Differential Mechanisms of Shedding of the Glycosylphosphatidylinositol (GPI)-anchored NKG2D Ligands

Abstract

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...

Comprehensive Analysis of NKG2D Ligand Expression and Release in Leukemia: Implications for NKG2D-Mediated NK Cell Responses

“…In our study, sNKG2DL were detectable in sera of patients without expression of the respective NKG2DL on the leukemic cells. NKG2DL can be released in soluble form by various mechanisms, including shedding from the cell surface by proteases, which may serve to explain this finding and indicates that leukemia cells may evade NKG2D-mediated immunosurveillance by reduction of cell surface NKG2DL density (19,27,(43)(44)(45)(46). Notably, all analyzed leukemia patient sera were positive for at least one of the five analyzed sNKG2DL, with sMICA being most frequently detectable and at significantly higher levels in acute as compared with chronic leukemias.…”

NK cells and cancer: you can teach innate cells new tricks

“…Furthermore, mice deficient for NKG2D (Klrk1 −/− ), or treated with an NKG2D-neutralizing antibody, are more susceptible to primary tumorigenesis 61,62 , confirming the crucial role of NKG2D in tumour immunosurveillance, although it has not yet been established whether NKG2D-mediated protection is conferred by NK cells, T cells or both. One of the mechanisms used by tumour cells to evade this surveillance is the shedding of NKG2DLs through cleavage by metalloproteinases, which decreases the amount of ligand at the tumour cell surface [63][64][65][66][67][68] (FIG. 2).…”

Tumor Suppressive MicroRNAs miR-34a/c Control Cancer Cell Expression of ULBP2, a Stress-Induced Ligand of the Natural Killer Cell Receptor NKG2D

“…Cancer cells often express a variety of NKG2DL, like melanoma cells that show a predominant expression of MICA and ULBP2 detectable in vitro and in situ (10)(11)(12). However, tumor cells can escape from NKG2D immune surveillance by an enhanced proteolytic shedding of NKG2DL (13)(14)(15). This shedding is most likely causative for the increased levels of soluble ligands in sera of cancer patients (16)(17)(18).…”