，我将使用MHC不匹配的GVHD模型， 研究Notch抑制如何影响同种异体T细胞的肠道向性。此外，我将使用一种RNA 测序、转座酶-可重复染色质测序（ATAC-seq）测定和下切割 使用核酸酶（CUT&RUN）染色质谱分析的靶点和释放，以提供基因组范围的视图， 在allo-T细胞引发/活化的早期阶段Notch信号传导的转录和表观遗传作用。 总之，这项建议将阐明细胞和分子机制的影响，早期 Notch信号在allo-BMT后形成同种免疫应答中的作用

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.