Tomotherapy and Hematopoietic Stem Cells For Tolerance to Kidney Transplants

描述(由申请人提供)：该项目旨在验证这样一种假设，即通过在非人类灵长类动物中建立稳定的免疫混合嵌合状态，在MOZOBIL G-CSF动员的供者造血细胞输注之后，使用一种新型的非清髓性、基于螺旋断层放射治疗的全淋巴照射(TLI)调节方案，可以有效和安全地实现对MHC不匹配的活体相关肾移植的耐受性。此外，我们将检验这样的假设，即该方案的成功将取决于供者和受者的“自然”母胎预适应，以及宿主Treg细胞在TLI/ATG受者中增加IL-10和IL-4产生的细胞因子偏向。我们建议通过两个特定的目标来验证这些假说：1)联合造血细胞/肾脏移植：确定在维持MHC-1半相合活体亲属供肾移植的正常移植物功能的同时，可以停用所有免疫抑制药物的猕猴的比例。我们将通过稳定的肾功能和供体嵌合体作为时间的函数来评估移植后的耐受性，这些受者接受了基于螺旋断层疗法的TLI和兔抗胸腺细胞球蛋白(ATG)，并给予了纯化的供体CD34造血干细胞(HSC)和特定剂量的供体T细胞。2)免疫监测、免疫病理及免疫活性：a)连续监测细胞内细胞因子IL-4、IL-10、干扰素-α、转化生长因子？在活化的NK T细胞、常规的CD4T细胞和造血干细胞联合移植受者的外周血单核细胞(PBMC)中，CD4 CD25 Treg细胞支持嵌合体和无GVHD动物的Th2偏向，从而允许移植后成功停用免疫抑制药物；b)确定从移植前开始，是否可以分别对间接和直接同种异体反应途径进行一系列的耐受分析，以预测嵌合体和无GVHD的受者是否可以成功停用免疫抑制药物。我们还将测试宿主和供者树突状细胞(DC)在停用免疫抑制药物后以及在与供者和第三方皮肤移植物一起挑战后的表型和功能，以评估耐受性与免疫原性抗原提呈细胞(APC)在移植物结果中的作用。C)通过监测微生物感染的发生率，检测移植后接种流感病毒疫苗后的Recall T细胞和抗体反应，以及Recall T细胞对带状疱疹、单纯疱疹、巨细胞病毒抗原和破伤风类毒素的反应，来确定免疫抑制药物停药后受者免疫能力的程度。此外，我们将通过检测移植后T细胞切除环和血液中幼稚T细胞、记忆T细胞以及调节性CD4和CD8T细胞的水平来确定移植后T细胞的恢复和胸腺功能。为了测试供体对供体第三方同种异体抗原的耐受性和免疫活性，供体皮肤移植将被放置在受者身上。

DESCRIPTION (provided by applicant): This project aims to test the hypothesis that tolerance to MHC mismatched living related kidney transplant can be effectively and safely achieved by establishing a stable immune mixed chimeric state in non-human primates using a novel non-myeloablative, helical tomotherapy-based total lymphoid irradiation (TLI) conditioning regimen followed by Mozobil + G-CSF mobilized donor hematopoietic cell infusions. Furthermore, we will test the hypotheses that the success of this protocol will depend upon "natural" maternal-fetal preconditioning of donor and recipient, and a cytokine bias of host Treg cells toward increased production of IL-10 and IL-4 in TLI/ATG recipients of HSCs. We propose to test these hypotheses by means of 2 specific aims: 1) Combined Hematopoietic Cell/Kidney Transplants: to determine the proportion of Rhesus macaques that can be withdrawn from all immunosuppressive drugs while maintaining normal graft function of MHC 1-haplotype mismatched living related donor kidney transplants. We will assess tolerance by stable kidney function and donor chimerism as a function of time after transplantation in recipients conditioned with helical tomotherapy-based TLI and rabbit anti-thymocyte globulin (ATG) and given purified donor CD34+ hematopoietic stem cells (HSC) and specified doses of donor T cells. 2) Immune Monitoring, Immunopathology and Immunocompetence: a) to determine if serial monitoring of intracellular cytokine expression including IL-4, IL-10 IFN-?, and TGF? in activated NK T cells, conventional CD4+ T cells, CD4+CD25+ Treg cells in the peripheral blood mononuclear cells (PBMCs) of recipients of combined kidney and HSC transplants supports a Th2 bias in animals with chimerism and no GVHD that allows successful withdrawal of immunosuppressive drugs after transplantation; b) to determine whether serial trans-vivo Delayed Type Hypersensitivity (tvDTH) and mixed lymphocyte reaction (MLR) analysis of tolerance on indirect and direct pathways of alloreactivity, respectively, beginning pre-transplant, can be used to predict recipients with chimerism and no GVHD allowing successful withdrawal of immunosuppressive drugs. We will also test the phenotype and function of host and donor dendritic cells (DCs) obtained after withdrawal of immunosuppressive drugs, and after a challenge with a donor and third party skin graft to assess the role of tolerogenic versus immunogenic antigen presenting cells (APCs) in graft outcome. c) to determine the degree of recipient immune competency after immunosuppressive drug withdrawal, by monitoring the incidence of microbial infection, testing the recall T cel and antibody responses to influenza virus vaccination after transplantation, as well as recall T cell responses to herpes zoster, herpes simplex, cytomegalovirus antigens, and tetanus toxoid. In addition, we will determine T cell recovery and thymic function after transplantation by measuring the levels of T cell excision circles, and naive, memory, and regulatory CD4+ and CD8+ T cells in the blood at serial time points. To test for donor-specific tolerance and immunocompetence to donor third party allo-antigens, donor skin grafts will be placed on the recipient.