Immune Pathophysiology of Aplastic Anemia and Immunosuppressive Treatments

再生障碍性贫血的免疫病理生理学和免疫抑制治疗

• 批准号: 8149485
• 负责人: NEAL S YOUNG
• 金额: $ 228.44万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8149485
• 关键词: Aplastic Anemia; Functional disorder; Immune; Immunosuppressive Agents

In aplastic anemia, the bone marrow is replaced by fat, and peripheral blood counts - - of white blood cells, red blood cells, and platelets - - fall to extremely low levels, leading to death from anemia, bleeding or infection. Aplastic anemia is a disease of young persons, and in its severe form is almost invariably fatal untreated. Historically, aplastic anemia has been linked to chemical exposures, in particular benzene; it is an idiosyncratic complication of some medical drug use; it occurs as a rare event in pregnancy and following seronegative hepatitis; and the diseases associated with certain immunologic conditions. The chance observation that some patients post-bone marrow transplant recovered their own marrow function led to the inference that the immunosuppressive conditioning regimen might have treated an underlying immune-mediated pathophysiology. Purposeful administration of antithymocyte globulin (ATG) has led to hematologic recovery in the majority of treated patients. Laboratory data have also revealed abnormalities of the immune system: lymphocyte populations that induce apoptosis in hematopoietic target cells by the Fas-mediated pathway, and oligoclones of effector T cells which express type 1 cytokines, especially gamma-interferon. The Hematology Branch has been a leader in both the scientific and medical studies of aplastic anemia pathophysiology and treatment. In the last year, we completed enrollment to our main clinical protocol for treatment of recent onset severe aplastic anemia. The study was initiated as a triple randomization among horse ATG (standard therapy in the United States for many years); rabbit ATG (newly introduced into the American market and used interchangeably with hATG); and Campath, a monoclonal antibody directed against CD52 with broad specificity to lymphocytes. The third arm was discontinued due to a low response rate after the treatment of about a dozen patients (see below). Entry into the hATG versus rATG protocol continued to completion, with 60 patients in each arm. The results of this study will be made public at the American Society of Hematology Meetings in December, when data analysis is complete, but they are likely to show marked and unexpected differences in hematologic response rates and possibly survival for two agents that have been employed indiscriminately for this disease - - therefore likely to change practice world-wide. Second, Campath, a highly immunosuppressive monoclonal antibody, has been employed in a variety of settings. The drug has been administered to 27 aplastic anemia patients refractory to hATG therapy; Campath appeared to be equivalent to rATG in inducing hematologic responses, in about 30% of refractory patients and > 50% of relapsed patients. However, only 3 of 16 patients with therapy-nave recent onset severe aplastic anemia responded, and Campath was judged to be inferior to standard hATG and therefore abandoned in this setting. The Branch has also pioneered another monoclonal antibody, daclizumab, which is directed to the interleukin 2 