Mechanisms in Induction of Immunological Tolerance

本研究计划的目的是研究导致心肾联合移植受者稳定免疫耐受的机制。在猪模型中，当来自同一供体的肾脏共移植时，接受异位移植的心脏。所有受体对两种移植物均产生稳定的耐受性。这种现象被称为肾诱导的心脏异体移植耐受（KICAT）。导致这种耐受性诱导长期成功的潜在机制尚不清楚。在计划的研究中，异位心脏和肾脏移植将在非人灵长类动物（NHP）中进行。Foxp3+CD25+CD4+调节性T细胞（Treg）似乎在同种异体移植接受过程中具有独特的作用。首先，研究Tregs在耐受性诱导过程中的作用。为了证明Treg的重要性，我们设想使用一种新的抗人ccr4免疫毒素来消耗CD4+CD25highFoxP3+细胞，这种毒素可以特异性地消耗食蟹猴（NHP）中的Treg效应物。这种Tregs的消耗被认为可以阻止心脏和肾脏同种异体移植物的耐受性诱导，证实了Tregs的重要作用。此外，我们计划通过研究新的表面标记CD49b和LAG-3来详细表征食蟹猴CD4+ 1型调节细胞耐受诱导过程中的Tregs。此外，浆细胞样树突状细胞（pDC）的作用有待进一步研究。初步数据表明，pDCs积极促进受体treg的激活和/或扩增，因此支持诱导KICAT。具体来说，CCR9+ pDCs能够将CD4+ naïve t细胞转化为treg，从而具有促耐受性功能。接受心脏/肾脏联合移植的NHP将接受抗cd303单抗治疗，导致pDC耗损，这可能会抑制KICAT的发作。最后，探讨促红细胞生成素（EPO）在KICAT发展中的作用。CD4+CD8+ T细胞表达促红细胞生成素受体（EPO- r）， EPO对同种异体CD4+ T细胞增殖呈剂量依赖性降低作用，提示其免疫抑制作用。为了确定高剂量促红细胞生成素治疗是否能提供与肾联合移植相似的耐受性诱导，动物要么在围手术期接受额外的促红细胞生成素，要么相反，通过给药一种已被证明能在体内减少促红细胞生成素的物质来实现促红细胞生成素的药理学下调。这些实验将进一步深入了解导致KICAT发展的机制。

Aim of this research proposal is to study mechanisms leading to a stable immunological tolerance in recipients of combined heart- and kidney transplantation. In a porcine model, heterotopic transplanted hearts were accepted when a kidney co-transplantation from the same donor was performed. All recipients developed stable tolerance toward both grafts.This phenomenon was termed kidney-induced cardiac allograft tolerance (KICAT). The underlying mechanisms leading to this long-term success of tolerance induction remain unclear. For the planned studies, heterotopic heart- and kidney transplantations will be performed in nonhuman primates (NHP).Foxp3+CD25+CD4+ regulatory T cells (Treg) seem to have a distinct role in the process of allograft acceptance. First, the role of Tregs within the tolerance induction process will be studied. In order to proof the importance of Tregs, a depletion of CD4+CD25highFoxP3+ cells is envisioned by using a novel anti-human CCR4-immunotoxin, which specifically deplete effector Treg in cynomolgus monkeys (NHP). This depletion of Tregs is assumed to prevent tolerance induction of both, heart and kidney allograft, confirming the substantial role of Tregs. Further, a detailed characterization of Tregs within the process of tolerance induction is planned by studying new surface markers, CD49b and LAG-3, which characterize specifically CD4+ type 1 regulatory cells in cynomolgus monkey. Further, the role of plasmacytoid dendritic cells (pDC) will undergo further research. Preliminary data suggests that pDCs actively promote the activation and/or expansion of recipient Tregs and therefore support the induction KICAT. In detail, CCR9+ pDCs are able to convert CD4+ naïve T-cells into Tregs, thereby having pro-tolerogenic function. NHP undergoing combined heart/kidney transplantation will receive a treatment with anti-CD303 mAB, leading to pDC depletion, which potentially inhibits the onset of KICAT.At last, the role of Erythropoetin (EPO) in the development of KICAT will be identified. CD4+CD8+ T cells express Erythropoetin receptor (EPO-R) and a dose-dependent decreasing effect of EPO on allogeneic CD4+ T-cell proliferation was detectable, thus indicating an immunosuppressive effect. In order to determine if high dose EPO treatment will provide similar tolerance induction as kidney co-transplantation, animals will either receive additional erythropoietin perioperatively or in contrast, a pharmacological downregulation of EPO will be achieved by administering a substance which has proven to diminish EPO in vivo, acriflavine. These experiments will provide further insight into mechanisms leading to the development of KICAT.