抗原特异性诱导中人Treg表型的分化动态及其保护异种胰岛移植物机制的研究

Xenotransplantation has an immense potential as an alternative source for clinical transplantation. However T cell mediated rejection remains as a barrier to the clinical application of xenotranplantation. Regulatory T cells (Treg) suppress immune responses to non-self antigen mediated by effector T cells and induce transplantation tolerance. Moreover, antigen-specifc Tregs are more effective and deplet xenoreactive effector T cells selectively reducing the potential risks of tumor and infection in recipients. This study aims to analyze the dynamics of human induced xenoantigen-specific Treg cell phenotype differentiation and determine the ability of human induced xenoantigen-specific Treg to prevent islet xenograft rejection and the mechanism(s) involved. Induced xenoantigen specific Treg in vitro suppressive activity will be assessed by measuring inhibition of proliferation of autologous peripheral blood mononuclear cells(PBMC.) stimulated with xenoantigen. Furthermore, NOD-SCID mice will be transplanted with porcine neonatal islet cell clusters (NICC) followed by reconstitution of human PBMC with or without induced xenoantigen-specific Treg labeled by firefly luciferase and renilla luciferase respectively. The T cells trafficking and homing to the xenograft in recipient mice will be longitudinally tracked by High-sensitivity optical molecular imaging and high-resolution digital X-ray. The engrafments and function of islet xenograft and human leukocytes will be determined by immunohistochemical staining and FACS analysis. This study will dissect the ability and the mechanisms of protection of islet xenograft from rejection by human induced xenoantigen-.specific Treg in NOD-SCID mice. In vitro antigen-specific Treg expansion is a effective strategy for generating autologous Treg and highlights a potential adoptive Treg cell therapy to suppress anti-xenongraft T cell responses and reduce the requirement for immunosuppression in xenotransplantation. The study will provide insight for improving outcomes of clinical xenotransplantation.

移植排斥是异种胰岛移植的一个主要障碍，诱导并维持移植免疫耐受是异种胰岛移植成功的有效措施与保障。调节性T细胞（Regulatory T cell，Treg）可通过抑制针对非自身抗原的免疫应答来诱导和维持移植免疫耐受，且抗原特异Treg能选择性清除对移植物有特异性反应的效应性T细胞，不仅增强疗效而且减少因过度免疫抑制而导致肿瘤和感染发生的潜在风险。本课题拟体外诱导扩增人异种抗原特异性Treg细胞，检测其表型的分化及其相应功能变化的动态；体内小动物活体成像技术与活体组织细胞检测技术等结合实时监控免疫细胞在受体小鼠体内的迁移以及在异种猪胰岛移植物部位的免疫应答，探究抗原特异性Treg诱导异种胰岛移植免疫耐受的分子机制，评估异种抗原特异性Treg诱导移植免疫耐受的效率，建立一种以经体外抗原诱导扩增培养的Treg细胞来特异性抑制T 细胞介导的异种胰岛移植排斥反应的细胞治疗，从而提高异种移植物的植入率

中文摘要:主要研究内容、重要结果及其科学意义等。.异种移植有望解决移植器官来源短缺的瓶颈问题，但移植免疫排斥反应比同种移植的更为严重。因此诱导异种移植免疫耐受是确保移植物能够存活并发挥正常生理功能重要的措施。调节性T细胞（Regulatory T cell，Treg）可通过抑制针对非自身抗原的免疫应答来诱导和维持移植免疫耐受，且抗原特异Treg能选择性清除对移植物有特异性反应的效应性T细胞，不仅增强疗效而且减少因非特异性免疫抑制而导致肿瘤和感染发生的潜在风险。本课题在体外将分离获得的人外周血Tregs与照射过的成体猪外周血单个核细胞共孵育培养，成功获得了异种抗原特异性Tregs，不仅细胞扩增倍数高于多克隆扩增的Tregs，而且抗原特异性Tregs在高表达CD25和Foxp3的基础上，其他表面分子ICOS，HLR-DR等的表达上调，表达CD45RO的细胞数量增加，且IL-10的表达亦增加，而IL-10的表达增加与IFR4上调Blimp-1的表达有关。抗原特异性Treg在功能上能抑制异种猪抗原刺激的CD25- T 细胞的增殖，且比多克隆扩增的Tregs 的抑制作用更明显。体内实验发现经体外诱导扩增的抗原特异性Tregs参与了抑制人PBMCs针对异种猪胰岛移植物的免疫排斥反应，异种胰岛移植物不仅具有内外分泌功能，而且存活时间明显延长达56天以上。Tregs在体内对异种胰岛移植物保护作用的机制与抗原特异性Tregs明显减少胰岛移植物周围浸润的CD45+，CD4+,CD8+T炎症细胞数量从而减轻对胰岛移植物的破坏，同时上调免疫抑制性细胞因子IL-10和下调免疫炎症性细胞因子IFN-有关。本项目成功地建立一种成熟的健康成人外周血分离及诱导扩增抗原特异性 Tregs的实验方法，该实验方法的建立为临床抗原特异性Tregs细胞治疗提供了细胞数量及质量的保证，并已经应用于临床异种胰岛移植患者自体抗原特异性Tregs细胞治疗来诱导移植免疫耐受的临床研究，为临床异种移植中免疫耐受的诱导带来了新的有效方法和希望。本项目成功建立了与国外NSG小鼠一样的NPG小鼠NICC移植耐受模型，利用该模型研究发现人抗原特异性Tregs具有较强抑制效应T细胞介导的NICC移植物排斥反应的能力，为人抗原特应性Tregs在临床移植耐受诱导的细胞治疗应用提供了的参考。