Elsevier

Cellular Immunology

Volume 239, Issue 1, January 2006, Pages 22-30
Cellular Immunology

Soluble ULBP suppresses natural killer cell activity via down-regulating NKG2D expression

https://doi.org/10.1016/j.cellimm.2006.03.002Get rights and content

Abstract

NKG2D is an activating receptor that is expressed on most natural killer (NK) cells and CD8+ T cells. MHC class I-related chain A(MICA) and UL16-binding protein (ULBP) 1, 2, and 3 are well-known ligands for NKG2D. Human gastric cancer cell lines, SNU216 and SNU638 cells which expressed UL16-binding protein (ULBP) were susceptible to NK cells in a NKG2D-dependent manner. However, SNU484 and SNU620 cells which had no ULBP on their surface were resistant to NK cells. ULBP 1, 2, and 3 are glycosylphosphatidylinositol (GPI)-anchored proteins which are sensitive to phosphatidylinositol-specific phospholipase C (PI-PLC). When SNU620 cells were treated with U73122, an inhibitor of PI-PLC, the surface expression of ULBP was elevated with increased NK susceptibility. Pre-incubating NK cells with culture supernatants of SNU620 or SNU638 cells, which contained soluble ULBP protein, reduced NK cell activity by decreasing surface expression of NKG2D in NK cells. Furthermore, recombinant ULBP-Fc induced the down-regulation of NKG2D expression in NK cells. Taken together, down-regulation of NKG2D by soluble ULBP provides a potential mechanism by which gastric cancer cells escape NKG2D-mediated attack by the immune cells.

Introduction

Natural killer (NK)1 cells are populations of lymphocytes that can recognize and lyse virally infected cells and tumor cells [1], [2], [3], [4]. NK killing activity against tumor cells is a process that results from counterbalance between inhibitory and positive signals. As the missing-self hypothesis postulates, loss of MHC class I molecules on target cells due to virus infection or tumor formation limits the inhibitory receptor function of NK cells to lyse the target cells [5], [6]. The immunoglobulin superfamily (KIR, killer cell immunoglobulin-like receptors) or the C-type lectin superfamily (Ly49 in mice and CD94/NKG2A in human) belongs to these inhibitory receptors [7], [8]. Natural cytotoxicity receptors (NCR), such as NKp46, NKp30, and NKp44 are Ig superfamilies and coupled to signal transducing adaptor proteins including CD3ζ, FcεRIγ, and KARAP/DAP12 that trigger cytolytic activity [9]. However, natural ligands recognized by NCR are still undefined, although putative viral ligands have been reported [10], [11]. Another NK receptor is NKG2D, a C-type lectin surface receptor which is associated with the transmembrane adapter protein DAP10 [12], [13]. DAP10 has a cytoplasmic domain with an YXXM motif, which binds to the p85 subunit of the PI-3 kinase [14]. In contrast to NCR, NKG2D is expressed on most NK cells, CD8 αβ T cells, and γδ T cells. NKG2D mediates NK cell activation by overcoming the inhibitory signals from self recognition [15], [16]. These characteristics demonstrate that NKG2D has a critical role in immune surveillance.

Recent studies reveal that MHC class I-like molecules are activating ligands for NKG2D receptor on NK cells. Among them, UL16-binding protein (ULBP) 1, 2, 3 and MHC class I-related chain molecule A (MICA) possess MHC class I-like alpha-1/alpha-2 and alpha-1/alpha-2/alpha-3 domains, respectively. In humans, stress-inducible MICA/B are expressed in certain epithelial origin tumors by stress signals [17], [18]. In contrast to the MICA/B, UL16-binding protein (ULBP) 1, 2, and 3 are expressed as glycosylphosphatidyl inositol (GPI)-linked proteins at cell surface of various type of cells and tissues [16], [19]. The ULBPs were identified to bind to a human cytomegalovirus protein, UL16 [15], [16], [30]. Recent studies indicate that ULBP mRNA is detected in a wide variety of tumors and cell lines, but is undetectable in several normal tissues [20]. In addition, soluble recombinant forms of ULBPs bind to human NK cells and stimulate NK cytotoxicity against tumor targets [21].

Overall NK cell activity is controlled by signal cross-talk from inhibitory and stimulatory receptors. Meanwhile, many malignant tumor cells express NKG2D ligands on their surface with reduced NKG2D expression on immune cells, suggesting that mechanisms of escaping from NKG2D/NKG2D ligand system may exist. Recently, it was reported that soluble MICA (sMICA) secreted by tumor cells down-modulated surface NKG2D expression on T cells to induce the functional impairment of anti-tumor immune effector cells [22], [23], [24]. Furthermore, another factor involved in NKG2D functional impairment is observed by TGF-β, secreted by tumor cells [25] even though the mechanisms for down-modulation of NKG2D by TGF-β are not clear yet. These data suggest that various proteins secreted by tumor cells are responsible for modulating NK cell activity by regulating surface expression of NKG2D.

In the present study, we analyzed gastric cancer cell lines for the expression of MICs and ULBPs. The functional consequences of ULBP expression by gastric tumor cells were evaluated in the interaction between tumor cells and NK cells. Soluble forms of ULBPs secreted by gastric tumor cells reduced NKG2D expression in NK cells with decreased NK cytotoxicity.

Section snippets

Cell culture and reagent

Human gastric tumor cell lines (SNU1, 16, 216, 484, 620, and SNU638) were purchased from Korea Cell Line Bank (Seoul, Korea). These cell lines were maintained in RPMI-1640 medium (Gibco, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Hyclone Laboratories, Inc., Logan UT), penicillin (100 U/ml), streptomycin (100 mg/ml), and l-glutamine (0.3 mg/ml) at 5% CO2 in a humidified incubator at 37 °C. Highly potent cytotoxic human NK cell line NK-92, originally isolated

Expression of ULBP in gastric tumor cells

To evaluate NKG2D ligands expression in gastric tumor cells, surface expression of ULBP was analyzed (Fig. 1A). SNU638 and SNU216 cells expressed low levels of ULBP1,2, and SNU638 cells expressed high level of ULBP3 on their surface. As a control, K562 cells expressed high levels of surface ULBP1,2 proteins. However, SNU484 and SNU620 cells did not express any ULBP protein. RT-PCR analysis showed that ULBP gene was expressed in all SNU gastric tumor cells (Fig. 1B). ULBP gene expression was

Discussion

Molecular regulation of NKG2D ligands is poorly understood. In human, stress-inducible MHC class I-related molecules, MICA/B are expressed in certain epithelial origin tumors by stress signals [27], [28]. Also, it was suggested that NKG2D ligands on tumor cells could be a target for immune recognition and elimination of malignant tumor cells. In contrast to the MICA/B, another NKG2D ligand, ULBP is expressed in a variety of tumors cell lines [16]. A recent report showed ULBP was also expressed

Acknowledgments

This work was partially supported by 21C Frontier Functional Human Genome Project (FG-3-1-04), 21C Frontier Stem Cell Research Project (SC13040) and M10416020004-05N1602-00420, from the Ministry of Science and Technology, Republic of Korea.

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