Characterization of a T cell dysfunctional state induced in mice with AML

描述(申请人提供)：肿瘤抗原特异性T细胞的初始激活由宿主树突状细胞(DC)调节。最近，人们观察到，垂死的癌细胞产生的“危险信号”激活了局部宿主DC，然后宿主DC能够启动适应性抗肿瘤T细胞反应。这些观察澄清了我们对肿瘤抗原特异性T细胞是如何被激活以对抗作为固体肿块生长的癌症的理解。然而，在血液病患者的宿主中，控制肿瘤抗原特异性T细胞激活和抑制的过程仍然难以捉摸。血液病患者以播散性模式生长，缺乏典型的“引流”淋巴。我们实验室的长期目标是在基础水平上解开这些机制，我们相信这将对指导未来白血病有效免疫疗法的发展具有重要意义。我们在小鼠急性髓系白血病(AML)模型中的工作表明，当白血病细胞通过静脉注射(IV)进入小鼠体内时，抗原特异性T细胞功能障碍迅速接踵而至，而皮下(SC)AML细胞接种导致T细胞成功启动，认为血液肿瘤和实体肿瘤激活宿主免疫系统的能力存在根本区别。因此，我们假设由耐受树突状细胞诱导的AML宿主体内存在与T细胞无能或缺失相一致的抗原特异性T细胞功能障碍状态，并提出以下具体目标：(1)确定在AML小鼠模型中诱导抗原特异性T细胞功能障碍的机制；(2)确定AML小鼠T细胞功能障碍的细胞介质；(3)确定针对宿主DC激活的策略，以预防或逆转AML小鼠T细胞功能障碍。为了达到这些目标，我们建立了一个临床前急性髓系白血病模型。C1498小鼠AML细胞系已被设计成表达一种模型抗原，该模型抗原可被T细胞受体转基因CD8 T细胞(2C)识别。将仔细分析静脉接种与SC C1498细胞接种后小鼠体内2C T细胞的增殖和功能，以确定T细胞功能障碍的机制。为了确定可能调节T细胞功能障碍的细胞(S)，将荧光标记的C1498细胞静脉接种到小鼠体内，并利用免疫荧光和共聚焦显微镜来鉴定哪个DC亚群(S)吞噬了次级淋巴器官中死亡的C1498细胞。然后，将针对特定的DC群体进行耗竭，以确定哪些DC群体最终导致AML小鼠T细胞功能障碍。最后，将探索针对宿主DC激活的临床相关策略，以确定哪些策略可以逆转急性髓细胞白血病小鼠的T细胞功能障碍。这些研究将有助于我们理解血液病是如何促进免疫逃避的。此外，还将测试旨在逆转免疫耐受的方法，可能会导致对临床翻译有用的策略。因此， 从生物学的角度来看，获得的知识很重要，也有望在未来为急性髓细胞白血病患者提供免疫治疗方法。

DESCRIPTION (provided by applicant): The initial activation of tumor antigen-specific T cells is regulated by host dendritic cells (DC). Recently, it has been observed that "danger signals" produced by dying cancer cells activate local host DC, which are then able to prime adaptive anti-tumor T cell responses. These observations have clarified our understanding of how tumor antigen-specific T cells are activated against cancers which grow as a solid mass. However, the processes which govern the activation versus suppression of tumor antigen-specific T cells in hosts with hematological cancers, which grow in a disseminated pattern and lack a classical "draining" lymph node, remain elusive. The long-term goal of our laboratory is to unravel these mechanisms at a basic level, which we believe will be important to guide the development of effective immunotherapy for leukemia in the future. Our work in a murine acute myeloid leukemia (AML) model has revealed that when leukemia cells are introduced into mice intravenously (IV), antigen-specific T cell dysfunction rapidly ensues, whereas a subcutaneous (SC) AML cell inoculation leads to successful T cell priming, arguing that a fundamental difference exists between the ability of hematological versus solid cancers to activate the host immune system. Thus, we hypothesize that a state of antigen-specific T cell dysfunction, consistent with T cell anergy or deletion, occurs in hosts with AML, which is induced by tolerogenic DC, and propose the following Specific Aims: (1) To identify the mechanism of antigen-specific T cell dysfunction induced in a murine AML model; (2) To identify the cellular mediators of T cell dysfunction in mice with AML; and (3) to identify strategies targeting activation of host DC to prevent or reverse T cell dysfunction in mice with AML. To address these aims, we have generated a pre-clinical AML model. The C1498 murine AML cell line has been engineered to express a model antigen which is recognized by a T cell receptor transgenic CD8+ T cell (2C). The proliferation and function of 2C T cells in mice following IV versus SC C1498 cell inoculation will be carefully analyzed to identify the mechanism of T cell dysfunction. To identify the cell(s) which may regulate T cell dysfunction, fluorescently-labeled C1498 cells will be inoculated IV into mice, and immunofluorescence and confocal microscopy will be utilized to identify which DC subset(s) engulf dying C1498 cells in secondary lymphoid organs. Specific DC populations will then be targeted for depletion to determine which is ultimately responsible for inducing T cell dysfunction in mice with AML. Lastly, clinically-relevant strategie targeting host DC activation will be explored to determine which can reverse T cell dysfunction in mice with AML. These studies will contribute to our understanding of how hematological cancers promote immune evasion. Additionally, approaches targeting the reversal of immune tolerance will be tested, possibly leading to strategies useful for clinical translation. Thus, the knowledge to be gained is important from a biological standpoint, and also is expected to lead to immunotherapeutic approaches for patients with AML in the future.