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, receptor synapses, lytic mechanisms, membrane dynamics, and regulation of gene expression. A 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. 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. We are curious to determine if there is a common theme to the trafficking a inhibitory receptors that might be related to their biological function? In our peliminary findings, as demonstrated for CD94/NKG2A, we have found that the leukocyte-associated Ig-like inhibitory receptor (LAIR)-1 and KIR2DL1 are continuously recycled between the cell surface and cytoplasm in a way that is independent of ligation. Despite the fact that these inhibitory receptors bind different ligands (collagen repeated motif for the former and HLA-CW4 for the latter), the fact that they appear to share a common trafficking pathway leads us to speculate that, in steady state conditions, this could represent a general mechanism that NK cells use to ensure the expression of the correct amount of inhibitory receptors on their surface. We are in the process of investgating this in great detail. 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 expression. In this study, for both primary NK cells and the NKL cell line, we show that NKG2D/DAP10 cell surface expression is differentially regulated by IL-2 or TGF-beta stimulation, and is correlated with DAP10 protein levels. IL-2 stimulation greatly increases DAP10 protein levels, but not mRNA. In contrast, the pleiotrophic cytokine, TGF-beta appears to function by suppressing DAP10 transcription by inhibiting recruitment of RNA polymerase II to the DAP10 promoter. Interestingly, when IL-2 plus TGF-beta are added to the cultures, TGF-beta has a dominant effect and the net result is a decrease in the levels of both DAP10 mRNA and protein. Moreover, we found that glycosylation of DAP10 serves to stabilize DAP10 and is necessary for its interaction with NKG2D. Therefore, collectively, cell surface expression of NKG2D is regulated by glycosylated DAP10 protein levels and IL-2 post-transcriptionally enhances DAP10 expression, whereas TGF-beta transcriptionally suppresses its expression (manuscript in preparation). The current emphasis is to understand first the role of LAMP1 in cytotoxicity of human NK cells. To investigate the role of LAMP1 protein, RNAi was used to disrupt LAMP1 expression and assess its effects on lytic granule exocytosis and NK cell cytotoxic potential. Stable NK cell lines carrying short hairpin (sh)RNA constructs specific for human LAMP1 were generated. We established that RNAi-mediated knock-down of LAMP1 completely inhibited NK cell cytotoxicity against susceptible target cells. Previous reports indicated that LAMP1 (and LAMP2) are involved in adhesion of peripheral blood mononuclear cell to the vascular endothelium. We found, however, that LAMP1 knock-down had no effect on conjugate formation between NK and target cells, indicating that LAMP1 do not contribute significantly to the adhesion of NK cells to target cells, and the inhibition of NK cell cytotoxicity in LAMP1 knock-down cells was not due to impaired adhesion and conjugation. Importantly, we discovered that despite normal conjugation, the amount of granzyme B delivered to target cells in LAMP1-deficient cells was substantially lower than in normal NK cells. This phenomenon was not due to decreased levels of granzyme B mRNA or protein, or the proteolytic activity of granzyme B, as these factors were not affected by the knock-down of LAMP1. Rather, the defective degranulation of LAMP1 RNAi cells and, consequently, impaired delivery of granzymes to target cell contributed to the inability of LAMP1 RNAi cells to lyse the susceptible targets. Furthermore, we observed that while LAMP1 RNAi cells were able to polarize the lytic granules toward the cell-cell contact site, the granules were substantially more dispersed and did not coalesce around the MTOC as the granules from untransduced or control shRNA-transduced cells. These results suggest that disruption of LAMP1 expression impairs the movement of the granules along the microtubules and therefore contributes to defective lytic activity of NK cells. Moreover, we found that LAMP1 RNAi cells had altered levels of perforin. Whereas mRNA levels of perforin in LAMP1 RNAi cells were normal, the protein level of perforin was decreased in these cells. At this point it is not clear whether the decrease of perforin protein levels is caused by deficient transport of perforin to lysosomes or increased degradation of the protein. Nevertheless, the decrease in the intracellular levels of perforin could provide an explanation for the defective delivery of granzyme B to target cells observed in LAMP1 RNAi cells. In addition, we found that knock-down of LAMP1 negatively affects the ability of NK cells to secrete IFN-gamma, indicating that LAMP1 is also involved in cytokine secretion in NK cells. However, the mechanism of this phenomenon is unknown and requires further experimentation. We postulate that LAMP1 is not only a convenient marker of NK cell degranulation, but plays a crucial role in NK cell activity and disruption of LAMP1 function has pleiotropic effects on NK cell lytic activity.

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Peruzzi, Giovanna; Femnou, Laurette; Gil-Krzewska, Aleksandra et al. (2013) Membrane-type 6 matrix metalloproteinase regulates the activation-induced downmodulation of CD16 in human primary NK cells. J Immunol 191:1883-94
Park, Yuk Pheel; Choi, Seung-Chul; Kiesler, Patricia et al. (2011) Complex regulation of human NKG2D-DAP10 cell surface expression: opposing roles of the ?c cytokines and TGF-?1. Blood 118:3019-27
Biassoni, Roberto; Coligan, John E; Moretta, Lorenzo (2011) Natural killer cells in healthy and diseased subjects. J Biomed Biotechnol 2011:795251
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Masilamani, Madhan; Narayanan, Sriram; Prieto, Martha et al. (2008) Uncommon endocytic and trafficking pathway of the natural killer cell CD94/NKG2A inhibitory receptor. Traffic 9:1019-34