Induction of cardiac allograft tolerance in a rat heart transplant model

尽管目前使用的免疫抑制药物在减少移植后急性排斥反应方面有效，但它们也使患者面临威胁生命的感染和癌症的风险。长期使用免疫抑制剂会导致发病率和死亡率的增加。此外，这些代理商的长期成本是一种财务负担。 在没有非特异性免疫抑制药物的情况下，建立对功能良好的移植的耐受性是器官移植治疗的主要目标。诱导受体对器官捐赠者的组织相容抗原的耐受性可以消除长期服用这些非特异性免疫抑制药物的需要。这将对长期存活的器官移植患者的生活质量和数量产生重大影响，因为它可以减少与长期免疫抑制治疗相关的免疫和非免疫并发症。 在动物心脏移植模型中诱导免疫耐受在很大程度上涉及到基于供者的耐受。一种成熟的模型是在移植前21天一次静脉注射供者脾细胞(25106个细胞)和一次腹膜内注射肋骨52(20毫克公斤)。RIB 52是一种不耗竭的抗大鼠CD4的单抗，能抑制辅助性T细胞1(Th1)，从而诱导强大的同种异体免疫耐受。最近的一项研究表明，供者特异性输血可以在淋巴组织和移植物中诱导同种异体抗原特异性辅助性T细胞2(Th2)的发育。这些体外产生的同种异体抗原特异性调节性T细胞过继转移(使用来自另一个个体的淋巴样细胞改变宿主免疫系统)在大鼠移植模型中诱导免疫耐受。这种方法也已用于骨髓移植，可作为基于受体的免疫耐受诱导的基础。在心脏移植中，基于受体的免疫耐受诱导比基于供体的免疫耐受诱导在临床上更适用，因为在移植前供体基因很少被知道。到目前为止，基于受体的免疫耐受诱导还没有在心脏移植中尝试过，但有来自骨髓移植文献的数据。 此外，尽管在实验动物中使用了多种基于供者的免疫干预已经实现了同种异体移植的耐受性，但还没有建立可靠的方法来确定心脏移植后的耐受性。实体器官移植领域的标准护理仍然是最初的三重免疫抑制治疗，然后是1-3种免疫抑制剂的长期维持治疗。即使移植看起来是宽容的，人们也不能自信地退出药物。 这项研究将在同一实验中同时应用基因组学和蛋白质组学，目的是研究基于供体和受体的免疫耐受诱导方案，并识别可能作为耐受候选生物标志物的蛋白质和基因变化。确定实验室方法将允许安全和准确地确认移植患者的免疫耐受，有可能显著改善结果。 自2006年该协议获得批准以来，已经有321只动物被使用。其中58个是在第一年使用的，263个是在本审查年度使用的。该协议分为两个部分：第一部分(基于接受者的容忍)和第二部分(基于捐赠者的容忍)。 在第一部分(基于受体的耐受)，第一阶段，我们能够成功地从受体BN大鼠中产生Th2.Rapa细胞。在第1、2阶段中，对这些过继转移的体外产生的BN Th2细胞进行了Flow和细胞因子表型试验，并确定了最佳雷帕霉素剂量。我们目前正在进行第1阶段第3阶段的研究，旨在确定Th2移位宿主(基于受体的耐受)是否减少了排斥反应。 在第二部分(基于供者的耐受)，阶段1(基于供者的诱导耐受)，我们已经成功地学习了在第二阶段将捐献心脏的大鼠(DA)的脾切除的技术。我们还成功地将处理过的脾细胞注射到将在第二阶段接受供体心脏的大鼠(BN)的胸腺中。我们目前正在进行第二部分，第二阶段的研究，旨在确定基于供体的耐受诱导是否可以减少排斥反应。

Although the immunosuppressive drugs currently in use are effective in reducing the incidence of acute rejection after transplantation, they also put the patient at risk for life threatening infections and cancers. The long term administration of immunosuppressive agents results in an increase in both morbidity and mortality. In addition, the long term cost of these agents represents a financial burden. Establishment of tolerance to a well-functioning transplant without nonspecific immunosuppressive drugs is a major goal of organ transplantation therapy. The induction of recipient tolerance to the histocompatibility antigens of the organ donor could eliminate the need for long term administration of these nonspecific immunosuppressive drugs. This would have a major impact on the quality and quantity of life of patients with long term surviving organ grafts by reducing the immunologic and non-immunologic complications associated with long term immunosuppressive therapy. Induction of immunotolerance in animal heart transplant models has for the most part involved donor-based tolerance. One well established model is a single intravenous injection of donor splenocytes (25106 cells) plus a single intraperitoneal injection of RIB 52 (20mgkg) 21 days prior to the transplantation. RIB 52 is a nondepleting anti-rat CD4 monoclonal antibody that suppresses T helper 1 (Th1) cells thus induces robust allospecific tolerance. A recent study has shown that donor-specific blood transfusion induces the development of alloantigen-specific T helper 2 (Th2) cells in lymphoid tissues and in the graft. Adoptive transfer (altering the hosts immune system using lymphoid cells from another individual) of these ex vivo generated alloantigen-specific regulatory T cells induces immunotolerance in a rat transplant model. This method has also been used in bone marrow transplantation and can be the basis for recipient-based immunotolerance induction. In heart transplantation, recipient-based immunotolerance induction is clinically more applicable than donor-based since donor genotype is rarely known prior to transplant. To date recipient-based immunotolerance induction has not been tried in heart transplantation but data exists from the bone marrow transplant literature. In addition, although allograft tolerance has been achieved using a wide variety of donor-based immunologic interventions in laboratory animals, no reliable method of confirming tolerance following cardiac transplantation has been established. Standard of care in the field of solid organ transplantation remains initial treatment with triple immunosuppression followed by long term maintenance therapy with 1-3 immunosuppressive agents. One cannot withdraw drugs confidently even if the graft seems to be tolerant. This study will apply both genomics and proteomics within the same experiment with the goal of studying donor and recipient-based immunotolerance induction protocols and identifying protein and gene changes that could serve as possible candidate biomarkers of tolerance. Identifying laboratory methods that will permit safe and precise confirmation of immune tolerance in the transplant patient has the potential to improve outcome substantially. There have been 321 animals used since the protocol was approved in 2006. Of these 58 were used in the first year and 263 in the year currently being reviewed. The protocol is divided into 2 parts: Part 1 (recipient-based tolerance); and Part 2 (donor-based tolerance). In part 1 (recipient-based tolerance), Stage 1 we were able to successfully generate Th2.rapa cells from recipient BN rats. In Part 1, Stage 2 these adoptively transferred ex vivo generated BN Th2 cells were tested in culture using flow and cytokine phenotype tests, and an optimal rapamycin dose has been determined. We are currently doing studies in Part 1 Stage 3, which is designed to determine if Th2-shifted hosts (recipient-based tolerance) have reduced rejection. In Part 2 (donor-base tolerance), Stage 1 (Induction donor-based tolderance) we have successfully learned the techniques to remove the spleen of the rat (DA) that will donate the heart in stage 2. We have also successfully injected its processed splenocytes into the thymus of the rat (BN) that will receive the donor heart in stage 2. We are currently doing studies in Part 2, Stage 2 which is designed to determine if donor-based tolerance induction reduces rejection.