Generating a model system to elucidate in vivo functions of soluble IL-15 complexes

生成模型系统来阐明可溶性 IL-15 复合物的体内功能

• 批准号: 9109342
• 负责人: KIMBERLY Sue SCHLUNS
• 金额: $ 8万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-02-15 至 2018-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9109342
• 关键词: Affinity; Amino Acids; Autoimmune Diseases; Autoimmunity; Automobile Driving; Biological Models; Breeding; CD8B1 gene; Cell Communication; Cell Survival; Cell physiology; Cell surface; Cells; Cleaved cell; Complex; DNA; Dendritic Cells; Development; Event; Generations; Genetically Engineered Mouse; Goals; Homeostasis; Human; Immune; Immune response; Infection; Inflammation; Inflammatory; Interferons; Interleukin-15; Knowledge; Laboratories; Lead; Lymphocyte; MHC Class I Genes; Malignant Neoplasms; Measures; Mediating; Memory; Metalloproteases; Modeling; Mus; Mutate; Natural Killer Cells; Phenotype; Process; Production; Proteins; Recombinants; Regulation; Resistance; Role; Serum; Site; Stimulus; Surface; T cell response; T-Cell Proliferation; T-Lymphocyte; TNFRSF5 gene; Testing; Therapeutic; Time; Toll-like receptors; Transgenes; Transgenic Mice; Transgenic Organisms; Vaccination; Virus Diseases; blastomere structure; cancer therapy; experience; immune activation; improved; in vivo; innovation; insight; interleukin-15 receptor; macrophage; migration; mouse model; neoplasm immunotherapy; novel; pathogen; prevent; promoter; protein complex; public health relevance; response; restoration; tool; tumor

 DESCRIPTION (provided by applicant): IL-15 is a major factor regulating the development and homeostasis of CD8 T cells and NK cells as well as their responses during immune stimulation. During the steady state, IL-15 responses are mediated via transpresentation, where cell surface IL-15, associated with IL-15Ra, stimulates neighboring cells during a cell- cell interaction. Conversely, studies have found that soluble (s) IL-15Ra/IL-15 complexes, with potent stimulatory activity, are produced by Toll-like receptor stimulation and after lymphodepletion in humans and mice. Additionally, we have recently shown that sIL-15Ra/IL-15 complexes are generated in response to viral infection, Interferon-a, and CD40 stimulation. Since cell surface IL-15Ra/IL-15 also increases under these conditions, the role of sIL-15Ra/IL-15 complexes is presently unclear. Because generation of sIL-15Ra/IL-15 complexes coincides with enhanced IL-15 responses, we hypothesize that sIL-15Ra/IL-15 complexes enhance lymphocyte responses during immune stimulation. Our long-term goals are to elucidate the role of sIL-15Ra/IL- 15 complexes generated in response to pathogen infections, vaccination, autoimmune diseases, and cancer so that we can identify novel ways to regulate CD8 T cells and NK cells. Previous studies suggest IL-15Ra/IL- 15 complexes are cleaved from the surface by the metalloproteinase ADAM17. Moreover, the cleavage site in the IL-15Ra and a critical amino acid required for cleavage has been identified. Therefore, the goal of these studies is to use this knowledge to generate transgenic (Tg) mice expressing a cleavage-resistant IL-15Ra that prevents production of sIL-15 complexes but leaves IL-15 transpresentation intact. This model system can then be used to distinguish the immunological roles of endogenously-produced soluble IL-15 complexes from those mediated by IL-15 transpresentation. To accomplish this goal, we propose the following two specific aims: 1. Develop genetically engineered mice expressing cleavage-resistant IL-15Ra. Constructs encoding cleavage-resistant IL-15Ra or Wt IL-15Ra driven by a MHC class I promoter will be inserted into IL- 15Ra-/- blastocytes for generation of Tg mice. Mice expressing the transgene at the DNA and protein level will be identified and bred to the IL-15Ra-/- background. 2. Determine effect of transgenic cleavage-resistant IL-15Ra on development of IL-15 responsive lymphocytes and expression of sIL-15 complexes. The general phenotype of T cells and NK cells will be examined in untreated cleavage-resistant IL-15Ra Tg+ and Wt IL-15Ra Tg mice to measure the restoration of IL-15 function in lymphocyte development and homeostasis. Expression of serum sIL-15Ra/IL-15 complexes will be measured in Tg+ mice after viral infection or other types of immune stimulation. Developing this model will allow us to identify the importance of sIL-15Ra/IL-15 complexes generated by various types of immune stimulation. The knowledge obtained will provide the critical insight needed to develop therapeutic strategies to promote IL-15 responses that can enhance immune responses to tumors or pathogens as well as block IL-15 responses that promote inflammatory events.

 描述（由申请人提供）：IL-15 是调节 CD8 T 细胞和 NK 细胞的发育和稳态及其在免疫刺激期间的反应的主要因素。在稳定状态下，IL-15 反应是通过转呈介导的。细胞表面 IL-15 与 IL-15Ra 相关，在细胞-细胞相互作用过程中刺激邻近细胞，研究发现可溶性 IL-15Ra/IL-15 复合物具有强效作用。此外，我们最近发现 sIL-15Ra/IL-15 复合物是响应病毒感染、干扰素-a 和 CD40 刺激而产生的。由于细胞表面 IL-15Ra/IL-15 在这些条件下也会增加，因此 sIL-15Ra/IL-15 复合物的作用目前尚不清楚。 sIL-15Ra/IL-15 复合物与增强的 IL-15 反应相一致，我们追求 sIL-15Ra/IL-15 复合物在免疫刺激过程中增强淋巴细胞反应，我们的长期目标是阐明 sIL-15Ra/IL 的作用。 - 因病原体感染、疫苗接种、自身免疫性疾病和癌症而产生的 15 种复合物，使我们能够找到调节 CD8 T 细胞和 NK 细胞的新方法。 IL-15Ra/IL-15复合物被金属蛋白酶ADAM17从表面裂解，而且，IL-15Ra中的裂解位点和裂解所需的关键氨基酸已被鉴定。这些知识可产生表达抗裂解 IL-15Ra 的转基因 (Tg) 小鼠，该小鼠可阻止 sIL-15 复合物的产生，但会导致 IL-15 转呈然后，该模型系统可用于区分内源产生的可溶性 IL-15 复合物与 IL-15 转呈介导的免疫学作用。 为了实现这一目标，我们提出以下两个具体目标： 1. 开发基因工程。表达抗切割 IL-15Ra 的小鼠将由 MHC I 类启动子驱动的编码抗切割 IL-15Ra 或 Wt IL-15Ra 的构建体插入 IL-15Ra 中。将鉴定在 DNA 和蛋白质水平上表达转基因的 15Ra-/- 胚细胞并培育至 IL-15Ra-/- 背景。 2. 确定转基因抗裂解 IL-15Ra 对发育的影响。 IL-15 反应性淋巴细胞的数量和 sIL-15 复合物的表达 将在未经处理的抗裂解 IL-15Ra 中检查 T 细胞和 NK 细胞的一般表型。 Tg+和Wt IL-15Ra Tg小鼠用于测量淋巴细胞发育和稳态中IL-15功能的恢复，将在病毒感染或其他类型的免疫刺激后测量Tg+小鼠中血清sIL-15Ra/IL-15复合物的表达。开发该模型将使我们能够确定各种类型的免疫刺激产生的 sIL-15Ra/IL-15 复合物的重要性，所获得的知识将为制定治疗策略提供关键的见解。促进 IL-15 反应，从而增强对肿瘤或病原体的免疫反应，并阻止促​​进炎症事件的 IL-15 反应。