Regulation, Expression, and Function of NK Cell Receptor

NK 细胞受体的调控、表达和功能

• 批准号: 6985736
• 负责人: JOHN COLIGAN
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6985736
• 关键词: MHC class I antigen; biological signal transduction; confocal scanning microscopy; enzyme linked immunosorbent assay; flow cytometry; genetic regulation; human tissue; immunologic receptors; immunoprecipitation; immunoregulation; interleukin 15; interleukin 2; intracellular transport; leukocyte activation /transformation; ligands; natural killer cells; protein structure function; receptor expression

Human CD94/NKG2A is an inhibitory receptor that recognizes HLA-E and is expressed by NK cells and a subset of T cells. To elucidate the cell surface dynamics of CD94/NKG2A receptors, we have expressed CD94/NKG2A-EGFP receptors in the rat basophilic leukemia (RBL) cell line. Photobleaching experiments revealed that CD94/NKG2A-EGFP receptors move freely within the plasma membrane and accumulate at the site of contact with ligand. The enriched CD94/NKG2A-EGFP is markedly less mobile than the nonligated receptor. We observed that not only are lipid rafts not required for receptor polarization, they are excluded from the site of receptor contact with the ligand. Furthermore, the lipid raft patches normally observed at the sites where FcepsilonR1 activation receptors are cross-linked were not observed when CD94/NKG2A was coengaged along with the activation receptor. These results suggest that immobilization of the CD94/NKG2A receptors at ligation sites not only promote sustenance of the inhibitory signal, but by lipid rafts exclusion prevent formation of activation signaling complexes. Also, we have shown that the orphan gene, NKG2F, can be expressed as protein, and that expression appears to be confined to intracellular compartments probably due to its inability to associate with CD94. It can however associate with DAP12 thereby providing activation signaling potential. In terms of gene regulation, we have identified the core promoters of human CD94, which contain conserved GAS/EBS elements that can form protein-DNA complexes. The two promoters differentially regulate expression of the CD94 gene in response to treatment with IL-2 or IL-15. Clarification of the precise roles of these two promoters and the factors that regulate them is under active investigation. Nk cells are notoriously difficult to transfect, a fact that has hampered NK cell biology studies. To overcome this, we developed procedures for the newly available Amaxa nucleofection system that allow for efficient transfection of NK cells.

人CD94/NKG2A是一种抑制性受体，识别HLA-E并由NK细胞和T细胞的子集表达。为了阐明CD94/NKG2A受体的细胞表面动力学，我们在大鼠嗜碱性白血病（RBL）细胞系中表达了CD94/NKG2A-EGFP受体。光漂白实验表明，CD94/NKG2A-EGFP受体在质膜内自由移动，并积聚在与配体接触位点。富集的CD94/NKG2A-EGFP明显少于非粘合受体。我们观察到，受体极化不仅需要脂质筏，还将它们排除在与配体接触的部位之外。此外，当CD94/NKG2A与激活受体一起促成CD94/NKG2A时，未观察到通常在FcePsilonR1激活受体与激活受体交联的位点上通常观察到的脂质筏斑。这些结果表明，在连接部位固定CD94/NKG2A受体不仅促进了抑制信号的寄生，而且还可以通过脂质筏排除来阻止形成激活信号复合物。另外，我们已经表明，孤儿基因NKG2F可以表示为蛋白质，并且该表达似乎仅限于细胞内室，这可能是由于其无法与CD94相关联。但是，它可以与DAP12相关联，从而提供激活信号电位。在基因调节方面，我们已经确定了人类CD94的核心启动子，这些核心启动子包含可以形成蛋白质-DNA复合物的保守气体/EBS元素。两个启动子对用IL-2或IL-15进行治疗的差异调节CD94基因的表达。阐明这两个启动子的精确作用以及调节它们的因素正在积极研究中。 NK细胞很难转染，这一事实阻碍了NK细胞生物学研究。为了克服这一点，我们开发了新近可用的Amaxa核反理系统的程序，以有效地转染NK细胞。