Regulation, Expression, and Function of NK Cell Receptor

人CD94/NKG2A是一种识别人类白细胞抗原-E的抑制性受体，由NK细胞和T细胞亚群表达。为了阐明CD94/NKG2A受体的细胞表面动力学，我们在大鼠嗜碱粒细胞白血病(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，并在与配体接触的部位聚集。富含CD94/NKG2A-EGFP的受体的流动性明显低于未连接的受体。我们观察到，不仅受体极化不需要脂筏，而且它们被排除在受体与配体接触的位置之外。此外，当CD94/NKG2A与激活受体共同作用时，通常在FcepsilonR1激活受体交联处观察到的脂筏斑块没有观察到。这些结果表明，CD94/NKG2A受体在连接部位的固定不仅促进抑制信号的维持，而且通过脂筏排斥防止激活信号复合体的形成。此外，我们已经证明了孤儿基因NKG2F可以以蛋白质的形式表达，这种表达似乎局限于细胞内的隔间，可能是因为它无法与CD94相关联。然而，它可以与DAP12结合，从而提供激活信号潜力。在基因调控方面，我们已经确定了人CD94的核心启动子，它含有保守的GAS/EBS元件，可以形成蛋白质-DNA复合体。这两个启动子以不同的方式调节CD94基因的表达，以响应IL-2或IL-15的处理。这两个启动子的确切作用以及调节它们的因素正在积极调查中。 众所周知，NK细胞很难转化，这一事实阻碍了NK细胞的生物学研究。为了克服这一点，我们为新推出的Amaxa核转移系统开发了程序，允许高效地转移NK细胞。

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.