Sample drift during acquisition was calculated and subtracted by reconstructing dSTORM images from subsets of frames (500 frames) and correlating these images to a reference frame (the initial time segment)

Sample drift during acquisition was calculated and subtracted by reconstructing dSTORM images from subsets of frames (500 frames) and correlating these images to a reference frame (the initial time segment). that NKG2DLs are not equivalent in their capacity to activate NKG2D and establish a previously unknown paradigm in how ligand-induced changes to the nanoscale organization of the cell surface can affect immune responses. Introduction Natural killer (NK) cells are lymphocytes that recognize and kill virally infected, stressed, or transformed cells and contribute to an immune response through the secretion of many cytokines and chemokines (1). Their activity is determined by the balance of signals from germ-line encoded inhibitory and activating receptors present around the cell surface (2C4). Inhibitory receptors include killer immunoglobulin (Ig)-like receptors (KIRs), which recognize self-major histocompatibility complex (MHC) class I molecules on healthy target cells and safeguard them from being killed by NK cells (5, 6). KIRs consist of a single polypeptide that contains immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in the cytoplasmic domain name that recruit tyrosine phosphatases, such as SHP-1, upon receptor ligation (7C10). One of the best characterized activating receptors on NK cells is the C-type, lectin-like, transmembrane receptor natural killer group 2D (NKG2D). In humans, NKG2D is usually a disulfide-linked homodimer present on the surface of all NK cells, most NKT cells, a subset of T cells, all CD8+ T cells, and a subset of CD4+ T cells (11C13). Signaling downstream of activating receptors commonly involves phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) that are either part of the receptor or present within associated adaptor proteins. However, NKG2D signals through the associated adaptor DAP10, which contains a costimulatory phosphatidylinositol-3-kinase (PI3K)Cbinding motif (YxNM) in its cytoplasmic domain name. DAP10 recruits PI3K and the adaptor Grb2 and then Grb2 recruits the guanine nucleotide exchange factor Vav1 (14, 15). In humans, NKG2D recognizes a Mouse monoclonal to ApoE set of ligands distantly related to MHC class I molecules that can be divided into two families: MHC class ICrelated protein A (MICA) and MICB, and UL16-binding proteins (ULBP1 to ULBP6) (11, 16C18). These ligands are rarely present on healthy cells but are induced or increase in abundance on infected or transformed cells rendering these cells susceptible to NK cellCmediated cytotoxicity (19C23). The abundance of NKG2D ligands is also increased on murine and human macrophages stimulated by a high dose of pathogen molecules, such as bacterial lipopolysaccharide (LPS) (24, 25). The different NKG2D ligands (NKG2DLs) are structurally diverse and have different patterns of expression and mechanisms of regulation. The reason for the presence of many NKG2DLs is not clear. One possibility is usually that they appeared during evolution of the immune system in response to selection pressures exerted by pathogens or cancer. Diversity in the ligands that activate NKG2D might make it harder for pathogens to interfere with this aspect of the immune response. Boc-D-FMK It is also possible that different ligands bind to NKG2D with varying affinities; thus, this diversity might fine-tune the extent of activation, perhaps in a tissue-specific manner. A major unknown for understanding NKG2D-mediated immune responses is whether the different ligands are qualitatively and quantitatively equivalent in their capacity to trigger cellular Boc-D-FMK activation. Interleukin-15 (IL-15) is an essential cytokine for the development, maintenance, and survival Boc-D-FMK of NK cells (26C29); some of its effects overlap with those of IL-2. IL-15 and IL-2 receptors have a similar structure: Both cytokine receptors form complexes that share a -chain (IL-2/IL-15R; also known as CD122), a common cytokine receptor -chain (c; also known as CD132), and distinct -chains. IL-15 increases the abundance of NKG2D and DAP10 in the NK cell surface membrane in vitro (17, 30C33). In mice, activation of the IL-15 receptor (IL-15R) results in phosphorylation of DAP10 by the kinase JNK3, thus priming DAP10 for NKG2D-dependent signaling (34). Thus, in addition to its well-established role in the survival and Boc-D-FMK proliferation of NK cells, IL-15 primes NK cell cytotoxicity induced by NKG2D. However, whether direct cross-talk between NKG2D and.