The MHC class I-related chain (MIC) A and MICB ligands for the activating receptor NKG2D can be shed from tumor cells, and the presence of these soluble molecules in sera is related with compromised immune response and progression of disease. Recently, thiol disulphide isomerases and members of the AD-AM (a disintegrin and metalloproteinase) gene family were identified as key enzymes in mediating MICA/B shedding from cells. Here, we report shedding of the most frequently expressed MICA allele in human populations (MICA*008) into exosomes, small membrane vesicles that are secreted upon fusion with the plasma membrane. Although similar to other MICA/B molecules in the extracellular domain, the predicted transmembrane and cytoplasmic domains of MICA*008 are quite different, and this difference seemed to be critical for the mode of release from tumor cells. Treatment of natural killer (NK) cells with exosomes containing MI-CA*008 molecules not only triggered downregulation of NKG2D from the cell surface but also provoked a marked reduction in NK cytotoxicity that is independent of NKG2D ligand expression by the target cell. Our findings reveal a mechanism of NK suppression in cancer that may facilitate immune escape and progression.

Tumor cells release NKG2D ligands to evade NKG2D-mediated immune surveillance. The purpose of our investigation was to explore the cellular mechanisms of release used by various members of the ULBP family. Using biochemical and cellular approaches in both transfectant systems and tumor cell lines, this paper shows that ULBP1, ULBP2, and ULBP3 are released from cells with different kinetics and by distinct mechanisms. Whereas ULBP2 is mainly shed by metalloproteases, ULBP3 is abundantly released as part of membrane vesicles known as exosomes. Interestingly, exosomal ULBP3 protein is much more potent for down-modulation of the NKG2D receptor than soluble ULBP2 protein. This is the first report showing functionally relevant differences in the biochemistry of the three members of the ULBP family and confirms that in depth study of the biochemical features of individual NKG2D ligands will be necessary to understand and manipulate the biology of these proteins for therapy.NKG2D is an activating immune receptor that can be expressed by most cytotoxic lymphocytes, including NK and CD8ϩ T cells (1). Engagement of NKG2D by its ligands leads to the activation or co-stimulation of lysis and cytokine secretion (for review, see Ref. 2). In humans, NKG2D ligands (NKG2D-L) 5 occur in two families of proteins: the polymorphic family of MHC-I-related chain A/B (MICA/B) and the multigene family of UL16-binding proteins (ULBPs, also known as RAET1A-E). In total, 10 members of this gene family have been described, of which six can be expressed as functional proteins (3). Two members of the ULBP family have a transmembrane region (ULBP4 and -5), like MICA/B, whereas the other ULBP molecules are linked to the cell membrane via glycosylphosphatidylinositol (GPI) anchors. The existence of such a large number of ligands for a single receptor is not fully understood but may reflect a differential role for different ligands in immune surveillance or an evolutionary response to selective pressures exerted by pathogens or cancer.In general, NKG2D-L are not expressed ubiquitously; instead, they are expressed in response to several types of cellular stress, such as pathogen infection (4), DNA damage (5), proteasome inhibition (6), and tumor transformation (7). For example, MICA/B are expressed in epithelial tumors, melanoma, neuroblastoma, various hematopoietic malignancies, and carcinomas; ULBPs are found in leukemia, gliomas and melanomas. An additional complication is that mRNA can be found in many cells that do not express protein suggesting post-transcriptional regulation of NKG2D-L expression (8 -10).Mice deficient in NKG2D expression show an enhanced susceptibility to the development of tumors (11). However, shedding NKG2D-L as soluble molecules allows tumor cells to evade NKG2D surveillance. Apart from reducing NKG2D-L expression on the tumor cell surface, the release of soluble molecules may also impair immune surveillance by promoting down-regulation of NKG2D (12, 13). In fact, the sustained presence in vivo of NKG2D-L down-modulates...

MHC class I-related chain (MIC)AT he MHC class I-related chain (MIC) 4 A and B proteins are polymorphic MHC-related molecules that bind the activating receptor NKG2D. Engagement of NKG2D by these ligands leads to the activation of lysis and cytokine secretion by NK cells and T cells and thus plays a central role in immune system activation (for review, see Refs. 1 and 2). The vast majority of healthy cells do not express MIC molecules and instead their expression at the cell surface is up-regulated in pathological situations like cancer and autoimmunity; so, broadly, MICA/B expression is related to stress, although much remains unclear about the molecular mechanisms regulating their expression (3).The interaction between NKG2D and its ligands plays an important role in the immunosurveillance of cancer and tumor control. Thus, it is not surprising that tumor cells have evolved mechanisms to inhibit this system. Cytokines such as TGF-␤ can provoke down-regulation of NKG2D and its ligands (4, 5). Moreover, NKG2D ligands can be shed from tumor cells and have been detected in sera from patients with various types of cancer. Soluble NKG2D ligands appears to impair immune surveillance by promoting down-regulation of NKG2D (6), and high levels of soluble MIC (sMIC) molecules in sera correlate strongly with poor clinical outcome in patients suffering from various types of cancer, including colon (7) and prostate cancers (8). The mechanisms that regulate shedding of MI-CA/B are not well understood, but inhibition of cellular metalloproteinase activity markedly interferes with their release (9 -12), and recently shedding of MICA has also been shown to depend on ERp5, a thiol isomerase (13).In this study, we report that the ADAM (a disintegrin and metalloproteinase) family member ADAM17, also known as TNF-␣-converting enzyme (TACE), is involved in the shedding of soluble MICB and that regions of the membrane enriched in cholesterol and sphingolipids, also known as detergent-resistant membrane microdomains (DRMs), are an important site for this proteolysis. Materials and Methods Experimental proceduresA detailed description of the experimental procedures is provided in the supplemental data online. 5ELISA sMICB was detected using a sandwich ELISA procedure. Plates were coated with an anti-MICB mAb from R&D Systems (5 g/ml) and blocked with 2% BSA-PBS. Tissue culture supernatant was added for 1 h at 37°C. Bound MICB protein was detected using biotinylated goat anti-MICB (R&D Systems) followed by streptavidin-HRP (Amersham) and developed using the peroxidase substrate system (ABTS; Roche). The absorbance was measured at 410 nm with a reference wavelength of 490 nm. Samples were analyzed in duplicates. Under these conditions, the cutoff for detection of recombinant sMICB (BioSupply

Cancer cells’ ability to migrate through constricting pores in the tissue matrix is limited by nuclear stiffness. MT1-MMP contributes to metastasis by widening matrix pores, facilitating confined migration. Here, we show that modulation of matrix pore size or of lamin A expression known to modulate nuclear stiffness directly impinges on levels of MT1-MMP-mediated pericellular collagenolysis by cancer cells. A component of this adaptive response is the centrosome-centered distribution of MT1-MMP intracellular storage compartments ahead of the nucleus. We further show that this response, including invadopodia formation in association with confining matrix fibrils, requires an intact connection between the nucleus and the centrosome via the linker of nucleoskeleton and cytoskeleton (LINC) complex protein nesprin-2 and dynein adaptor Lis1. Our results uncover a digest-on-demand strategy for nuclear translocation through constricted spaces whereby confined migration triggers polarization of MT1-MMP storage compartments and matrix proteolysis in front of the nucleus depending on nucleus-microtubule linkage.