Induction of cardiac allograft tolerance in a rat heart transplant model

尽管目前使用的免疫抑制药物可以有效降低移植后急性排斥反应的发生率，但它们也使患者面临危及生命的感染和癌症的风险。长期服用免疫抑制剂会导致发病率和死亡率增加。此外，这些代理的长期成本也构成了经济负担。 在不使用非特异性免疫抑制药物的情况下建立对功能良好的移植物的耐受性是器官移植治疗的主要目标。诱导受体（宿主）对器官供体的组织相容性抗原的耐受性可以消除长期施用这些非特异性免疫抑制药物的需要。通过减少与长期免疫抑制治疗相关的免疫学和非免疫学并发症，这将对长期存活器官移植患者的生活质量和数量产生重大影响。 动物心脏移植模型中免疫耐受的诱导大部分涉及基于供体的耐受。一种完善的模型是注射供体脾细胞加上单次注射 T 细胞抑制剂抗大鼠淋巴细胞血清或 RIB 5/2。离体产生的同种异体抗原特异性调节 T 细胞的过继转移（使用来自另一个个体的淋巴细胞改变宿主免疫系统）可在大鼠移植模型中诱导免疫耐受。该方法也已用于骨髓移植，并且可以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在临床上比基于供体的免疫耐受诱导更适用，因为移植前很少知道供体基因型。迄今为止，关于心脏移植中基于受体的免疫耐受诱导的数据有限，但骨髓移植文献中存在数据。 此外，尽管在实验动物中使用各种基于供体的免疫干预措施已经实现了同种异体移植物耐受，但尚未建立确认心脏移植后耐受性的可靠方法。实体器官移植领域的护理标准仍然是采用三重免疫抑制剂进行初始治疗，然后采用 1-3 种免疫抑制剂进行长期维持治疗。即使移植物看起来具有耐受性，人们也不能自信地撤药。 本研究将应用高通量表达谱，目的是研究基于供体和受体的免疫耐受诱导方案，并鉴定可作为可能的候选耐受生物标志物的蛋白质和基因变化。确定能够安全、精确地确认移植患者免疫耐受的实验室方法有可能显着改善结果。 该方案于 2006 年获得批准，在方案期间总共使用了 403 只大鼠，该方案于 2012 年结束。该方案分为 2 部分：第 1 部分（基于接受者的耐受性）；和第 2 部分（基于捐助者的耐受性）。在第 1 部分（基于受体的耐受性）的第 1 阶段，我们能够从受体 BN 大鼠中成功生成 Th2.rapa 细胞。 在第 1 部分第 2 阶段中，使用流式和细胞因子表型测试在培养物中对这些过继转移的离体产生的 BN Th2 细胞进行了测试，并确定了最佳雷帕霉素剂量。 2009 年，我们完成了第 1 部分第 3 阶段的研究，旨在确定 Th2 转移宿主（基于受体的耐受性）是否减少了排斥反应。 在第 2 部分（基于供体的耐受性）的第 1 阶段（诱导基于供体的耐受性）中，我们成功学习了切除将在第 2 阶段捐献心脏的大鼠 (DA) 脾脏的技术。我们还成功地将其处理过的脾细胞注射到将在第 2 阶段接受供体心脏的大鼠 (BN) 的胸腺中。2009 年，我们完成了第 2 部分第 2 阶段的研究，旨在确定基于供体的耐受性诱导是否会减少排斥反应。 2010年，从外周血单核细胞制备总RNA并用于高密度寡核苷酸微阵列。 2011 年发表的一篇手稿 (PloS One, 6(4): e18885, 2011) 确立了宿主型 Th2 的能力。移植前给予 Rapa 细胞疗法，与短期环孢菌素疗法联合使用时，可将移植后细胞因子转变为 Th2 表型，并延长同种异体移植物的活力。 如上所述，该协议于 2012 年关闭；然而，我们将继续报告该项目，因为随着新的基因组技术的出现，我们可能会对这些已完成的研究及其存储的样本进行进一步的数据处理和分析。

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 (host) 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 injection of donor splenocytes plus a single injection of the T-cell supressing agents anti-rat lymphocyte serum or RIB 5/2. Adoptive transfer (altering the hosts immune system using lymphoid cells from another individual) of 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 there is limited data on recipient-based immunotolerance induction 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 high throughput expression profiling 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. The protocol was approved in 2006 and a total of 403 rats were used over the duration of the protocol which was closed in 2012. The protocol was 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 was determined. In 2009, we completed studies in Part 1 Stage 3, which was designed to determine if Th2-shifted hosts (recipient-based tolerance) have reduced rejection. In Part 2 (donor-base tolerance), Stage 1 (Induction donor-based tolerance) we successfully learned the techniques to remove the spleen of the rat (DA) that will donate the heart in stage 2. We also successfully injected its processed splenocytes into the thymus of the rat (BN) that will receive the donor heart in stage 2. In 2009, we completed studies in Part 2, Stage 2 which were designed to determine if donor-based tolerance induction reduces rejection. In 2010, total RNA was prepared from peripheral blood mononuclear cells and used for high density oligonucleotide microarrays. A manuscript was published in 2011 (PloS One, 6(4): e18885, 2011) establishing the ability of host-type Th2. Rapa cell therapy given pre-transplant to shift post-transplant cytokines towards a Th2 phenotype and prolong allograft viability when used in combination with a short course of cyclosporine therapy. As mentioned above, in 2012 the protocol was closed; however, we continue to report on the project, since as new genomic techniques become available we may do further data processing and analysis of these completed studies and their stored samples.