Molecular Mechanism of Immune Therapy for Bone Marrow Failures

翻译后摘要：克隆性疾病的大颗粒淋巴细胞（LGL）的特点是淋巴细胞增多和T 骨髓腔室的细胞介导的血细胞减少。T细胞介导骨髓的几种综合征 失败具有相似的病理学，包括再生障碍性贫血（AA），阵发性睡眠性血红蛋白尿（PNH）， 和骨髓增生异常综合征（MDS）患者的子集。所有这些综合征的特点是 通过免疫抑制治疗（IST）的临床改善，如低剂量甲氨蝶呤（MTX），低剂量甲氨蝶呤（MTX）， 剂量环磷酰胺（CY）或环孢素A（CyA），以及在再生障碍性贫血和MDS的情况下，T细胞 用抗胸腺细胞球蛋白（ATG）耗竭。这项建议的广泛的长期目标是改善 LGL白血病和其他骨髓衰竭疾病患者的诊断和治疗。 在这些T细胞介导的骨髓衰竭综合征患者中，尚不清楚T细胞是否 淋巴细胞在骨髓中局部活化以对抗特定的骨髓抗原，或在 外周淋巴系统然后回到骨髓。在过去的一段时间里， 一种抗原，可能是骨髓祖细胞上的一种共同抗原， 抑制骨髓生成这被认为是由于这种“假定的” 骨髓特异性抗原”和扩增的抗原特异性T细胞克隆。在进度报告中，我们的数据 强烈表明，这种疾病过程的多步骤自身免疫模型是基于 最后一次融资。我们的初步结果表明，淋巴结归巢受体L-选择素 在LGL白血病患者中，CD 8 + T细胞中的CD 62 L显著丢失（手稿发表在Blood 2009年）。在这项提议中要解决的主要假设是，改变骨髓归巢是 对LGL白血病发病机制至关重要。我们假设，急性激活的初级淋巴细胞， 系统导致肿瘤坏死因子转换酶切割和脱落CD 62 L （TACE），也称为ADAM-17。这种基质金属蛋白酶（MMP）不仅切割CD 62 L， 控制T细胞从淋巴结的出口，但它也是必要的活动， 已知在LGL白血病和其它自身免疫性疾病中具有重要性的炎性细胞因子。在 具体目标1，我们将证实我们的假设，即CD 62 L表达的丧失主要是由 胞外区脱落的ADAM-17基质金属蛋白酶。因为CD 62 L是一种淋巴归巢， 受体，我们假设这种受体的缺失，然后允许这些活化的T细胞退出淋巴 但CD 62 L的单独缺失预期不会触发骨髓中的迁移和定殖 这些细胞抑制造血。具体目标2的重点是确定 使用体外系统在LGL白血病中通过T细胞的骨髓归巢。归巢和迁徙是 复杂的事件在体内得到最佳监测，并且没有LGL白血病或骨的小鼠模型 骨髓衰竭疾病目前可用。此外，归巢到骨髓的调节甚至 在正常情况下是不完全理解的。由于激活LGL中T细胞的抗原 白血病是未知的，我们将使用一个明确的转基因小鼠模型来研究的重要方面， 骨髓归巢和抗原特异性T细胞的血液抑制。这将使我们能够测试 CD 62 L、VLA-4和ADAM-17在体内的重要性。此外，药理学抑制剂的功效 将对阻断骨髓归巢的药物进行测试，以提供关键的临床前数据， 在未来的临床研究中。

Abstract: Clonal diseases of large granular lymphocytes (LGL) are characterized by lymphocytosis and T cell-mediated cytopenias of the myeloid compartment. Several syndromes of T cell-mediated bone marrow failure have similar pathology including aplastic anemia (AA), paroxysmal nocturnal hemaglobinuria (PNH), and a subset of patients with Myelodysplastic Syndrome (MDS). All of these syndromes are characterized by clinical improvement with immunosuppressive therapy (IST) such as low-dose methotrexate (MTX), low- dose cyclophosphamide (CY) or cyclosporine A (CyA) and, in the case of aplastic anemia and MDS, T cell depletion with anti-thymocyte globulin (ATG). The broad long-term goal of this proposal is to improve the diagnosis and treatment of patients with LGL leukemia and these other bone marrow failure diseases. In patients with these T-cell mediated bone marrow failure syndromes, it is not clear whether the T lymphocytes become activated locally in the bone marrow against specific bone marrow antigens, or within the peripheral lymphoid system and then home to the bone marrow. The prevailing idea during the last funding period was that an antigen, presumably a common antigen on myeloid progenitors was responsible for suppression of myelopoiesis. This was thought to be due to the direct interaction between this "putative myeloid-specific antigen" and the expanded antigen-specific T cell clone. In the Progress Report, our data strongly suggests that a multi-step model of autoimmunity for this disease process based on work during the last funding period. We show in preliminary results that the lymph node homing receptor L-selectin (CD62L) is dramatically lost from CD8+ T cells in LGL leukemia patients (manuscript published in Blood 2009). The main hypothesis to be addressed in this proposal is that altered bone marrow homing is critical for LGL leukemia pathogenesis. We hypothesize that acute activation in the primary lymphoid system leads to cleavage and shedding of CD62L by the Tumor Necrosis Factor-¿ Converting Enzyme (TACE), which also known as ADAM-17. This matrix metalloproteinase (MMP) cleaves not only CD62L to control the egress of T cells from the lymph node but it is also necessary for the activity of several inflammatory cytokines with known importance in LGL leukemia and other autoimmune diseases. In Specific Aim 1, we will confirm our hypothesis that loss of CD62L expression is mediated primarily by ectodomain shedding by the ADAM-17 matrix metalloproteinase. Because CD62L is a lymphoid homing receptor, we hypothesize that loss of this receptor then allows these activated T cells to exit the lymph node but loss of CD62L alone is not expected to trigger migration and colonization in the bone marrow where these cells suppress hematopoiesis. The focus of Specific Aim 2 is to determine the mechanism of bone marrow homing by T cells in LGL leukemia using an in vitro system. Homing and migration are complex events that are optimally monitored in vivo and there is no mouse model of LGL leukemia or bone marrow failure disease currently available. Moreover, the regulation of homing to the bone marrow even under normal conditions is incompletely understood. Since the antigen that activates T cells in LGL leukemia is unknown, we will use a well defined transgenic mouse model to study important aspects of bone marrow homing and hemosuppression by antigen-specific T cells. This will allow us to test the importance of CD62L, VLA-4, and ADAM-17 in vivo. Additionally, the efficacy of pharmacological inhibitors to block homing to the bone marrow will be tested to provide critical pre-clinical data for application to clinical studies in patients in the future.