HER2/HER3 signaling regulates NK cell-mediated cytotoxicity via MHC class I chain-related molecule A and B expression in human breast cancer cell lines

Overexpression of the receptor tyrosine kinases HER2 and HER3 is associated with a poor prognosis in several types of cancer. Presently, HER2- as well as HER3-targeted therapies are in clinical practice or evaluated within clinical trials, including treatment with mAbs mediating growth inhibition and/or activation of Ab-induced innate or adaptive cellular immunity. A better understanding of how HER2/HER3 signaling in tumors influences cellular immune mechanisms is therefore warranted. In this study, we demonstrate that HER2/HER3 signaling regulates the expression of MHC class I-related chain A and B (MICA and MICB) in breast cancer cell lines. The MICA and MICB (MICA/B) molecules act as key ligands for the activating receptor NK group 2, member D (NKG2D) and promote NK cell-mediated recognition and cytolysis. Genetic silencing of HER3 but not HER2 downregulated the expression of MICA/B, and HER3 overexpression significantly enhanced MICA expression. Among the major pathways activated by HER2/HER3 signaling, the PI3K/AKT pathway was shown to predominantly regulate MICA/B expression. Treatment with the HER3-specific ligand neuregulin 1β promoted the expression in a process that was antagonized by pharmacological and genetic interference with HER3 but not by the ataxia-telangiectasia-mutated (ATM) and ATM and Rad3-related protein kinases inhibitor caffeine. These observations further emphasize that HER2/HER3 signaling directly, and not via genotoxic stress, regulates MICA/B expression. As anticipated, stimulating HER2/HER3 enhanced the NKG2D-MICA/B-dependent NK cell-mediated cytotoxicity. Taken together, we conclude that signaling via the HER2/HER3 pathway in breast carcinoma cell lines may lead to enhanced NKG2D-MICA/B recognition by NK cells and T cells.     

Alternatively spliced forms of <Emphasis Type="Italic">MICA</Emphasis> and <Emphasis Type="Italic">MICB</Emphasis> lacking exon 3 in a human cell line and evidence of presence of similar RNA in human peripheral blood mononuclear cells

MICA and MICB genes encode MHC class I chain-related proteins, which are polymorphic, do not appear to present peptides or associate with beta(2)-microglobulin, and are expressed predominantly in epithelial cells, endothelial cells, fibroblasts and several cultured cell lines. Alternatively spliced isoforms are known to exist for HLA-A and B, as well as HLA-G and the MHC class I-related gene, MR1. In the course of cloning MICA and MICB cDNA from the colon carcinoma cell line HCT 116, it was observed that two kinds of cDNAs were obtained: a 1161-bp cDNA, representing full-length MICA or MICB, and a shorter variant of 873 bp. The sequences of these short cDNAs were those of the correct MICA or MICB alleles but lacking exon 3. They were found in 7 of 72 clones examined or about 10% and were called MICA2 and MICB2. MICA1 and MICA2 were transfected into Chinese hamster ovary cells and found to be expressed both in the cells and on their surface. PCR with a primer based on a sequence formed by the joining of exons 2 and 4 allowed detection of the isoform RNA in different cells including freshly prepared normal PBMC.     

Intracellular retention of the MHC class I-related chain B ligand of NKG2D by the human cytomegalovirus UL16 glycoprotein

Infection by human CMV induces expression of the cellular MHC class I-related chain A (MICA) and chain B (MICB) surface proteins, which function as ligands for the activating NKG2D receptor. Engagement of NKG2D triggers NK cells and costimulates Ag-specific effector CD8 alphabeta T cells. The potency of MHC class I-related chain-NKG2D in stimulating these anti-viral immune responses may be countered by a CMV-encoded transmembrane glycoprotein, UL16, which specifically binds MICB as well as two of the UL16-binding proteins that are ligands of NKG2D. However, the function and significance of these interactions are undefined. Using a stably transfected B cell line, we show that expression of UL16 results in loss of surface MICB. This effect is caused by the failure of newly synthesized MICB to mature and transit the secretory pathway due to physical association with UL16. The intracellular retention of these protein complexes is mediated by a tyrosine-based motif in the cytoplasmic tail sequence of UL16, which determines localization to or retrieval from the trans-Golgi network. Deletion of this motif restores surface expression of MICB, whereas UL16 may be redirected to endosomal compartments. Predictably, the retention of MICB abrogates the stimulatory function of NKG2D. These results suggest a potential mechanism of viral immune evasion. However, this activity remains to be confirmed with CMV-infected fibroblasts or endothelial cells, in particular because MICB is normally coexpressed with MICA, which is not retained by UL16.     

