Transplantation tolerance and immune function following mTOR inhibition.

描述(申请人提供)：非清髓性骨髓移植为非恶性血液病，如镰状细胞性贫血提供了一种安全、有潜在疗效的治疗方法。不幸的是，由于免疫介导的移植物排斥反应，成功的非清髓性移植治疗镰状细胞性贫血受到了限制。我们的研究表明，雷帕霉素可以促进调节性T细胞(Treg)对初始T细胞的分化和T细胞活化后Th1细胞的无能。我们利用这些观察结果开发了一种新的制备方案，通过促进T细胞耐受来抑制排斥反应和移植物抗宿主病(GVHD)。我们的策略减少了使用阿伦珠单抗的同种异体反应T细胞的频率，为低剂量全身照射的植入创造了“空间”，并允许在延长雷帕霉素治疗的情况下淋巴细胞恢复。在配对的兄弟姐妹中，这种方法取得了巨大的成功，导致了稳定的混合嵌合体，纠正了他们的镰状细胞性贫血的血液学表型，并逆转了肺动脉高压。在这一成功的基础上，第二次临床试验被开发出来，以扩大潜在的供者池，包括半相合的亲属供者，极大地增加了这种疗法对患者的潜在可获得性。这项新的试验在选择准备方案时采用了相同的基本原则，并增加了移植后环磷酰胺的剂量，以进一步减少可能导致排斥或GVHD的同种异体反应性T细胞。这项试验的患者和捐献者的外周血液样本将提供一个独特的机会，系统地研究T细胞耐受在促进稳定嵌合体方面的作用。我们建议通过检测混合淋巴细胞反应(MLRs)和细胞内细胞因子染色(ICS)从移植前、移植后使用雷帕霉素和完成雷帕霉素治疗后的移植后获得的样本来实现这一点。我们将确定雷帕霉素的持续存在是否是抑制体外同种异体反应所必需的，以及MLR中测量的耐受性是否依赖于Tregs的存在。我们将测试加入雷帕霉素或Tregs是否能够抑制在接受雷帕霉素治疗的同时发生移植物排斥反应或移植物抗宿主病的患者的MLR。我们将确定临床上以排斥或移植物抗宿主病的形式对雷帕霉素的耐药性是否与雷帕霉素在mTOR靶向磷酸化水平上的生化耐药性相对应，以及一种新的mTOR激酶抑制剂是否可以在体外克服这种生化耐药性。最终目的是确定长期抑制mTOR是否会干扰抗原特异性T细胞细胞因子的产生或导致与临床相关的巨细胞病毒或甲型流感相关抗原特异性Tregs的产生。我们相信，更好地了解抑制mTOR的免疫学后果将导致更安全和更成功的骨髓移植，从而使这一潜在的治疗方法扩展到更多患有慢性血液病的患者。 公共卫生相关性(由申请者提供)：镰状细胞性贫血和其他慢性贫血给患者带来了巨大的疼痛和痛苦负担，以及与住院和慢性输血/螯合治疗相关的巨额医疗费用。在这项建议中，我们试图通过非清髓性骨髓移植治疗镰状细胞病的新方案，确定促进T细胞耐受的可操作的细胞和生化过程。希望这项研究的结果将允许这种治疗方法更安全地应用于更多的患者，并有助于改善自身免疫和实体器官移植的耐受治疗。

DESCRIPTION (provided by applicant): Nonmyeloablative bone marrow transplant offers a safe, potentially curative treatment for non-malignant hematological diseases such as sickle cell anemia. Unfortunately, successful nonmyeloablative transplant to treat sickle cell anemia has been limited due to immune-mediated graft rejection. Our research has demonstrated that rapamycin can promote regulatory T cell (Treg) differentiation of naive T cells and anergy of Th1 cells following T cell activation. We used these observations to develop a novel preparative regimen to inhibit rejection and graft versus host disease (GVHD) by promoting T cell tolerance. Our strategy reduces the frequency of alloreactive T cells with alemtuzumab, creates "space" for engraftment with low dose total body irradiation, and allows lymphocyte recovery under extended rapamycin treatment. In the matched sibling setting this approach has had great success, resulting in stable mixed chimerism that corrects their hematological phenotype of sickle cell anemia and reverses pulmonary hypertension. Based on this success, a second clinical trial was developed to expand the potential donor pool to include haploidentical related donors, greatly increasing potential availability of this therapy to patients. The new trial employs the same fundamental principles in the choice of preparative regimen with the addition of dose escalation of post transplant cyclophosphamide to further reduce alloreactive T cells that could contribute to rejection or GVHD. Peripheral blood samples from patients and donors on this trial will provide a unique opportunity to systematically investigate the role of T cell tolerance in promoting stable chimerism. We propose to do this by examination of mixed lymphocyte reactions (MLRs) and intracellular cytokine staining (ICS) from samples obtained pretransplant, posttransplant on rapamycin, and posttransplant after completion of rapamycin therapy. We will determine whether the continued presence of rapamycin is necessary to suppress allogeneic responses in vitro and whether the tolerance measured in the MLR is dependent on the presence of Tregs. We will test whether addition of rapamycin or Tregs is able to suppress the MLR from a patient who develops graft rejection or GVHD while receiving rapamycin. We will determine if clinical resistance to rapamycin in the form of rejection or GVHD corresponds to biochemical resistance to rapamycin at the level of mTOR target phosphorylation and whether a novel mTOR kinase inhibitor can overcome such biochemical resistance in vitro. A final aim is to determine whether prolonged mTOR inhibition interferes with antigen specific T cell cytokine production or leads to generation of antigen specific Tregs to clinically relevant CMV or influenza A. We believe that a better understanding of the immunologic consequences of mTOR inhibition will result in safer and more successful bone marrow transplantation, allowing expansion of this potentially curative therapy to a wider number of patients with chronic hematologic illnesses. PUBLIC HEALTH RELEVANCE (provided by applicant): Sickle cell anemia and other chronic anemias impose a huge burden of pain and suffering for patients and large health care costs related to hospitalizations and chronic transfusion/chelation therapy. In this proposal we seek to define the operative cellular and biochemical processes that promote T cell tolerance in a novel protocol to cure sickle cell disease with non-myeloablative bone marrow transplantation. It is hoped that the results of this study will allow safer application of this curative therapy to a greater number of patients and aid in improving tolerogenic therapy for autoimmunity and solid organ transplantation as well.