Establishing Mixed Hematopoietic Chimerism in a Canine Model

项目1：建立稳定的混合造血嵌合体INA犬模型 我们已经建立了稳定的狗白细胞抗原(DLA)相合的骨髓移植物，使用亚致死条件 术前2Gy全身照射(TBI)加短程免疫抑制 莫非替尔和环孢素在移植后应用。该方法已成功应用于临床。 治疗恶性(见项目3和4)和非恶性血液疾病患者。项目1将使用 解决临床异基因造血细胞移植三大问题的犬模型 (HCT)，避免调理方案的长期后遗症，控制移植物抗宿主病(GVHD) 没有移植后长期免疫抑制的需要和副作用，并增强了移植物对 在不增加移植物抗宿主病风险的情况下对肿瘤产生影响。至于第一个目标，我们有临床前和临床两个目标 有证据表明，细胞毒性调节不是同种异体移植物回家的强制性条件。我们将延长这些措施 观察并确定特异性和无毒性的移植前耐受诱导，探索六个 犬特异性T细胞共刺激阻断剂和两种影响调节通路的药物，开发于 我们的实验室可以取代脑损伤所带来的广泛的免疫抑制，并通过这种方式避免 短期和长期的辐射毒性。至于第二个目标，我们将评估三种可供选择的移植后 更好地控制GVHD和HVG反应的操作，如果成功，将避免需要 用于延长移植后的免疫抑制。为此，我们将研究免疫抑制剂 阿司他丁-211(211At)标记的单抗移植后3天给环磷酰胺 抗T细胞活化抗原CD70的抗体和T细胞共刺激阻滞剂的拮抗剂 CD28与下调分子CTLA-4的激动剂结合。建议在 前两个目标可能会产生稳定的供者/宿主混合造血嵌合体。持久化主机 造血可以作为HCT后持续恶性血液病的模型，这是一个常见的问题 在项目3和项目4下移植的患者中遇到的。我们打算与项目2合作， 识别特定于造血细胞(造血细胞抗原)的微小非DLA抗原差异 使用基因组学方法的捐赠者/接受者对。这些知识将为今后的研究提供基础。 解决第三个目标，即捐赠者淋巴细胞对宿主造血抗原具有免疫力 输注以转变为混合供者嵌合体，无GVHD或最低限度GVHD。如果成功，我们将测试 这种方法在实验诱导的急性白血病犬模型中的有效性。这些措施的结果 临床前研究将与项目3和4下的未来临床试验相关，并可能增加 同种异体红细胞移植在人类患者中的成功和安全性。

PROJECT 1: ESTABLISHING STABLE MIXED HEMATOPOIETIC CHIMERISM INA CANINE MODEL We have established stable dog leukocyte antigen (DLA)-identical marrow grafts using sublethal conditioning with 2 Gy total body irradiation (TBI) before and a short course of immunosuppression with mycophenolate mofetil and cyclosporine after transplantation. The approach has been translated successfully into the clinic to treat patients with malignant (see Projects 3 and 4) and nonmalignant blood disorders. Project 1 will use the canine model to address three major issues of clinical allogeneic hematopoietic cell transplantation (HCT), avoiding long-term sequelae from conditioning regimens, controlling graft-vs.-host disease (GVHD) without need for and side effects from extended postgrafting immunosuppression, and enhancing graft-vs.- tumor effects without increasing the risk of GVHD. As for the first goal, we have both preclinical and clinical evidence that cytotoxic conditioning is not mandatory for allogeneic grafts to home. We will extend these observations and determine whether specific and non-toxic pretransplant tolerance induction, exploring six canine-specific blockers of T-cell co-stimulation and two agents affecting regulatory pathways, developed in our laboratory, can be substituted for the broad immunosuppression imparted by TBI and, this way, avoid short- and long-term radiation toxicities. As for the second goal, we will assess three alternative postgrafting manipulations for better control of both GVHD and HVG reactions which, if successful, would avert the need for extended postgrafting immunosuppression. To that end, we will study the immunosuppressive agent cyclophosphamide administered 3 days after marrow grafting, an astatine-211 (211At)-labeled monoclonal antibody against the T-cell activation antigen CD70, and an antagonist to the T-cell costimulatory blocker CD28 combined with an agonist to the down-regulatory molecule CTLA-4. The studies proposed under the first two goals are likely to generate stable mixed donor/host hematopoietic chimeras. Persistent host hematopoiesis can serve as model of persistent hematologic malignancy after HCT, a frequent problem encountered in patients transplanted under Projects 3 and 4. In collaboration with Project 2, we intend to identify minor non-DLA antigen disparities specific for hematopoietic cells (hematopoietic antigens) in given donor/recipient pairs using genomics approaches. This knowledge will provide the basis for future studies addressing the third goal in which donor lymphocytes rendered immune to host hematopoietic antigens will be infused in order to shift mixed to all-donor chimerism with no or minimal GVHD. If successful, we will test the efficacy of this approach in a canine model of experimentally induced acute leukemia. Results of these preclinical studies will be relevant for future clinical trials under Projects 3 and 4 and are likely to increase success and safety of allogeneic HCT in human patients.