Immunology and Cell Biology (2014) 92, 230–236 & 2014 Australasian Society for Immunology Inc. All rights reserved 0818-9641/14 www.nature.com/icb

REVIEW

Advances in NKG2D ligand recognition and responses by NK cells Nina Le Bert and Stephan Gasser The natural killer (NK) group 2 member D (NKG2D) is an activating immune receptor expressed on NK cells, cytotoxic T cells and a subset of other T cells. It has an important role in the recognition and lysis of a variety of infected and tumor cells. Despite significant gains in our understanding of NKG2D, the relevance of NKG2D and its ligands in human diseases has only recently started to emerge. Here, we present an overview of the recent advances in NKG2D biology, discuss the expression of NKG2D ligands in cancer patients and evaluate the diagnostic and prognostic potential of NKG2D ligands. Immunology and Cell Biology (2014) 92, 230–236; doi:10.1038/icb.2013.111; published online 21 January 2014 Keywords: NK cells; NKG2D; innate immunity; cancer

Natural killer (NK) cells are lymphocytes of the innate immune system, which can directly eliminate target cells and secrete cytokines that assist in establishing an adaptive immune response.1 NK cell activation is regulated by a balance between inhibitory and activating receptors. Inhibitory receptors engage molecules present on normal host cells including class I major histocompatibility complex (MHC), human class I leukocyte antigen and Clr-b/Ocil, a member of a distinct family of lectin-like cell-surface glycoproteins.2,3 Ligands for inhibitory receptors have a crucial role in the recognition of tumor cells and infected cells that often lose or downregulate the expression of class I MHC molecules, referred to as ‘missing self-recognition’, presumably as a means for these cells to evade an adaptive immune response.4 In contrast, activating receptors engage with host-encoded ligands that are induced on diseased cells (induced self-recognition), but are absent or expressed at low levels on healthy cells.5 Tumorigenesis, infection and other insults activate molecular stress pathways, which regulate expression of self-ligands. This review will focus on the best-characterized activating receptor, the NK group 2 member D (NKG2D), which is expressed on all NK cells, CD8 þ T cells in humans, activated CD8 þ T cells in mice, CD4 þ T cells under some conditions and subsets of both gd and NK T cells.6,7 NKG2D The activating immune receptor NKG2D is a lectin-like type 2 transmembrane glycoprotein.8 It lacks signaling elements in the cytoplasmic domain, but forms a stable complex with the adapter molecule DNAX-activating protein (DAP) 10, carrying the activation sequence YXXM.9 Disulfide-linked homodimers of NKG2D associate with four chains of DAP10, which are required for stable cell-surface expression of the receptor.10 Upon receptor ligation, DAP10 recruits the p85 subunit of phosphatidylinositol 3-kinase (PI3K) and the growth

factor receptor-bound protein 2, both triggering signaling cascades leading to NK cell degranulation and cytotoxicity, as well as activation of survival pathways and cytokine production.9,11,12 In activated mouse NK cells, NKG2D associates with DAP10 and DAP12, which triggers Syk family protein tyrosine kinases.13–15 Binding of NKG2D to its ligands is sufficient to induce NK cell activation even in the presence of inhibitory signals, such as MHC molecules, although naive human NK cells require the engagement of additional activating receptors.16 Several cytokines, such as interleukin (IL)-2, IL-7, IL-15 and type I interferons (IFNs), upregulate the expression of NKG2D on the surface of NK cells and some of these proinflammatory cytokines were shown to change NKG2D-mediated signaling.12,17–20 In contrast, IL-21, IFN-g, transforming growth factor microRNAs and engagement of NKG2D by its ligands (see below) were shown to downregulate the expression of NKG2D.21–26 NKG2D LIGANDS NKG2D binds to a number of structurally diverse ligands with affinities ranging from 6 to 9000 nM.6,7,27,28 In normal cells, NKG2D ligands are absent or poorly expressed, although expression of MHC class-I-related chains A (MICA) was reported on some gastrointestinal epithelial cells. In contrast, broad expression of NKG2D ligands is found in a wide range of malignancies, including cancer, infection and autoimmunity.1 In humans, two families of NKG2D ligands have been described: MIC proteins and retinoic acid early transcripts-1 (RAET1), also called UL-16 binding proteins. The RAET1 family is shared between mice and humans, while no orthologs of the MIC genes MICA or MICB have been found in the mouse genome. The mouse Raet1 genes consist of five Rae-1 isoforms (Rae-1a, Rae-1b, Rae-1g, Rae-1d and Rae-1e), three H60 isoforms (H60a, b and c) and one Mult1 gene. All NKG2D ligands