Interaction of monocytes with NK cells upon Toll-like receptor-induced expression of the NKG2D ligand MICA

Reciprocal interactions between NK cells and dendritic cells have been shown to influence activation of NK cells, maturation, or lysis of dendritic cells and subsequent adaptive immune responses. However, little is known about the crosstalk between monocytes and NK cells and the receptors involved in this interaction. We report in this study that human monocytes, upon TLR triggering, up-regulate MHC class I-Related Chain (MIC) A, but not other ligands for the activating immunoreceptor NKG2D like MICB or UL-16 binding proteins 1-3. MICA expression was associated with CD80, MHC class I and MHC class II up-regulation, secretion of proinflammatory cytokines, and apoptosis inhibition, but was not accompanied by release of MIC molecules in soluble form. TLR-induced MICA on the monocyte cell surface was detected by autologous NK cells as revealed by NKG2D down-regulation. Although MICA expression did not render monocytes susceptible for NK cell cytotoxicity, LPS-treated monocytes stimulated IFN-gamma production of activated NK cells which was substantially dependent on MICA-NKG2D interaction. No enhanced NK cell proliferation or cytotoxicity against third-party target cells was observed after stimulation of NK cells with LPS-activated monocytes. Our data indicate that MICA-NKG2D interaction constitutes a mechanism by which monocytes and NK cells as an early source of IFN-gamma may communicate directly during an innate immune response to infections in humans.     

Soluble MICB in malignant diseases: analysis of diagnostic significance and correlation with soluble MICA

Expression of ligands of the immunoreceptor NKG2D such as MICA and MICB has been proposed to play an important role in the immunosurveillance of tumors. Proteolytic shedding of NKG2D ligands from cancer cells therefore constitutes an immune escape mechanism impairing anti-tumor reactivity by NKG2D-bearing cytotoxic lymphocytes. Serum levels of sMICA have been shown to be of diagnostic significance in malignant diseases of various origins. Here, we investigated the potential of soluble MICB, the sister molecule of MICA, as a marker in cancer and its correlation with soluble MICA. Analysis of MICB in sera of 512 individuals revealed slightly higher MICB levels in patients with various malignancies (N = 296; 95th percentile 216 pg/ml; P = 0.069) than in healthy individuals (N = 62; 95th percentile 51 pg/ml). Patients with benign diseases (N = 154; 95th percentile 198 pg/ml) exhibited intermediate MICB levels. In cancer patients, elevated MICB levels correlated significantly with cancer stage and metastasis (P = 0.007 and 0.007, respectively). Between MICB and MICA levels, only a weak correlation was found (r = 0.24). Combination of both markers resulted only in a slightly higher diagnostic power in the high specificity range. The reduction of MICA and MICB surface expression on cells by shedding and the effects of sMICA and sMICB in serum on host lymphocyte NKG2D expression might play a role in late stages of tumor progression by overcoming the confining effect of NK cells and CD8 T cells. While MICB levels are not suited for the diagnosis of cancer in early stages, they may provide additional information for the staging of cancer disease.Alexander Steinle and Helmut R. Salih contributed equally to this work.     

Human cytomegalovirus-encoded UL16 discriminates MIC molecules by their alpha2 domains

Human CMV infection results in MHC class I down-regulation and induction of NKG2D ligand expression favoring NK recognition of infected cells. However, human CMV-encoded UL16 counteracts surface expression of several NKG2D ligands by intracellular retention. Interestingly, UL16 interacts with MICB, but not with the closely related MICA, and with UL16-binding proteins (ULBP) ULBP1 and ULBP2, which are only distantly related to MICB, but not with ULPB3 or ULBP4, although all constitute ligands for NKG2D. Here, we dissected the molecular basis of MICA-MICB discrimination by UL16 to elucidate its puzzling binding behavior. We report that the UL16-MICB interaction is independent of glycosylation and demonstrate that selective MICB recognition by UL16 is governed by helical structures of the MICB alpha2 domain. Transplantation of the MICB alpha2 domain confers UL16 binding capacity to MICA, and thus, diversification of the MICA alpha2 domain may have been driven by the selective pressure exerted by UL16.     

