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

 描述(申请人提供)：IL-15是调节CD8T细胞和NK细胞的发育和动态平衡以及它们在免疫刺激过程中的反应的主要因素。在稳态中，IL-15的反应是通过转压介导的，其中细胞表面的IL-15与IL-15ra相关，在细胞与细胞的相互作用中刺激邻近细胞。相反，研究发现，具有强大刺激活性的(S)可溶性IL-15Ra/IL-15复合体是在人和小鼠淋巴枯竭后由Toll样受体刺激产生的。此外，我们最近发现，sIL-15Ra/IL-15复合体是在病毒感染、干扰素-a和CD40刺激下产生的。由于在这些条件下细胞表面IL-15Ra/IL-15也增加，sIL-15Ra/IL-15复合体的作用目前尚不清楚。由于sIL-15Ra/IL-15复合体的产生与增强的IL-15反应相一致，我们假设sIL-15Ra/IL-15复合体在免疫刺激过程中增强了淋巴细胞反应。我们的长期目标是阐明在病原体感染、疫苗接种、自身免疫性疾病和癌症中产生的sIL-15Ra/IL-15复合体的作用，以便我们能够找到调节CD8T细胞和NK细胞的新途径。以前的研究表明，IL-15Ra/IL-15复合体被金属蛋白酶ADAM17从表面切割。此外，还确定了IL-15Ra的切割位点和切割所需的关键氨基酸。因此，这些研究的目标是利用这一知识来产生表达抗切割的IL-15Ra的转基因(TG)小鼠，该基因可以阻止sIL-15复合体的产生，但保持IL-15的转压不变。这个模型系统可以用来区分内源性产生的可溶性IL-15复合体和由IL-15转压介导的免疫作用。为了实现这一目标，我们提出了以下两个具体目标：1.建立表达抗切割IL-15ra的基因工程小鼠。编码MHC-I类启动子驱动的抗切割IL-15Ra或Wt IL-15Ra的构建体将被插入到IL-15Ra-/-囊胚细胞中，用于生成TG小鼠。在DNA和蛋白质水平表达转基因的小鼠将被鉴定并培育成IL-15Ra-/-背景。2.检测转基因抗切割IL-15ra对IL-15反应性淋巴细胞发育和sIL-15复合体表达的影响。对未经处理的抗切割IL-15Ra TG+和Wt IL-15Ra TG小鼠的T细胞和NK细胞的一般表型进行检测，以衡量IL-15在淋巴细胞发育和动态平衡方面的功能恢复。在病毒感染或其他类型的免疫刺激后，将在TG+小鼠中检测血清sIL-15Ra/IL-15复合体的表达。开发这一模型将使我们能够识别由各种类型的免疫刺激产生的sIL-15Ra/IL-15复合体的重要性。所获得的知识将为开发促进IL-15反应的治疗策略提供必要的关键洞察力，这些反应可以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。

 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.