Role of NKG2 Family Receptors in Regulating the Immune Response

必须严格调节免疫反应，一方面避免低反应性，另一方面避免过度炎症和自身免疫（高反应性）的发展。这种平衡至少部分是通过抑制信号对激活信号的抑制来实现的。理想情况下，这导致了对入侵者的充分免疫反应，没有过度和延长的炎症信号，促进自身免疫的发展。CD94/NKG2受体家族由具有激活或抑制潜能的成员组成。这些受体主要在NK细胞和CD8 T细胞亚群上表达，它们已被证明在调节对感染和致瘤细胞的反应中发挥重要作用。我们的研究探索了这些受体生物学的各个方面，包括配体和受体相互作用、信号传导、膜动力学和基因表达调控。我们目前的重点是在细胞生物学和分子水平上了解CD94/NKGA抑制受体如何以主导方式失活激活受体产生的信号，以及该受体通过何种机制运输以保持在细胞表面的持续存在。

Immune responses must be tightly regulated to avoid hypo-responsiveness on one hand or excessive inflammation and the development of autoimmunity (hyper-responsiveness) on the other. This balance is at least partially attained through the throttling of activating signals by inhibitory signals. This ideally leads to an adequate immune response against an invader without excessive and extended inflammatory signals that promote the development of autoimmunity. The CD94/NKG2 family of receptors is composed of members with activating or inhibitory potential. These receptors are expressed predominantly on NK cells and a subset of CD8 T cells, and they have been shown to play an important role in regulating responses against infected and tumorigenic cells. Our studies explore all aspects of the biology of these receptors, including ligand and receptor interaction, signaling, membrane dynamics, and regulation of gene expression. Our current emphasis is to understand, at the cell biology and molecular levels, how the the CD94/NKGA inhibitory receptor inactivates signals generated by activation receptors in a dominating manner and by what mechanism this receptor traffics so as to maintain constant presence on the cell surface. Expression of abundant levels of inhibitory receptors, one of which is CD94/NKG2A, must be maintained to suppress unwarranted activation in normal circumstances and to help regulate potentially overzealous responses in combating disease. To maintain cell surface CD94/NKG2A expression levels, NK cells must deal with the fact that CD94/NKG2A is constantly exposed to its ligand, HLA-E, expressed by surrounding cells. In many cases, ligand exposure tends to induce receptor downregulation. We know from previous studies that CD94/NKG2A is long lived and continuously recycles to the cell surface and that the interaction with ligand does not lead to its downregulation. We investigated CD94/NKG2A endocytosis and found that it occurs by an amiloride-sensitive, Rac1-dependent pinocytic process; however, it does not require clathrin, dynamin, ADP ribosylation factor-6, phosphoinositide-3 kinase or the actin cytoskeleton. Once endocytosed, CD94/NKG2A traffics to early endosomal antigen 1+, Rab5+ early endosomes. It does appear in Rab4+ early/sorting endosome, but, in the time period examined, fails to reach Rab11+ recycling or Rab7+ late endosomes or lysosome-associated membrane protein-1+ lysosomes. These results indicate that CD94/NKG2A utilizes a previously undescribed endocytic mechanism coupled with an abbreviated trafficking pattern, perhaps to insure surface expression. The uptake of solutes and particles by cells, as well as most surface receptors, occurs through pinocytosis. Macro- and micropinosomes share the characteristic of engulfment of the extracellular fluid, but are morphologically distinguished by their size, greater or lesser than 0.2 um, respectively. It is not yet clear that how many types of pinocytic vesicles exist and how many pathways are involved in their endocytosis; however, it is clear that distinguishing these pathways by the size of the endocytic vesicle utilized is somewhat arbitrary. Keeping this in mind, the endocytosis of CD94/NKG2A is clearly a fluid-phase pinocytic process as it is co-endocytosed with fluid-phase markers. The fact that CD94/NKG2A endocytic vesicles are much greater in diameter (0.5-1.5 um) than micropinosomes (<0.2 um) clearly lead us to morphologically characterize them as macropinosomes. The fact that internalized CD94/NKG2A colocalizes with dextran and lucifer yellow, and that its endocytosis is amiloride sensitive and Rac1 dependent supports this conclusion. The cell adhesion molecules ICAM-1 and platelet endothelial cell adhesion molecule-1 are internalized by a macropinocytic mechanism that is independent of clathrin, caveolin and PI3K activity; however, this internalization process requires actin and dynamin activity. The fact that CD94/NKG2A internalization does not require actin, dynamin or Arf6 and is insensitive to PI3K inhibition and PKC stimulation clearly distinguishes it from previously discussed macropinocytic mechanisms. However, despite the fact that CD94/NKG2A-containing endocytic vesicles are too large to fit the definition of a micropinocytosis, they do share, in addition to PI3K independence, the notable feature of actin independency. We arbitrarily chose to term CD94/NKG2A endocytosis as macropinocytic-like realizing that it is clearly biochemically distinguishable from previously described macropinocytic mechanisms. The CD94-NKG2 receptor family that regulates NK and T cells is unique among the lectin-like receptors encoded within the natural killer cell complex. The function of the CD94-NKG2 receptors is dictated by the pairing of the invariant CD94 polypeptide with specific NKG2 isoforms to form a family of functionally distinct heterodimeric receptors. However, the structural basis for this selective pairing and how they interact with their ligand, HLA-E, is unknown. We described the 2.5 resolution crystal structure of CD94-NKG2A in which the mode of dimerization contrasts with that of other homodimeric NK receptors. Despite structural homology between the CD94 and NKG2A subunits, the dimer interface is asymmetric, thereby providing a structural basis for the preferred heterodimeric assembly. Structure-based sequence comparisons of other CD94-NKG2 family members, combined with extensive mutagenesis studies on HLA-E and CD94-NKG2A, allowed for a model of the interaction between CD94-NKG2A and HLA-E to be established, in which the invariant CD94 chain plays a more dominant role in interacting with HLA-E in comparison to the variable NKG2 chain. Human NKG2D/DAP10 is an activation receptor expressed by NK and subsets of T cells, whose ligands include MHC class I chain-related (MIC) protein A and protein B and UL16-binding proteins that are often up-regulated by stress or pathological conditions. DAP10 is required for NKG2D/DAP10 cell surface expression and signaling capacity. Little is known about the mechanisms that regulate DAP10 gene expression. We described the existence of multiple transcriptional start sites upstream of DAP10 exon 1 and identified the location of the basic promoter upstream of these starting sites. The promoter is active in NK and CD8+ T cells, but not in CD4+ T cells. We demonstrated TCR-mediated up-regulation of DAP10 transcription and found that a 40 bp region within the DAP10 promoter, containing an Ap-1 binding site, is largely responsible for this increased transcription. Using pull-down and chromatin immunoprecipitation assays, we showed that the DAP10 promoter interacts with Ap-1 transcription factors in primary CD8+ T and NK cells in vitro and in vivo. Our data indicate that Ap-1 is an important transcription factor for regulating DAP10 expression in human NK and T cells.