Immunology Programme, Department of Microbiology, National University of Singapore, Singapore Correspondence: Dr S Gasser, Immunology Programme, Department of Microbiology, National University of Singapore, Singapore 117456, Singapore. E-mail: [email protected] Received 13 October 2013; revised 9 December 2013; accepted 9 December 2013; published online 21 January 2014

Advances in NKG2D biology N Le Bert and S Gasser 231

identified to date are distant homologs of class I MHC molecules and are located on human chromosome six and mouse chromosome ten. Unlike classical class I MHC proteins, ligands for NKG2D do not present antigen to T cells or bind b2-microglobulin. MICA and MICB are located within the MHC region and are transmembrane molecules with three a-domains analogous to class I MHC. Both MICA and MICB are highly polymorphic; 80 alleles of MICA and 13 alleles of MICB have been described so far.29 The second family of human NKG2D ligands consisting of six members of the ULBP family (ULBP1-6) is located on the other side of the centromere on chromosome six. In contrast to MICA and MICB, UL16 binding proteins only contain two domains similar to a1 and a2 of class I MHC proteins, while the a3 domain is absent. ULBP4 and 5 are transmembrane molecules, whereas ULBP1, ULBP3 and ULBP6 are glycophosphatidylinositol-linked cell membrane proteins. ULBP2 has been shown to exist as a transmembrane protein and a glycophosphatidylinositol-anchor molecule on the same cell.30 REGULATION OF NKG2D LIGAND EXPRESSION IN TUMOR CELLS Regulation of NKG2D ligand expression occurs at different levels including transcription, mRNA stability, translation and protein turnover (Regulation of NKG2D ligand expression was recently reviewed and we refer the interested reader to Raulet et al.7). Transcription of mouse and human RAET1/ULBP family members and possibly MICA and MICB is induced by activating E2F transcription factors in response to hyperproliferation.31 Heat shock was also shown to regulate the transcription of MICA and MICB, while no upregulation was observed for ULBP members. In agreement with this observation, heat-shock response elements were characterized in the promoter of both MICA and MICB.32,33 In contrast, the DNA damage response induces the transcription of ULBP1 and ULBP2, while also stabilizing Rae-1 transcripts and possibly transcripts of other NKG2D ligands.7,34–36 Our data suggest that regulation of NKG2D ligands in response to DNA damage depends on the accumulation of cytosolic DNA and the induction of the nucleotide sensing pathways, which activate the TANK-binding kinase 1 and the IFN regulatory factor 3.37 Numerous instances have been reported in which MICA, MICB and ULBP3 transcripts are targeted by viral and cellular microRNAs.38–42 Inhibition of PI3K, a kinase often activated in virus-infected cells and tumor cells, impaired Raet1 transcript levels and an even stronger effect was observed at the protein level suggesting that PI3K regulates Raet1 at the transcriptional and post-transcriptional stage.43 In support of posttranscriptional regulation of Raet1 by PI3K, we recently demonstrated that H-RASV12 induced RAE-1 translation depended on PI3K.44 In addition to its role in transcriptional regulation of MICA and MICB, heat shock counteracts ubiquitin-mediated degradation of MULT1.45,46 Moreover, NKG2D ligands including MICA, MICB and ULBP2 are cleaved from the cell surface by ADAM10, ADAM17 and MMP14, members of the disintegrin and metalloproteinase (ADAM) family, the levels of which correlate with poor survival of cancer patients.47–53 Cleavage of MICA also depends on the membrane-associated disulfide isomerase endoplasmic reticulum protein 5 (ERP5), which is also upregulated in cancer patients and may induce conformational changes required for efficient cleavage by proteases.54,55 ADAM family members cleave the entire ectodomain of MICA, and the shedded form of MICA is soluble and highly glycosylated.51 In addition to proteolytic cleavage from the cell surface, NKG2D ligands have also been found to be excreted in exosomes, and exosomes containing ULBP3 or