The endogenous danger signals HSP70 and MICA cooperate in the activation of cytotoxic effector functions of NK cells

Although natural killer (NK) cells are often described as first line defence against infected or malignant cells which act without the need of prior activation, it is known now that the NK cell activity is tightly regulated by other cells and soluble factors. We show here that the stress‐inducible heat shock protein (HSP) 70 activates human NK cells to kill target cells expressing major histocompatibility complex class I chain‐related molecule A (MICA) in a natural killer group 2 member D (NKG2D‐) dependent manner. The HSP70‐derived peptide TKD (TKDNNLLGRFELSG) was able to replace the full‐length HSP70 and to exert the same function. Interestingly, the expression of the cytotoxic effector protease granzyme B in NK cells was increased after TKD stimulation. When MICA and MICB expression was induced in human tumour cells by a histone deacetylase inhibitor and NK cells were activated by HSP70 or TKD, both treatments jointly improved the killing of the tumour cells. Thus, the synergistic activity of two stress‐inducible immunological danger signals, HSP70 and MICA/B, leads to activation and enhanced cytotoxicity of human NK cells against tumour cells.     

Epigenetic Mechanisms Regulate MHC and Antigen Processing Molecules in Human Embryonic and Induced Pluripotent Stem Cells

Background

Human embryonic stem cells (hESCs) are an attractive resource for new therapeutic approaches that involve tissue regeneration. hESCs have exhibited low immunogenicity due to low levels of Mayor Histocompatibility Complex (MHC) class-I and absence of MHC class-II expression. Nevertheless, the mechanisms regulating MHC expression in hESCs had not been explored.

Methodology/Principal Findings

We analyzed the expression levels of classical and non-classical MHC class-I, MHC class-II molecules, antigen-processing machinery (APM) components and NKG2D ligands (NKG2D-L) in hESCs, induced pluripotent stem cells (iPSCs) and NTera2 (NT2) teratocarcinoma cell line. Epigenetic mechanisms involved in the regulation of these genes were investigated by bisulfite sequencing and chromatin immunoprecipitation (ChIP) assays. We showed that low levels of MHC class-I molecules were associated with absent or reduced expression of the transporter associated with antigen processing 1 (TAP-1) and tapasin (TPN) components in hESCs and iPSCs, which are involved in the transport and load of peptides. Furthermore, lack of β2-microglobulin (β2m) light chain in these cells limited the expression of MHC class I trimeric molecule on the cell surface. NKG2D ligands (MICA, MICB) were observed in all pluripotent stem cells lines. Epigenetic analysis showed that H3K9me3 repressed the TPN gene in undifferentiated cells whilst HLA-B and β2m acquired the H3K4me3 modification during the differentiation to embryoid bodies (EBs). Absence of HLA-DR and HLA-G expression was regulated by DNA methylation.

Conclusions/Significance

Our data provide fundamental evidence for the epigenetic control of MHC in hESCs and iPSCs. Reduced MHC class I and class II expression in hESCs and iPSCs can limit their recognition by the immune response against these cells. The knowledge of these mechanisms will further allow the development of strategies to induce tolerance and improve stem cell allograft acceptance.     

The Traffic of the NKG2D/Dap10 Receptor Complex during Natural Killer (NK) Cell Activation

