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

 描述（由申请人提供）：IL-15是调节CD 8 T细胞和NK细胞的发育和稳态以及它们在免疫刺激期间的应答的主要因子。在稳态期间，IL-15应答经由转表达介导，其中与IL-15 Ra相关的细胞表面IL-15在细胞-细胞相互作用期间刺激邻近细胞。相反，研究发现，具有有效刺激活性的可溶性IL-15 Ra/IL-15复合物通过Toll样受体刺激和在人和小鼠中淋巴细胞耗竭后产生。此外，我们最近发现，病毒感染、干扰素-a和CD 40刺激会产生sIL-15 Ra/IL-15复合物。由于在这些条件下细胞表面IL-15 Ra/IL-15也增加，因此sIL-15 Ra/IL-15复合物的作用目前尚不清楚。由于sIL-15 Ra/IL-15复合物的产生与增强的IL-15应答一致，我们假设sIL-15 Ra/IL-15复合物在免疫刺激期间增强淋巴细胞应答。我们的长期目标是阐明sIL-15 Ra/IL- 15复合物在病原体感染、疫苗接种、自身免疫性疾病和癌症中的作用，以便我们能够确定调节CD 8 T细胞和NK细胞的新方法。先前的研究表明IL-15 Ra/IL- 15复合物被金属蛋白酶ADAM 17从表面切割。此外，已经鉴定了IL-15 Ra中的切割位点和切割所需的关键氨基酸。因此，这些研究的目的是利用这些知识来产生表达抗切割IL-15 Ra的转基因（Tg）小鼠，该抗切割IL-15 Ra可阻止sIL-15复合物的产生，但保持IL-15转表达完整。然后，该模型系统可用于区分内源性产生的可溶性IL-15复合物的免疫作用与IL-15转表达介导的免疫作用。为实现这一目标，我们提出以下两个具体目标：1.开发表达抗切割IL-15 Ra的基因工程小鼠。将编码由MHC I类启动子驱动的抗切割IL-15 Ra或Wt IL-15 Ra的构建体插入IL-15 Ra-/-胚细胞中以产生Tg小鼠。将鉴定在DNA和蛋白质水平表达转基因的小鼠，并将其繁殖至IL-15 Ra-/-背景。2.确定转基因抗切割IL-15 Ra对IL-15应答性淋巴细胞发育和sIL-15复合物表达的影响。将在未处理的抗裂解IL-15 Ra Tg+和Wt IL-15 Ra Tg小鼠中检查T细胞和NK细胞的一般表型，以测量IL-15在淋巴细胞发育和稳态中功能的恢复。在病毒感染或其他类型的免疫刺激后，将在Tg+小鼠中测量血清sIL-15 Ra/IL-15复合物的表达。开发该模型将使我们能够确定由各种类型的免疫刺激产生的sIL-15 Ra/IL-15复合物的重要性。所获得的知识将提供开发治疗策略所需的关键见解，以促进IL-15反应，从而增强对肿瘤或病原体的免疫反应，并阻断促进炎症事件的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.