MICA*008, one the most frequently expressed alleles of MICA, strongly suppress NKG2D expression and NK cell cytotoxicity.56,57 Finally, the expression of NKG2D ligands on tumors may also be modulated by IFN-g, IFN-a, transforming growth factor-b and IL-10, although the mechanisms underlying the downregulation have not been elucidated in detail.23,58,59 Tumors may use combinations of mechanisms to downregulate the expression of NKG2D ligands. Malignant gliomas were shown to use transforming growth factor-b and metalloproteinases to reduce the expression of NKG2D ligands.58 In summary, various signals and pathways, many of which are associated with tumorigenesis and infections, regulate the gene expression of NKG2D ligands at different levels. NKG2D LIGAND EXPRESSION IN HUMAN CANCER Most of the current knowledge regarding the regulation and expression of NKG2D ligands has been gained through the use of cancer cell lines and mouse models of cancer. One of the first studies on NKG2D ligand expression in human cancer specimens found that MICA/B was expressed by freshly isolated epithelial carcinomas of lung (2/10), breast (2/10), kidney (2/2), ovary (5/6), prostate (2/4) and colon (4/5), whereas healthy tissue samples were negative (Table 1).60 MICA/B expression correlated with increased numbers of Vd1 gd T cells that were cytotoxic against MICA- and MICB-transfected cell lines. In subsequent studies, MICA or MICB was found to be expressed by 100% of colorectal tumors, 95% of renal cell carcinomas, 97% of breast cancers, 20% of squamous cervical carcinomas, 30% of CD34 þ cells in patients with myelodysplastic syndrome, 77% of primary cutaneous and 50% of primary uveal melanomas, but was reduced or absent from metastatic melanoma lesions.61–67 Interestingly, the study of uveal melanoma patients suggested that NKG2D ligands might be re-expressed on metastatic lesions after the patients received chemotherapy, possibly due to upregulation of the DNA damage response.61 Where tested, the authors found no MICA or MICB expression in normal tissue from which the tumors originated. In squamous cervical carcinomas, breast cancers and renal cell carcinomas, the number of tumor infiltrating NK cells did not correlate with MICA levels, which was in line with a recent report showing that recruitment of NK cells to mouse liver carcinoma cells depends on p53-induced expression of chemokines, but not NKG2D ligands.64,65,68 Another study found that glioma cells express ULBP1-3, in addition to MICA and MICB, whereas normal brain tissue did not express detectable NKG2D ligands.58 The expression of MICA and ULBP2, but not MICB, ULBP1 or ULBP3, decreased with tumor grade. In vitro studies suggested that MICA and ULBP2, but not other NKG2D ligands, are susceptible to downregulation by transforming growth factor-b and cleavage by metalloproteinase. ULBP2 was also detected in 82.9% of ovarian cancer samples, 97.6% of which also expressed MICA.69 Leukemia cells expressed multiple NKG2D ligands, including MICA, MICB and ULBP1-3. At least one ligand was expressed by 75% of 205 analyzed samples, out of which about 60% stained positive for two to five different NKG2D ligands. Interestingly, acute myeloid leukemia cells expressed four to five different NKG2D ligands and were previously shown to be more susceptible to NK cell lysis than acute lymphoid leukemia cells, which mostly expressed one or two, but never more than three different NKG2D ligands.70 Overall, MICA was the most frequently expressed ligand (56%), whereas ULBP2 was the least expressed ligand (23%) on leukemia cells. A few studies have linked the expression of NKG2D ligands to survival of cancer patients.50,52,63,66,69,71,72 The levels of MICA expression in 449 colorectal cancer patients correlated positively Immunology and Cell Biology

Advances in NKG2D biology N Le Bert and S Gasser 232

Table 1 NKG2D ligand expression on tumors or presence of soluble ligands in serum of cancer patients correlates with disease progression Ligand

Patient

expression

number

Cell surface

530

Correlation with clinical features

Ref

Breast

97% of tumors were MICA þ . MICA expression levels correlated with poor prognosis. MICA þ .

63

449

Colorectal

100% of tumors were Improved disease-specific survival correlated with higher expression levels of MICA. Independent prognostic significance in multivariate analysis.

66

205

Leukemia

75% expressed MICA, MICB, ULBP1, ULBP2 and/or ULBP3. Levels did not correlate with survival or disease stage.

83

148 140

Pancreatic Renal cell carcinoma

89.3% of tumors were MICA þ . Prognostic factor for resected pancreatic cancer. 495% of tumors were MICA/B þ .

87

Ovarian Myelodysplastic syndrome

97.6% of tumors were MICA/B þ , 82.9% of tumors were ULBP2 þ . 30% were MICA þ and/or MICB þ . Impaired NKG2D function in all patients.