NKG2D is an important activating receptor for triggering the NK cell cytotoxic activity, although chronic engagement of specific ligands by NKG2D is also known to provoke decreased cell surface expression of the receptor and compromised NK cell function. We have studied the dynamics of surface NKG2D expression and how exposure to the specific ligand major histocompatibility complex class I chain-related molecule B (MICB) affects receptor traffic and fate. While in the NKL cell line and “resting” NK cells NKG2D was found principally at the cell surface, in activated primary NK cells an intracellular pool of receptor could also be found recycling to the plasma membrane. Exposure of NK cells to targets expressing MICB resulted in degradation of ∼50% of total NKG2D protein and lysosomal degradation of the DAP10 adaptor molecule. Consistent with these observations, confocal microscopy experiments demonstrated that DAP10 trafficked to secretory lysosomes in both transfected NKL cells and in activated primary NK cells upon interaction with MICB-expressing target cells. Interestingly, polarization to the synapse of secretory lysosomes containing DAP10 was also observed. The implications of the intracellular traffic of the NKG2D/DAP10 receptor complex for NK cell activation are discussed. We propose that the rapid degradation of NKG2D/DAP10 observed coincident with recruitment of the receptor to the cytotoxic immune synapse may explain the loss of NKG2D receptor expression after chronic exposure to NKG2D ligands.The killer cell lectin-like receptor NKG2D is one of the best characterized NK³ cell-activating receptors. Signaling via NKG2D depends on its association with DAP10, a transmembrane adaptor molecule containing the sequence YINM, which signals via recruitment of phosphatidylinositol 3-kinase and Grb2 (growth factor receptor-bound protein 2) (1, 2). Effector cell activation mediated by NKG2D has been described as immune recognition of the “induced self,” because the cellular ligands for NKG2D (NKG2D-L): the polymorphic MHC class I chain-related molecules (MIC) A and MICB and the UL16-binding proteins are not normally expressed but instead are up-regulated on target cells after pathogen infection or tumor transformation to render these cells susceptible to NK cell lysis (3). Strikingly, however, although induced expression of NKG2D-L acts as a danger signal to provoke an immune response, a number of studies performed in mouse models have shown that chronic exposure to NKG2D-L can also lead to down-modulation of the surface expression of NKG2D and impaired NK cell cytotoxic function (4–6).In humans, a common feature of patients with multiple different tumors is the presence in the serum of high levels of soluble MICA and -B or UL16-binding proteins, released by tumor cells, that are associated with an impairment of CTL and NK cell cytotoxic function (7–10). These observations have been interpreted as suggesting that the release of soluble NKG2D-L is a strategy of tumor immune evasion (11). However, recent data show that receptor interaction with cell membrane-anchored MICB can also lead to impaired NKG2D function. We have shown that brief cytotoxic interactions between NK cells and MICB-expressing target cells trigger a synaptic interchange of NKG2D and MICB as well as a rapid down-modulation of surface NKG2D and compromised NK cell cytotoxicity suggesting that NKG2D traffic is rapidly altered upon recognition of MICB expressed on target cell (12).The surface level of a receptor is dictated by the relative rates of synthesis and transport to the plasma membrane and endocytosis, recycling, and degradation. The loss of cell surface NKG2D observed after NKG2D-L binding (7–10, 12) raises the question of what is the intracellular fate of the receptor on interaction with NKG2D-L. However, the traffic of this receptor has not been previously studied. Here we describe the dynamics of surface NKG2D expression and examine how cytotoxic interactions between NK cells and the MHC class I- 721.221 (here called 221) cells that express MICB (here called 221B) affect the traffic and fate of the NKG2D/DAP10 receptor complex. In NKL cells and resting primary NK cells NKG2D is mainly expressed at the cell surface; however, in activated primary NK cells an intracellular pool of receptor recycling to the cell surface is detected. During cytotoxic interactions the recognition of MICB expressed on target cells results in a rapid degradation of NKG2D/DAP10 that is associated with the traffic of DAP10 to secretory lysosomes (SLs) (13, 14). Our data provide new insights into the dynamics of NKG2D receptor expression in NK cells and suggest a plausible model to explain how chronic exposure to NKG2D-L could lead to NKG2D down-modulation and compromised NK cell function.     

Soluble Ligands for the NKG2D Receptor Are Released during Endometriosis and Correlate with Disease Severity

Background

Endometriosis is a benign gynaecological disease. Abundant bulk of evidence suggests that patients with endometriosis have an immunity dysfunction that enables ectopic endometrial cells to implant and proliferate. Previous studies show that natural killer cells have a pivotal role in the immune control of endometriosis.

