Establishing Mixed Hematopoietic Chimerism in a Canine Model

项目1：建立稳定的混合造血嵌合体犬模型 我们已经建立了稳定的狗白细胞抗原（DLA）相同的骨髓移植使用亚致死条件 全身照射（TBI）前给予2戈伊剂量的全身照射（TBI），并短期应用霉酚酸酯进行免疫抑制 移植后给予吗替洛尔和环孢素。该方法已成功地应用于临床 治疗恶性（见项目3和4）和非恶性血液病患者。项目1将使用 犬模型的建立解决了临床异基因造血细胞移植的三大问题 (HCT)，避免预处理方案的长期后遗症，控制移植物与宿主病（GVHD） 而不需要延长移植后免疫抑制，也不需要延长移植后免疫抑制的副作用， 肿瘤效应而不增加GVHD的风险。至于第一个目标，我们有临床前和临床 有证据表明，细胞毒性预处理不是同种异体移植到家庭的强制性。我们将把这些 观察和确定是否特异性和无毒的移植前耐受诱导，探索六个 犬特异性T细胞共刺激阻断剂和两种影响调节途径的药物， 我们的实验室，可以取代广泛的免疫抑制所赋予的TBI，这样，避免 短期和长期辐射毒性。至于第二个目标，我们将评估三种替代移植后 更好地控制GVHD和HVG反应的操作，如果成功，将避免需要 用于移植后免疫抑制为此，我们将研究免疫抑制剂 骨髓移植后3天给予环磷酰胺，一种211 At标记的单克隆抗体， 抗T细胞活化抗原CD 70的抗体和T细胞共刺激阻断剂的拮抗剂 CD 28与下调分子CTLA-4的激动剂组合。根据《公约》建议的研究 前两个目标可能产生稳定的混合供体/宿主造血嵌合体。持久主机 造血可以作为HCT后持续性恶性血液病模型，这是一个常见问题 在项目3和项目4下移植的患者中遇到的问题。与项目2合作，我们打算 在给定的造血细胞中鉴定对造血细胞（造血抗原）特异性的次要非DLA抗原差异 使用基因组学方法的供体/受体对。这些知识将为今后的研究提供基础 第三个目标是使供体淋巴细胞对宿主造血抗原免疫， 输注以将混合的嵌合体转变为没有或最小限度的GVHD的全供体嵌合体。如果成功，我们将测试 这种方法在实验诱导的急性白血病的犬模型中的有效性。结果进行 临床前研究将与项目3和项目4下的未来临床试验相关， 人类患者中同种异体HCT成功性和安全性。

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.