Molecular Mechanism of Immune Therapy for Bone Marrow Failures

摘要：大颗粒淋巴细胞(LGL)克隆性疾病以淋巴细胞增多和T淋巴细胞增多为特征。 髓系间室细胞介导性细胞减少症。T细胞介导的骨髓的几种综合征 衰竭的病理相似，包括再生障碍性贫血(AA)、阵发性夜间血球蛋白尿(PNH)、 以及一组骨髓增生异常综合征(MDS)患者。所有这些综合征的特征都是 通过免疫抑制治疗(IST)的临床改善，如小剂量甲氨蝶呤(MTX)，低剂量 剂量环磷酰胺(CY)或环孢素A(CyA)，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。这项建议的广泛长期目标是改善 LGL白血病和其他骨髓衰竭疾病的诊断和治疗。 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 淋巴细胞在骨髓中被局部激活，对抗特定的骨髓抗原，或在 外周淋巴系统，然后是骨髓的归宿。上一年的主流观点 资助期是一种抗原，可能是髓系祖细胞上的一种共同抗原 用来抑制骨髓生成。这被认为是由于这一推定的 和扩增的抗原特异性T细胞克隆。在进展报告中，我们的数据 强烈建议，这种疾病过程的自身免疫的多步骤模型是基于 最后一个资助期。我们的初步结果表明，淋巴归巢受体L-选择素 LGL白血病患者CD8+T细胞中(CD62L)显著丢失(手稿发表在血液中 2009年)。这项提议中要解决的主要假设是，改变的骨髓归巢是 对LGL白血病的发病至关重要。我们假设初级淋巴系统中的急性激活 系统导致肿瘤坏死因子转换酶对CD62L的切割和脱落 (TACE)，也被称为ADAM-17。这种基质金属蛋白酶不仅能将CD62L裂解为 控制T细胞从淋巴结中流出，但这也是几种活动所必需的 炎性细胞因子在LGL白血病和其他自身免疫性疾病中具有已知的重要性。在……里面 具体目标1，我们将确认我们的假设，即CD62L表达的丧失主要是通过 ADAM-17基质金属蛋白酶的胞外结构域脱落。因为CD62L是一种淋巴归巢 受体，我们假设这种受体的丢失会使这些激活的T细胞离开淋巴。 但CD62L单独丢失不会在骨髓中引发迁移和定植 在那里这些细胞会抑制造血。具体目标2的重点是确定 LGL白血病患者T细胞骨髓归巢体外系统的研究归宿和迁徙是 体内最佳监测的复杂事件，没有LGL白血病或骨骼的小鼠模型 目前可获得的是骨髓衰竭疾病。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，我们将使用定义良好的转基因小鼠模型来研究 抗原特异性T细胞的骨髓归巢和血液抑制。这将允许我们测试 CD62L、VLA-4和ADAM-17在体内的重要性。此外，药物抑制剂的疗效 将对阻断骨髓归巢进行测试，以提供关键的临床前数据 在未来的患者临床研究中。

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.