69

82 48 40 38

MICA/B þ .

65

67 62

Cutaneous melanoma Glioma

31 of 40 primary tumors and 13/20 metastatic lesions were MICA, MICB and ULPB1-3 were upregulated in tumor cells. MICA and ULBP2

HPV-induced squamous

expression decreased with increasing WHO grade of malignancy. Increased MICA levels in 3/15 patients. ULBP2 expression decreased with tumor

9/11

cervical carcinoma Uveal melanoma

development. 50% of primary tumors expressed MICA/B. Cutaneous metastases were MICA/B.

512

Various

Levels of sMICA were significantly increased (Po0.0001) in patients with various malignancies and correlated with cancer stage (Po0.015) and metastasis (Po0.007).

73

512

Various

Levels of sMICB were significantly increased (Po0.069) in patients with various malignancies and correlated with cancer stage (Po0.007) and metastasis (Po0.007).

74

183

Leukemia

100% of patient sera contained sMICA, sMICB, sULBP1, sULBP2 and/or sULBP3. In AML sMICB and sULBP2 correlated negatively with survival. In CLL, sULBP2 correlated

83

148

Pancreatic

with advanced disease. 78.6% of cancer patient sera were sMICA þ . Level correlated with cancer

Stage IV oral squamous cell

progression and metastasis. Patients with stage IV disease and/or regional lymph node metastasis exhibited

15

Soluble

Type of cancer

113

58

64

61

87

78

carcinoma

significantly elevated levels of sMICA. Inverse correlation between overall survival rates and sMICA levels.

98

Chronic lymphocytic leukemia

Levels of sMICA, sMICB and sULPB2 were increased (Po0.0001). High levels correlated with poor survival.

77

97

Multiple myeloma

Levels of sMICA were significantly increased in patients. sMICA was an independent predictive factor for overall (P ¼ 0.007) and progression-free survival (P ¼ 0.002).

76

49 49

Pancreatic carcinoma Chronic myeloid leukemia

Elevated sMICA and sMICB levels correlated with tumor stage and differentiation. Significant upregulation of sMICA levels (P ¼ 0.01). sMICA levels correlated with white

81

42

Stomach, colon and rectum

blood cell count (P ¼ 0.01). 24% sMICA þ ; 29% sMICB þ ; 33% sMICA þ and sMICB þ .

27

Cervical

26

Hepatocellular carcinomas

25

Various leukemia

56% of leukemia cells expressed MIC or ULBP molecules. sMICA and sMICB levels were upregulated (Po0.03).

79

24

Gastric cancer

sMICA and sMICB were significantly increased in patient sera (Po0.05) and correlated with the concentrations of growth factor midkine.

80

24

Gastric, colon and rectum carcinoma

Significant upregulation of sMICA levels (Po0.001).

51

23 NI

Prostate Breast

sMICA/B levels correlated with disease progression. Levels of sMICA were significantly increased in advanced patients.

82

99

50

carcinoma Levels of sMICA were significantly increased in cancer patients (P40.001). Correlation between sMICA and legion stage (P40.001). Negative correlation between sMICA and NKG2D expressing T cells. sMICA was preferentially released from patients with advanced HCC.

89

88

75

Abbreviations: AML, acute myeloid leukemia; CLL, chronic lymphoid leukemia; HCC, human hepatocellular carcinoma; NI, not indicated; sMICA, soluble MICA; WHO, world health organization.

with favorable prognosis of survival, especially for stage 3 tumor patients, where expression correlated with a good prognosis.63 In the ovarian cancer patients, high expression of ULBP2 correlated with a poor prognosis for overall and progression-free survival, whereas no correlation between MICA or MICB expression and disease development was found.69 Interestingly, the authors also provided evidence that intra-stromal infiltration of NK cells indicated poor overall survival, whereas high T-cell intra-epithelial infiltration Immunology and Cell Biology

correlated with a good prognosis for patients. Serum analysis of a small subgroup of patients failed to detect soluble ULBP2, suggesting that ligand shedding is not responsible for the poor prognosis. This is in line with other studies demonstrating that only soluble MICA, but not soluble MICB or ULBP2, is able to downregulate the expression of NKG2D on NK cells.50,52 In contrast, membrane-bound MICA, MICB and ULBP2 all reduced NKG2D receptor expression. In summary, the data suggest that the expression of NKG2D ligands