Methods and Findings

This is a prospective laboratory study conducted in a tertiary-care university hospital between January 2011 and April 2013. We investigated non-pregnant, younger than 42-year-old patients (n= 202) during surgery for benign gynaecological conditions. After complete surgical exploration of the abdominopelvic cavity, 121 women with histologically proven endometriosis and 81 endometriosis-free controls women were enrolled. Patients with endometriosis were classified according to a surgical classification in three different types of endometriosis: superficial peritoneal endometriosis (SUP), ovarian endometrioma (OMA) and deep infiltrating endometriosis (DIE). Peritoneal fluid samples were obtained from all study participants during the surgery in order to detect soluble NKG2D ligands (MICA, MICB and ULBP-2). When samples with undetectable peritoneal fluid levels of MICA, MICB and ULBP-2 were excluded, MICA ratio levels were significantly higher in endometriosis patients than in controls (median, 1.1 pg/mg; range, 0.1–143.5 versus median, 0.6 pg/mg; range, 0.1–3.5; p=0.003). In a similar manner peritoneal fluid MICB levels were also increased in endometriosis-affected patients compared with disease-free women (median, 4.6 pg/mg; range, 1.2–4702 versus median, 3.4 pg/mg; range, 0.7–20.1; p=0.001). According to the surgical classification, peritoneal fluid soluble MICA, MICB and ULBP-2 ratio levels were significantly increased in DIE as compared to controls (p=0.015, p=0.003 and p=0.045 respectively). MICA ratio levels also correlated with dysmenorrhea (r=0.232; p=0.029), total rAFS score (r=0.221; p=0.031) and adhesions rAFS score (r=0.221; p=0.031).

Conclusions

We demonstrate a significant increase of peritoneal fluid NKG2D ligands in women with endometriosis especially in those cases presenting DIE. This study suggests that NKG2D ligands shedding is a novel pathway in endometriosis complex pathogenesis that impairs NK cell function.     

Reduced immune effector cell NKG2D expression and increased levels of soluble NKG2D ligands in multiple myeloma may not be causally linked

Background

There is limited understanding of the dysregulation of the innate immune system in multiple myeloma (MM). We analysed the expression of the activating receptor NKG2D on NK cells and T cells of MM patients and investigated the impact of soluble versus membrane-bound NKG2D ligands on the expression of NKG2D.

Design

NKG2D expression on NK cells and CD8+ αβ T cells from patients with MM or monoclonal gammopathy of uncertain significance and healthy controls was examined flow-cytometrically. Sera from patients and controls were analysed for soluble NKG2D ligands (sNKG2D ligands).

Results

Significantly fewer NK cells and CD8+ αβ T cells from patients expressed NKG2D compared to healthy controls (NK cells: median 54% interquartile range (IQR) 32–68 versus 71% IQR 44–82%, P = 0.017, CD8+ αβ T cells: median 63% IQR 52–81 versus 77% IQR 71–90%, P = 0.018). The sNKG2D ligand sMICA was increased in patients [median 175 (IQR 87–295) pg/ml] versus controls [median 80 (IQR 32–129) pg/ml, P < 0.001], but levels of sMICA did not correlate with NKG2D expression on effector cells. To elucidate the mechanism of NKG2D down-regulation, we incubated lymphocytes from healthy donors in the presence of sNKG2D ligands or in co-culture with MM cell lines. sNKG2D ligands in clinically relevant concentrations did not down-regulate NKG2D expression, but co-culture of effector cells with myeloma cells with high surface expression of NKG2D ligands reduced NKG2D expression significantly.

Conclusions

These results indicate that MM is associated with a significant reduction in NKG2D expression which may be contact-mediated rather than caused by soluble NKG2D ligands.     

A hypoxia-induced decrease of either MICA/B or Hsp70 on the membrane of tumor cells mediates immune escape from NK cells

Recent findings suggest that hypoxia of the tumor microenvironment contributes to immune escape from natural killer (NK) cell-mediated cytotoxicity. Heat shock protein 70 (Hsp70) and the stress-regulated major histocompatibility class I chain-related protein A and B (MICA/B) both serve as ligands for activated NK cells when expressed on the cell surface of tumor cells. Herein, we studied the effects of hypoxia and hypoxia-inducible factor-1α (HIF-1α) on the membrane expression of these NK cell ligands in H1339 with high and MDA-MB-231 tumor cells with low basal HIF-1α levels and its consequences on NK cell-mediated cytotoxicity. We could show that a hypoxia-induced decrease in the membrane expression of MICA/B and Hsp70 on H1339 and MDA-MB-231 cells, respectively, is associated with a reduced sensitivity to NK cell-mediated lysis. A knockdown of HIF-1α revealed that the decreased surface expression of MICA/B under hypoxia is dependent on HIF-1α in H1339 cells with high basal HIF-1α levels. Hypoxia and HIF-1α did not affect the MICA/B expression in MDA-MB-231 cells but reduced the Hsp70 membrane expression which in turn also impaired NK cell recognition. Furthermore, we could show that the hypoxia-induced decrease in membrane Hsp70 is independent of HIF-1α in MDA-MB-231. Our data indicate that hypoxia-induced downregulation of both NK cell ligands MICA/B and Hsp70 impairs NK cell-mediated cytotoxicity, whereby only MICA/B appears to be regulated by HIF-1α.     

