Tolerance In Organ Transplantation And Autoimmune Diseas

本项目的总体目标是研究免疫毒素诱导的瞬时T细胞耗竭对T细胞从免疫应答向耐受应答的调节的影响，并将这些知识用于T细胞驱动的自身免疫性疾病、移植物抗宿主病以及诱导对错配器官和细胞移植的耐受的实验和临床治疗。我们先前表明，用CRM 9（白喉毒素的结合位点突变体）构建的抗恒河猴CD 3免疫毒素的2-3天疗程在48小时滞后期后消耗99%的淋巴结和血液T细胞。这一过程与不匹配的功能性恒河猴肾移植的生存期显著延长相关，在没有进一步免疫抑制治疗的情况下，9个月的生存率为50%。排斥反应之前出现了抗移植物抗体（用S。Knechtle和J.托马斯）。为了避免这种不利的T细胞介导的B细胞应答，其显然是在免疫毒素诱导的T细胞杀伤延迟期间启动的，我们加入了短疗程的被认为阻断抗原呈递的试剂，如脱氧精胍菌素（DSG）。5例接受双侧肾切除术的受者中，3例移植物存活4年或更长时间，达到长期耐受，未进一步免疫抑制。在患有自发性胰岛素依赖性糖尿病的猴中进行同源胰岛细胞移植已显示出逆转糖尿病，如通过在不存在外源性胰岛素的情况下使非空腹血糖和糖化血红蛋白值恢复正常所判断的。短期免疫毒素，甲基强的松龙和环孢素提供诱导稳定的操作耐受胰岛，已持续一年以上的情况。这是第一次报道使用不依赖于慢性免疫抑制的耐受方案逆转糖尿病，慢性免疫抑制是一个先前显示损害移植胰岛功能的过程（由J.托马斯完成）。通过使用抗T细胞免疫毒素联合14天的DSG和同种异体移植的胰岛（与J.托马斯一起完成），已成功逆转了链脲霉素诱导的猴糖尿病。七分之三是长期耐受的幸存者（6-7年）。已经开发了重组二价抗人抗T细胞免疫毒素，其具有先前化学缀合物的20倍效力。在毕赤酵母中表达。这种材料目前正在生产中，用于T细胞白血病和再生障碍性贫血（一种T细胞驱动的自身免疫性疾病）的I期临床试验。为了帮助在巴斯德毕赤酵母中产生免疫毒素和其他生物活性蛋白，已经构建了新的菌株，其在存在活性未折叠蛋白应答的情况下表现出毒素抗性和增加的异源蛋白产量。

The general aim of this project is to study the effects of immunotoxin induced transient T-cell depletion on the modulation of T cells from immunization responses towards tolerizing responses, and to use this knowledge for the experimental and clinical treatment of T cell driven autoimmune diseases, graft-versus-host disease, and the induction of tolerance to mismatched organ and cell transplants. We previously showed that a 2-3 day course of anti-rhesus CD3 immunotoxin constructed with CRM9, a binding site mutant of diphtheria toxin, depletes lymph node and blood T cells by 99% after a 48 hour lag period. This process was associated with a marked prolongation of survival of mismatched functioning rhesus kidney transplants, 50% over 9 months without further immunosuppressive therapy. Rejections, were preceded by the appearance of anti-graft antibodies (done with S. Knechtle and J. Thomas). To circumvent this adverse T cell mediated B cell response that is apparently initiated during the delay in immunotoxin induced T cell killing, we have added a short course of agents that are believed to block antigen presentation such as deoxyspergualin (DSG). Out of 5 bilaterally rephrectomized recipients long term tolerance was achieved in 3 without further immunosuppresion as judged by a graft survival of 4 years or longer. Congeneic pancreatic islet cell transplants in monkeys suffering from spontaneous insulin dependent diabetes have been shown to reverse diabetes as judged by returning non-fasting blood glucose and glycosylated hemoglobin values to normal in the absence of exogenous insulin. A short course of immunotoxin, methyl prednisolone and cyclosporine provide induction of stable operational tolerance to islets that has lasted over one year in one case. This is the first reported reversal of diabetes using a tolerizing protocol that does not depend on chronic immunosuppression, a process previously shown to compromise transplanted islet function (done with J. Thomas). Streptozotocin induced diabetes in monkeys has been successfully reversed by using anti-T cell immunotoxin in combination with 14 days of DSG and allografted pancreatic islets (done with J. Thomas). Three out of seven are long term tolerant survivors (6-7 years). A recombinant divalent anti-human anti-T cell immunotoxin has been developed that has 20-fold the potency of the previous chemical conjugates. This is expressed in Pichia pastoris. This material is now under production for a phase I clinical trial in T cell leukemia and aplastic anemia, a T cell driven autoimmune disease. In order to aid the production of immunotoxin and other bioactive proteins in Pichia pastoris, new strains have been constructed that exhibit toxin resistance and increased heterologous protein production in the presence of an active unfolded-protein response.