Transplantation tolerance and immune function following mTOR inhibition.

描述（由申请人提供）：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、潜在的治愈性治疗。不幸的是，由于免疫介导的移植排斥反应，成功的非清髓性移植治疗镰状细胞性贫血的效果受到限制。我们的研究表明，雷帕霉素可以促进初始T细胞的调节性T细胞（Treg）分化和T细胞活化后的Th 1细胞无能。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）。我们的策略降低了Alemtuzumab的同种异体反应性T细胞的频率，为低剂量全身照射的植入创造了“空间”，并允许在延长的雷帕霉素治疗下淋巴细胞恢复。在配对的同胞中，这种方法取得了巨大的成功，产生了稳定的混合嵌合体，纠正了他们的镰状细胞贫血的血液学表型，并逆转了肺动脉高压。基于这一成功，开发了第二项临床试验，以扩大潜在的供体库，包括单倍体相合的相关供体，大大增加了这种疗法对患者的潜在可用性。新试验在准备方案的选择中采用了相同的基本原则，并增加了移植后环磷酰胺的剂量递增，以进一步减少可能导致排斥反应或GVHD的同种异体反应性T细胞。来自本试验患者和供体的外周血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中的作用。我们建议这样做的混合淋巴细胞反应（MLR）和细胞内细胞因子染色（ICS）从移植前，移植后的雷帕霉素，和移植后完成雷帕霉素治疗后获得的样本检查。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，以及在MLR中测量的耐受性是否依赖于TMF的存在。我们将测试加入雷帕霉素或TMF是否能够抑制在接受雷帕霉素时发生移植物排斥或GVHD的患者的MLR。我们将确定排斥反应或GVHD形式的雷帕霉素临床耐药性是否对应于mTOR靶点磷酸化水平上对雷帕霉素的生化耐药性，以及新型mTOR激酶抑制剂是否可以在体外克服这种生化耐药性。最终目的是确定延长的mTOR抑制是否干扰抗原特异性T细胞细胞因子的产生或导致产生针对临床相关CMV或甲型流感的抗原特异性T细胞因子。我们相信，更好地了解mTOR抑制的免疫学后果将导致更安全和更成功的骨髓移植，从而将这种潜在的治愈性治疗扩展到更广泛的慢性血液病患者。 公共卫生相关性（由申请人提供）：镰状细胞性贫血和其他慢性贫血给患者带来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保健费用。在这个建议中，我们试图定义一个新的协议，以治疗镰状细胞病与非清髓性骨髓移植的细胞和生化过程，促进T细胞耐受。希望这项研究的结果将允许更安全地将这种治愈性治疗应用于更多的患者，并有助于改善自身免疫和实体器官移植的致耐受性治疗。

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.