Oxidative stress increases MICA and MICB gene expression in the human colon carcinoma cell line (CaCo-2)

The human major histocompatibility complex class I chain-related A gene (MICA) and the MICB gene are newly identified members of the major histocompatibility complex class I chain-related gene family. We demonstrate here that oxidative stress, induced by H(2)O(2), promoted MICA (2.2-fold) and MICB (3.8-fold) gene expression using the human colon carcinoma cell line (CaCo-2) and semi-quantitative RT-PCR.     

Generation of Cell Lines to Complement Adenovirus Vectors using Recombination-Mediated Cassette Exchange

Background

Adenovirus serotype 5 (Ad5) has many favourable characteristics for development as a gene therapy vector. However, the utility of current Ad5 vectors is limited by transient transgene expression, toxicity and immunogenicity. The most promising form of vector is the high capacity type, which is deleted for all viral genes. However, these vectors can only be produced to relatively low titres and with the aid of helper virus. Therefore a continuing challenge is the generation of more effective Ad5 vectors that can still be grown to high titres. Our approach is to generate complementing cell lines to support the growth of Ad5 vectors with novel late gene deficiencies.

Results

We have used LoxP/Cre recombination mediated cassette exchange (RMCE) to generate cell lines expressing Ad5 proteins encoded by the L4 region of the genome, the products of which play a pivotal role in the expression of Ad5 structural proteins. A panel of LoxP parent 293 cell lines was generated, each containing a GFP expression cassette under the control of a tetracycline-regulated promoter inserted at a random genome location; the cassette also contained a LoxP site between the promoter and GFP sequence. Clones displayed a variety of patterns of regulation, stability and level of GFP expression. Clone A1 was identified as a suitable parent for creation of inducible cell lines because of the tight inducibility and stability of its GFP expression. Using LoxP-targeted, Cre recombinase-mediated insertion of an L4 cassette to displace GFP from the regulated promoter in this parent clone, cell line A1-L4 was generated. This cell line expressed L4 100K, 22K and 33K proteins at levels sufficient to complement L4-33K mutant and L4-deleted viruses.

Conclusions

RMCE provides a method for rapid generation of Ad5 complementing cell lines from a pre-selected parental cell line, chosen for its desirable transgene expression characteristics. Parent cell lines can be selected for high or low gene expression, and for tight regulation, allowing viral protein expression to mirror that found during infection. Cell lines derived from a single parent will allow the growth of different vectors to be assessed without the complication of varying complementing protein expression.     

Intracellular retention of the NKG2D ligand MHC class I chain-related gene A in human melanomas confers immune privilege and prevents NK cell-mediated cytotoxicity

Most tumors grow in immunocompetent hosts despite expressing NKG2D ligands (NKG2DLs) such as the MHC class I chain-related genes A and B (MICA/B). However, their participation in tumor cell evasion is still not completely understood. Here we demonstrate that several human melanomas (cell lines and freshly isolated metastases) do not express MICA on the cell surface but have intracellular deposits of this NKG2DL. Susceptibility to NK cell-mediated cytotoxicity correlated with the ratio of NKG2DLs to HLA class I molecules but not with the amounts of MICA on the cell surface of tumor cells. Transfection-mediated overexpression of MICA restored cell surface expression and resulted in an increased in vitro cytotoxicity and IFN-gamma secretion by human NK cells. In xenografted nude mice, these melanomas exhibited a delayed growth and extensive in vivo apoptosis. Retardation of tumor growth was due to NK cell-mediated antitumor activity against MICA-transfected tumors, given that this effect was not observed in NK cell-depleted mice. Also, mouse NK cells killed MICA-overexpressing melanomas in vitro. A mechanistic analysis revealed the retention of MICA in the endoplasmic reticulum, an effect that was associated with accumulation of endoH-sensitive (immature) forms of MICA, retrograde transport to the cytoplasm, and degradation by the proteasome. Our study identifies a novel strategy developed by melanoma cells to evade NK cell-mediated immune surveillance based on the intracellular sequestration of immature forms of MICA in the endoplasmic reticulum. Furthermore, this tumor immune escape strategy can be overcome by gene therapy approaches aimed at overexpressing MICA on tumor cells.