receptor of T cells. Long-term outcomes in patients with moderate aplastic anemia and pure red cell aplasia were reported: 7/28 moderate aplastic anemia patients achieved long-term hematologic recovery, and 38% of patients with pure red cell aplasia showed long-term transfusion independence. The toxicity profile for dacluzimab is excellent; unfortunately, the monoclonal antibody is no longer produced for the American market. In other clinical studies, retrospective examination of mortality in severe aplastic anemia has disclosed a marked decrease in deaths overall, especially in patients who failed to respond to an initial course of hATG. Multi-parameter analysis suggested that this improvement was mainly due to the introduction of superior anti-fungal therapies, their greater effectiveness and earlier implementation in severely neutropenic patients. Additionally, patients have been retreated with immunosuppression and referred earlier and more often to stem cell transplant due to decreased risks from this procedure. Also reported from the Clinical Center was the effectiveness of granulocyte transfusions in the setting of severe aplastic anemia; this product has been life-saving in patients who were severely neutropenic but not yet responded to ATG. Laboratory investigation of aplastic anemia has exploited several different modalities. We have created a mouse model of immune-mediated bone marrow failure, based on runt disease: lymph node cells from a parental strain are infused into F1 hybrid animals, resulting in rapid destruction of the host bone marrow, pancytopenia, and death from infection and bleeding. This model is dependent on lymphocytes, type 1 cytokines, and a potent innocent bystander effect. In recent work, we have demonstrated that T-bet, an important transcription factor that polarizes T-cells to type 1 cytokine production, and dysregulation of which has been implicated in human aplastic anemia, also plays a role in this animal model, as demonstrated in genetically defective animals. Lymph node cells from T-bet knock-out animals failed to induce bone marrow failure, although other cytokines were increased and associated with mild blood count abnormalities. In other murine experiments, the perforin/granzyme pathway was shown to be less important than the Fas/FasL pathway in inducing bone marrow cell destruction. Aplastic anemia in men and mouse was also addressed in imaging studies. We developed a novel methodology employing confocal microscopy to examine intact bone marrow to unprecendented depths, approximately 200 microns, in combination with multicolor antibody or flourochrome staining. This method allows digital reconstruction of the three dimensional architecture of a tissue. Confocal imaging reveals marked expansion in the number and size of fat cells in both experimental immune mediated bone marrow failure in the mouse and in humans with aplastic anemia, as well as rapid invasion of the murine bone marrow by CD8 effector T-cells. Imaging also allows visualization of the boney and vascular architecture of the marrow and of engraftment over time after marrow transplantation in animals. Aplastic anemia has also been investigated utilizing conventional and advanced flow cytometric methods. We have characterized the role of T-helper type 17 (Th17) lymphocytes, which have been associated with