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的发生率方面是有效的，但它们也使患者处于威胁生命的感染和癌症的风险中。免疫抑制剂的长期施用导致发病率和死亡率的增加。此外，这些药剂的长期费用也是一种财政负担。 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可以消除长期施用这些非特异性免疫抑制药物的需要。这将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对具有长期存活器官移植物的患者的生活质量和数量产生重大影响。 在动物心脏移植模型中诱导免疫耐受在很大程度上涉及基于供体的耐受。一种良好建立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。体外产生的同种异体抗原特异性调节性T细胞的连续转移（使用来自另一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。该方法也已用于骨髓移植，并可作为基于免疫耐受诱导的基础。在心脏移植中，由于供体基因型在移植前很少知道，因此基于供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。迄今为止，心脏移植中基于免疫耐受诱导的数据有限，但骨髓移植文献中有数据。 此外，尽管在实验室动物中使用各种基于供体的免疫干预已经实现了同种异体移植物耐受性，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用三重免疫抑制剂进行初始治疗，然后用1-3种免疫抑制剂进行长期维持治疗。即使移植物似乎是耐受的，人们也不能自信地撤回药物。 本研究将应用高通量表达谱分析，目的是研究基于供体和宿主的免疫耐受诱导方案，并鉴定可作为耐受性可能的候选生物标志物的蛋白质和基因变化。确定实验室方法，将允许安全和准确的确认移植患者的免疫耐受性，有可能大大改善结果。 该方案于2006年获得批准，在2012年关闭的方案期间共使用了403只大鼠。该方案分为2部分：第1部分（基于供体的耐受性）和第2部分（基于供体的耐受性）。在第1部分（基于免疫耐受），第1阶段，我们能够成功地从受体BN大鼠产生Th2.rapa细胞。 在第1部分第2阶段，使用流动和细胞因子表型测试在培养物中测试这些过继转移的离体产生的BN Th 2细胞，并确定最佳雷帕霉素剂量。 2009年，我们完成了第1部分第3阶段的研究，旨在确定Th 2移位宿主（基于免疫耐受性）是否降低了排斥反应。 在第2部分（供体-基础耐受性），第1阶段（诱导供体-基础耐受性）中，我们成功地学习了在第2阶段切除将捐献心脏的大鼠（DA）脾脏的技术。 我们还成功地将其处理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诱导是否会减少排斥反应。 2010年，从外周血单核细胞中制备总RNA并用于高密度寡核苷酸微阵列。 2011年发表了一份手稿（PloS One，6（4）：e18885，2011），建立了宿主型Th 2的能力。在移植前给予Rapa细胞治疗，以使移植后细胞因子向Th 2表型转变，并在与短期环孢素治疗联合使用时延长同种异体移植物存活率。 如上所述，2012年该方案关闭;然而，我们继续报告该项目，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储存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.