Tomotherapy and Hematopoietic Stem Cells For Tolerance to Kidney Transplants

描述（申请人提供）：本项目旨在验证以下假设：通过使用一种新型非清髓性，基于螺旋断层治疗的全淋巴照射（TLI）预处理方案，随后进行释倍灵+ G-CSF动员的供体造血细胞输注。 此外，我们将测试该方案的成功将取决于供体和受体的“天然”母胎预处理，以及宿主Treg细胞对HSC的TLI/ATG受体中IL-10和IL-4产生增加的细胞因子偏好的假设。 我们建议通过2个具体目标来测试这些假设：1）联合造血细胞/肾移植：确定可以从所有免疫抑制药物中撤出的恒河猴的比例，同时维持MHC 1-单倍型不匹配的活体亲属肾移植的正常移植功能。我们将通过稳定的肾功能和供体嵌合体作为移植后时间的函数，在接受基于螺旋断层治疗的TLI和兔抗胸腺细胞球蛋白（ATG）并给予纯化的供体CD 34+造血干细胞（HSC）和指定剂量的供体T细胞的受体中评估耐受性。 2)免疫监测、免疫病理学和免疫活性：a）确定是否连续监测细胞内细胞因子表达，包括IL-4、IL-10 IFN-？， 而TGF？ 在肾和HSC联合移植的受体的外周血单核细胞（PBMC）中，活化的NK T细胞、常规的CD 4 + T细胞、CD 4 + CD 25 + Treg细胞支持在具有嵌合状态且无GVHD的动物中的Th 2偏好，其允许在移植后成功撤回免疫抑制药物; B）确定在移植前开始，分别对同种异体反应性的间接和直接途径的耐受性进行连续的经体内迟发型超敏反应（tvDTH）和混合淋巴细胞反应（MLR）分析，是否可用于预测 具有嵌合体且没有GVHD的受体允许成功地撤回免疫抑制药物。我们还将测试宿主和供体树突状细胞（DC）的表型和功能，这些树突状细胞是在免疫抑制药物停药后以及供体和第三方皮肤移植物激发后获得的，以评估致耐受性与免疫原性抗原呈递细胞（APC）在移植物结局中的作用。c）通过监测微生物感染的发生率，测试移植后对流感病毒疫苗接种的回忆T细胞和抗体应答，以及对带状疱疹、单纯疱疹、巨细胞病毒抗原和破伤风类毒素的回忆T细胞应答，确定免疫抑制药物停药后受体免疫能力的程度。此外，我们将通过在连续时间点测量血液中T细胞切除环以及幼稚、记忆和调节性CD 4+和CD 8 + T细胞的水平来确定移植后T细胞恢复和胸腺功能。为了测试供体特异性耐受性和对供体第三方同种异体抗原的免疫活性，将供体皮肤移植物放置在受体上。

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aims: 1) Combined Hematopoietic Cell/Kidney Transplants: to determine the proportion of Rhesus macaques that can be withdrawn from all immunosuppressive drugs while maintaining normal graft function of MHC 1-haplotype mismatched living related donor kidney transplants.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.