other autoimmune diseases. These cells are increased in patients with aplastic anemia at presentation and their presence correlated with disease activity; furthermore, Th17 and CD4 regulatory T-cells were inversely correlated. Th17 cell numbers also rise in the lymph node infusion model of bone marrow failure, although in the mouse model they appear to be of secondary importance to type 1 cytokine lymphocytes. We have also utilized multiplex cytokine analysis to examine bone marrow failure. When many dozens of cytokines levels are determined in serum or plasma, distinct signatures appear for aplastic anemia and a diagnostically confusing entity, hypocellular myelodysplastic syndrome. Multiplex cytokine analysis has also been applied to serum sickness, and certain cytokines correlate both with the concentrations of human antibody directed against xenoprotein and with the occurrence of clinical serum sickness. Certain cytokines are only produced in the presence of adequate platelet and megakaryocyte numbers, and in vitro, these cells can be shown to be the source of the proteins.

在再生障碍性贫血中，骨髓被脂肪取代，外周血计数（白细胞、红细胞和血小板）降至极低水平，导致因贫血、出血或感染而死亡。 再生障碍性贫血是年轻人的一种疾病，严重时如果不治疗几乎总是致命的。 从历史上看，再生障碍性贫血与化学物质接触有关，特别是苯；它是某些药物使用的特殊并发症；它在怀孕期间和血清阴性肝炎后发生为罕见事件；以及与某些免疫状况相关的疾病。 一些患者在骨髓移植后恢复了自己的骨髓功能，这一偶然观察导致了这样的推断：免疫抑制调理方案可能治疗了潜在的免疫介导的病理生理学。 有目的地施用抗胸腺细胞球蛋白（ATG）已使大多数接受治疗的患者的血液学恢复。 实验室数据还揭示了免疫系统的异常：通过Fas介导的途径诱导造血靶细胞凋亡的淋巴细胞群，以及表达1型细胞因子（尤其是γ-干扰素）的效应T细胞的寡克隆。 血液学分支一直是再生障碍性贫血病理生理学和治疗的科学研究和医学研究的领导者。 去年，我们完成了治疗近期发作的严重再生障碍性贫血的主要临床方案的注册。 该研究是在马 ATG（美国多年来的标准疗法）中进行三重随机分组开始的；兔ATG（新引入美国市场，与hATG互换使用）； Campath，一种针对 CD52 的单克隆抗体，对淋巴细胞具有广泛的特异性。 第三组由于在治疗大约十几名患者后反应率低而终止（见下文）。 hATG 与 rATG 方案的进入持续完成，每组有 60 名患者。 这项研究的结果将于 12 月的美国血液学会会议上公布，届时数据分析完成，但它们可能会显示出两种不加区别地使用的药物在血液学反应率和可能的生存方面存在显着且意想不到的差异。对于这种疾病 - 因此可能会改变世界范围内的做法。 其次，Campath 是一种高度免疫抑制的单克隆抗体，已被用于多种场合。 该药物已用于 27 名 hATG 治疗难治性再生障碍性贫血患者；在大约 30% 的难治性患者和 > 50% 的复发患者中，Campath 在诱导血液学反应方面似乎与 rATG 相当。 然而，16 名新近发病的重度再生障碍性贫血患者中，只有 3 名有反应，Campath 被认为不如标准 hATG，因此被放弃。 该分公司还首创了另一种单克隆抗体 daclizumab，它针对 T 细胞的白细胞介素 2 受体。 据报道，中度再生障碍性贫血和纯红细胞再生障碍性贫血患者的长期结局：7/28的中度再生障碍性贫血患者实现了长期血液学恢复，38%的纯红细胞再生障碍性贫血患者表现出长期输血独立性。 dacluzimab 的毒性特征非常好；不幸的是，单克隆抗体不再为美国市场生产。 在其他临床研究中，对严重再生障碍性贫血死亡率的回顾性检查显示，总体死亡率显着下降，特别是对 hATG 初始疗程没有反应的患者。 多参数分析表明，这种改善主要归功于优质抗真菌疗法的引入、其更有效的治疗以及在严重中性粒细胞减少患者中更早实施。 此外，由于干细胞移植的风险降低，患者已接受免疫抑制治疗，并更早、更频繁地转诊至干细胞移植。 临床中心还报告了粒细胞输注在严重再生障碍性贫血中的有效性；该产品可以挽救严重中性粒细胞减少症但尚未对 ATG 产生反应的患者的生命。 再生障碍性贫血的实验室研究采用了几种不同的方式。 我们基于矮小症创建了一种免疫介导的骨髓衰竭小鼠模型：将来自亲本品系的淋巴结细胞注入 F1 杂交动物体内，导致宿主骨髓快速破坏、全血细胞减少，并因感染和死亡而死亡。流血。 该模型依赖于淋巴细胞、1 型细胞因子和有效的无辜旁观者效应。 在最近的工作中，我们证明了 T-bet 是一种重要的转录因子，可使 T 细胞极化为 1 型细胞因子的产生，其失调与人类再生障碍性贫血有关，也在该动物模型中发挥作用，如所证明的在有遗传缺陷的动物中。 尽管其他细胞因子增加并与轻度血细胞计数异常相关，但 T-bet 敲除动物的淋巴结细胞未能诱导骨髓衰竭。 在其他小鼠实验中，穿孔素/颗粒酶途径在诱导骨髓细胞破坏方面的重要性不如 Fas/FasL 途径。 影像学研究也解决了男性和小鼠的再生障碍性贫血问题。 我们开发了一种新颖的方法，采用共聚焦显微镜结合多色抗体或荧光染料染色，以前所未有的深度（约 200 微米）检查完整的骨髓。 该方法允许对组织的三维结构进行数字重建。 共聚焦成像显示，在实验性免疫介导的小鼠和患有再生障碍性贫血的人类骨髓衰竭中，脂肪细胞的数量和大小显着增加，以及 CD8 效应 T 细胞对小鼠骨髓的快速侵袭。 成像还可以使骨髓的骨和血管结构以及动物骨髓移植后随时间的植入情况可视化。 还利用传统和先进的流式细胞术方法对再生障碍性贫血进行了研究。 我们已经描述了 17 型辅助性 T (Th17) 淋巴细胞的作用，该淋巴细胞与其他自身免疫性疾病有关。 这些细胞在再生障碍性贫血患者就诊时有所增加，并且它们的存在与疾病活动相关；此外，Th17 和 CD4 调节性 T 细胞呈负相关。 在骨髓衰竭的淋巴结输注模型中，Th17 细胞数量也有所增加，尽管在小鼠模型中，它们似乎对 1 型细胞因子淋巴细胞而言次要。 我们还利用多重细胞因子分析来检查骨髓衰竭。 当测定血清或血浆中数十种细胞因子水平时，再生障碍性贫血和细胞少性骨髓增生异常综合征这一诊断上令人困惑的疾病会出现明显的特征。 多重细胞因子分析也已应用于血清病，某些细胞因子与针对异种蛋白的人类抗体的浓度以及临床血清病的发生相关。 某些细胞因子只有在足够的血小板和巨核细胞数量存在的情况下才会产生，并且在体外，这些细胞可以被证明是蛋白质的来源。