miRNA-28/150调控耗竭性T细胞诱导免疫耐受在心脏移植中的作用及机制研究

Heart transplantation is the only treatment for terminal cardiac diseases; unfortunately, immune tolerance which would result in permanent graft acceptance remains an elusive goal. In the past decade, our lab has developed various regimens to promote graft acceptance through immune modulation and gene silencing. However, attempts to establish tolerance have had limited success in animal models. Thus, more efficient and robust regimens of tolerance induction are highly clinically demanding..T cell exhaustion is emerging as a new mechanism for immune tolerance, however, its impact on cardiac graft acceptance remains to be evaluated2. In recent studies, we found that heart graft acceptance, induced by blocking mTOR/TLR pathways, is correlated with upregulation of multiple iRs such as PD1 and BTLA. We have defined novel "exhaustion-like" Treg cells in tolerant recipients who accepted cardiac allografts. We have also identified two miRNAs (miR-28 and miR-150) that were significantly upregulated while displayed repressed iRs and less exhausted T cells in rejecting recipients of heart transplantation. However, miRNA modulating T cell exhaustion in transplantation has never been tested..Based on these new findings, we are proceeding with these hypotheses: T cell exhaustion is critical for transplant tolerance; T cell exhaustive differentiation can be regulated by "exhaustion-promoting" DCs and epigenetically by miRNA; and manipulating exhaustive differentiation of T cells is beneficial for heart graft acceptance..To achieve our research goals, we will conduct the research with these specific Aims: 1) Evaluate the impact of T cell exhaustion in mTOR/TLR-mediated tolerance in heart transplantation; 2) Validate the role of mTOR/TLR signaling in T cell exhaustion. 3) Identify miRNAs in modulating T cell exhaustion and enhancing tolerance induction in heart transplantation..Our ultimate objective is to understand the role of T cell exhaustion in transplant tolerance, dissect the mechanisms and regulatory pathways of T cell exhaustion, and explore tolerance-promoting protocols.

器官移植中亟待解决的一个问题是免疫排斥反应。近二十年来申请人应用各种免疫调控手段，诱导器官移植的免疫耐受。尽管在动物实验中取得了较好的效果，但是迄今为止，开发适用于临床治疗的免疫耐受方法尚未建立。目前，免疫耐受的诱导产生及其调控机制尚未完全认知。近年提出了一种免疫抑制的新概念，即T细胞衰竭（T cell exhaustion)，引起了人们的广泛关注， 但其在器官移植中的作用、意义及调控机制尚不清楚。我们的前期工作发现了两种miRNA（即miR-28和miR-150）可以同时调控三种以上iRs，进而可能影响T细胞功能。但是应用miRNA在移植免疫中调控T细胞衰竭的研究尚未见报道。本研究将对衰竭性T细胞产生和调控的机制及其在心脏移植中的作用和意义进行系统深入研究，以期进一步完善认知移植免疫调控机制、为实现移植免疫耐受奠定理论基础，为防止器官移植免疫排斥探索新治疗手段。

T细胞对于调节移植物抗宿主急性排斥免疫反应至关重要。 T细胞耗竭是一种免疫调节现象，有助于降低T细胞活性，防止移植物排异。然而，在移植耐受中T细胞耗竭的机制仍然未知。我们假设，调控DC中抑制性受体（iR）配体相关的miRNA可诱导T细胞衰竭并延长心脏同种异体移植的存活时间。在本研究中，我们评估了同种异体心脏移植的耐受小鼠DC中iR配体的表达。使用miRNA基因芯片筛选来自同种异体移植耐受小鼠的PD-L1阳性耐受性DC中的上调/下调的miRNA, 发现miR-5119显著降低。将miR-5119的模拟物或抑制剂转染到DC中以进行体外和体内实验，然后进行同种异体（C57BL / 6至BALB / c）心脏移植后，我们发现，通过miRNA阵列分析和RT-qPCR验证，在雷帕霉素治疗的耐受心脏移植的小鼠中，miRNA-5119显着下调。生物信息学分析表明，miR-5119可以靶向调控多个免疫调节分子，例如PD-L1和IDO2。用miRNA-5119抑制的DC治疗心脏受体，可显着延长同种异体移植的存活时间。耐受性与体内PD-L1和IDO2表达的上调，T细胞耗竭的促进以及Treg细胞的产生有关。因此，miRNA可以靶向DC中的多个免疫分子并产生免疫耐受，从而防止心脏移植后的同种异体移植排斥。