Establishing Stable Mixed Hematopoietic Chimerism

在犬白细胞抗原(DLA)相合的骨髓移植中，采用亚致死性预适应联合全身照射(TBI)，联合应用霉酚酸酯(MMF)和环孢菌素(CsA)短程免疫抑制，建立了稳定的供体/宿主混合造血嵌合体。这使得移植不会出现传统高剂量调节方案所特有的严重器官毒性和骨髓清除术。该方案已成功转化为临床治疗恶性和非恶性疾病患者。在上一次资助期间，我们表明200cgytbi的主要作用是提供宿主免疫抑制，因为在接受仅限于颈部、胸部和上部照射的狗身上也可以实现稳定的混合嵌合体。 腹部淋巴结链。此外，我们能够将供体外周血单个核细胞(PBMC)预刺激并给予共刺激阻滞剂细胞毒性T淋巴细胞抗原-4免疫球蛋白的受体犬的条件化所需的TBI剂量减少到100cGy.研究结果表明，细胞毒性药物为同种异体移植物创造骨髓空间的历史观念已不再有效，移植物完全可以在免疫抑制剂的帮助下建立。随后在T细胞功能受损的患者中成功地进行了无需条件处理的移植，这进一步支持了通过将免疫平衡转移到供体细胞来植入的新概念。在这里，我们试图验证 新的同种异体移植概念，并确定是否可以用没有或几乎没有毒性的特异性免疫抑制来替代广泛的免疫抑制以及与脑损伤相关的短期和长期毒性。我们将采取两种主要方法来实现我们的目标。第一种是在骨髓移植前减少宿主对移植物的免疫反应，方法是用供体PBMC预置受者，然后通过抗T细胞激活抗原CD70的抗体、抗代谢的甲氨蝶呤或阻断两条共刺激通路B7：CD28和CD40：CD154来消除激活的供体反应性T细胞。第二种方法是使用对MMF具有抵抗力的转基因供体T细胞，将免疫平衡转向移植物。

We have established stable mixed donor/host hematopoietic chimerism in dogs by combining sublethal conditioning with 200 cGy total body irradiation (TBI) before and a short course of immunosuppression with mycophenolate mofetil (MMF) and cyclosporine after dog leukocyte antigen (DLA)-identical marrow transplantation. This has allowed transplantation without the severe organ toxicities and myeloablation characteristic of traditional high-dose conditioning regimens. The regimen has been translated successfully into the clinic to treat patients with malignant and nonmalignant diseases. During the last grant period, we showed that the major role of the 200 cGy TBI was to provide host immunosuppression since stable mixed chimerism could also be accomplished in dogs given irradiation limited to the cervical, thoracic, and upper abdominal lymph node chain. Further, we were able to decrease the TBI dose needed for conditioning to 100 cGy in recipient dogs primed with donor peripheral blood mononuclear cells (PBMC) and given the costimulatory blocker cytotoxic T lymphocyte antigen-4 immunoglobulin. Findings suggested that the historic concept of creation of marrow space for allografts by cytotoxic agents was no longer valid and grafts could be established solely with the help of immunosuppressive agents. Subsequent successful transplantation without conditioning in patients with impaired T-cell function lent further support to the new concept of engraftment through shifting of the immunological balance toward donor cells. Here, we seek to validate the new allograft concept and to determine whether specific immunosuppression without or with little toxicity can be substituted for both the broad immunosuppression and the short- and long-term toxicities associated with TBI. Two principal approaches will be taken to accomplish our goals. The first is to reduce host-versus-graft immune responses before marrow grafting by priming recipients with donor PBMC and then eliminating activated donor-reactive T cells by either an antibody against the T-cell activation antigen CD70, the antimetabolite methotrexate, or blockade of two costimulatory pathways, B7:CD28 and CD40:CD154. The second approach is to shift the immunological balance toward the graft using genetically-modified donor T cells which express resistance to MMF.