Dendritic Cells, Rapamycin and Transplant Tolerance

描述（由申请人提供）：可预测和安全的人体器官移植耐受诱导仍然是一个难以捉摸的，备受追捧的目标。在大多数患者中，急性排斥反应可以有效地控制，但许多同种异体移植物屈服于慢性排斥反应的破坏。骨髓来源的树突状细胞（DC）是独特的装备良好的抗原（Ag）呈递细胞，现在被认为是免疫反应性的关键调节因子。未成熟/半成熟DC，表达诱导效应T细胞反应所需的低水平共刺激分子，具有固有的耐受性。这些细胞是一种很有希望改善移植结果的方法。挑战在于确定最适合促进治疗耐受性的成熟抗性DC。我们最近的数据显示，rapamycin （RAPA）是一种免疫亲素结合的免疫抑制前药物，因其对T细胞生长的抑制作用而被广泛认可，稳定地抑制DC成熟，抑制它们对toll受体和CD40连接以及促炎细胞因子的反应。RAPA处理的DC （RAPA-DC）在体外和体内诱导同种异体抗原特异性T细胞低反应性，并在体外扩增CD4+CD25*Foxp3+ T调节细胞（Treg）。此外，术前输注mhc不匹配的器官移植受体与宿主来源的RAPA-DC，脉冲供体Ag，促进供体特异性移植物存活。反复输注，或短期最小的免疫抑制，提高长期移植生存，与移植物内Treg相关。基于这些令人鼓舞的数据，我们假设一种基于受体衍生的、药理学修饰的、成熟抗性DC （RAPA-DC）与供体Ag脉冲的治疗方案，能够影响同种异体识别的间接途径，将促进稳定的长期同种异体移植存活。我们进一步假设这种方法将导致通过T reg进行免疫调节的发展。基于组织特异性抗原自身免疫在器官移植排斥反应中的作用的新证据，我们还将探索RAPA-DC与组织特异性抗原的脉冲作用可能增强其耐受性的新概念。我们有四个具体目标。在AIM I中，我们将阐明RAPA对DC成熟抑制作用的分子机制，并优化体外成熟抗性小鼠DC的生成。我们将评估RAPA如何通过Toll和细胞因子受体影响信号传导以最大化其耐受性；在AIM II中，我们将确定使用RAPA-DC诱导长期无排斥的同种异体器官移植存活的最佳方案，包括使用共刺激阻断。在AIM III中，我们将通过重点关注T reg作用的RAPA-DC确定alloAg呈递和调控alloAg特异性T细胞反应的机制。在AIM IV中，我们将产生成熟耐药的人RAPA-DC，用于潜在的临床应用。这些研究将为将来使用DC作为移植耐受的治疗载体铺平道路。操纵树突状细胞以更好地控制器官移植排斥反应，减少患者对抗排斥药物的依赖，有望降低移植人群的发病率和死亡率，并最大限度地减少损耗

DESCRIPTION (provided by applicant): The predictable and safe induction of human organ transplant tolerance remains an elusive, much sought-after goal. In most patients, acute rejection can be managed effectively, but many allografts succumb to the ravages of chronic rejection. Bone marrow-derived dendritic cells (DC) are uniquely well-equipped antigen (Ag)-presenting cells, now regarded as critical regulators of immune reactivity. Immature/semi-mature DC, that express low levels of costimulatory molecules necessary for the induction of effector T cell responses, have inherent tolerogenic potential. These cells are a promising means of improving transplant outcome. The challenge is to identify the maturation-resistant DC best-suited to promote therapeutic tolerance. Our recent data show that rapamycin (RAPA), an immunophilin- binding immunosuppressive pro-drug, well-recognized for its inhibitory action on T cell growth, stably suppresses DC maturation, inhibiting their responses to Toll-receptor and CD40 ligation and pro-inflammatory cytokines. These RAPA- treated DC (RAPA-DC) induce alloAg-specific T cell hyporesponsiveness in vitro and in vivo and expand CD4+CD25*Foxp3+ T regulatory cells (Treg) in vitro. Furthermore, pre-operative infusion of MHC-mismatched organ graft recipients with host-derived RAPA-DC, pulsed with donor Ag, promotes donor-specific graft survival. Repeated infusion, or a short course of minimal immunosuppression, enhances long-term transplant survival, associated with intra-graft Treg. Based on these encouraging data, we hypothesize that a regimen based on treatment of allograft recipients with recipient-derived, pharmacologically-modified, maturation-resistant DC (RAPA-DC) pulsed with donor Ag, and capable of impacting the indirect pathway of allorecognition, will promote stable long-term allograft survival. We further hypothesize that this approach will lead to development of immune regulation via T reg. Based on emerging evidence of the role of autoimmunity to tissue-specific Ag in organ graft rejection, we will also explore the novel concept that pulsing of RAPA-DC with tissue-specific Ag may enhance their tolerogenicity. We have four specific Aims. In AIM I, we will elucidate molecular mechanisms underlying the inhibitory action of RAPA on DC maturation and optimize generation of maturation-resistant murine DC in vitro. We will assess how RAPA affects signaling via Toll and cytokine receptors to maximize their tolerogenic potential; in AIM II, we will determine the optimal protocol for induction of long-term, rejection-free, organ allograft survival using RAPA-DC, including the use of co-stimulation blockade. In AIM III, we will ascertain mechanisms underlying alloAg presentation and regulation of alloAg-specific T cell responses by RAPA-DC focusing on the role of T reg. In AIM IV, we will generate maturation-resistant human RAPA-DC for potential clinical application. These studies will pave the way for the future use of DC as therapeutic vectors of transplant tolerance. Manipulation of dendritic cells to better control organ transplant rejection and to reduce patients' dependency on anti-rejection drugs, promises to reduce morbidity and mortality in the transplant population and to minimize the attrition