Advances in NKG2D biology N Le Bert and S Gasser 233

on tumor cells only poorly correlates with NK cell infiltration and survival of patients. It is possible that the limited number of patients often used in the studies confounded the analysis, or that NKG2D ligand expression correlates with certain types of cancer at specific stages of cancer development. EXPRESSION OF SOLUBLE NKG2D LIGANDS IN HUMAN CANCER As mentioned earlier, NKG2D ligands can be proteolytically cleaved from the cell surface, excreted or secreted from cancer cells. Several studies have explored the presence of NKG2D ligands in the serum of cancer patients, although few studies distinguish between the different forms. In 2006, a study carried out by Holdenrieder et al.73,74 analyzed the serum samples of 296 patients with malignant tumors, 154 patients with benign tumors and 62 healthy controls for levels of soluble MICA and MICB by enzyme-linked immunosorbent assay. Overall, cancer patients had significantly (Po0.0001) higher serum levels of soluble MICA, but not soluble MICB, than healthy donors or patients with benign disease. However, soluble MICA levels were similar in patients with gastrointestinal benign or malignant tumors, probably due to the predisposition of gastrointestinal epithelium to express MICA in situations of cell stress.32 The diagnostic power of soluble MICA levels present in the serum of cancer patients was greatest for lung malignancies, although many cancer patients were seronegative for soluble MICA, which may limit the usefulness of soluble MICA for screening purposes in a clinical setting. Lung cancer patients showed high levels of soluble MICA already at an early stage of disease, whereas in all other cancers tested, the serum level increased with progressing tumor stages and correlated with metastasis. A number of other smaller studies of different cancers also reported correlations between soluble MICA levels and cancer stage.75–80 In addition to soluble MICA, the presence of cancer in patients also correlated with the levels of soluble MICB and ULBP1-3.74,79–84 It was suggested that release of soluble ligands represents a form of immune evasion strategy for tumor cells, as soluble NKG2D ligands were reported to induce downregulation of NKG2D and impair NK cell function and T-cell activation.83,85 The neutralization of soluble NKG2D ligands with NKG2D-Fc fusion proteins in serum of leukemia patients revealed that soluble ligands contributed strongly to reduced NKG2D expression and impaired cytotoxicity of NK cells against ligand expressing target cells.83 Reduced levels of NKG2D were recovered in acute myeloid leukemia patients following complete remission.83 Release of membrane-bound NKG2D ligands also reduced the density of ligands on the surface of target cells, which directly correlated with their capacity to stimulate tumor immunity in vivo.86 Furthermore, high concentration of soluble ligands in the serum of patients due to shedding and exosomal release may compete with receptor binding to ligands expressed on the surface of cancer cells. If soluble ligands contribute to immune evasion, soluble ligands are expected to increase with progression of cancer. In support of this role of soluble NKG2D ligands in immune escape, augmented levels of MICA and MICB were found in the sera of pancreatic carcinoma patients and the levels correlated with tumor stage.81 The lytic activity of NK cells and gd T cells against pancreatic tumor cells was almost entirely abrogated in patients with MICA and MICB in the serum. Similarly, in another cohort of pancreatic cancer patients, serum levels of soluble MICA were significantly augmented in advanced stages of cancer and correlated with metastasis.87 Surface expression of MICA was lost in high-grade pancreatic cancers. Overall survival was higher in patients with an increased MICA surface expression and low soluble MICA serum concentrations. Importantly, NKG2D expression

on NK cells and their cytotoxicity were reduced in patients with soluble MICA. Furthermore, in a small study of prostate cancer patients, tumor progression correlated with decreased MICA and MICB surface expression, diminished NKG2D expression on NK cells, reduced NK cell cytotoxicity against cancer and increased levels of soluble MICA and MICB.82 Encouragingly, in vitro stimulation of NK cells from advanced prostate cancer patients with IL-2 and IL-15 restored NKG2D surface expression and NK cell function, suggesting that the effects of soluble NKG2D ligands on NKG2D expression and NK cell function are reversible. In patients with advanced human hepatocellular carcinomas, the levels of soluble MICA were frequently elevated and the increased soluble MICA levels were correlated with lower NKG2D expression and impaired activation of NK cells.88 In patients with cervical cancer the levels of soluble MICA were augmented in the serum when compared with patients with precursor lesions, whereas the levels of soluble MICA were associated with the severity of lesions.89 The levels of soluble MICA correlated weakly with NKG2D levels on T cells, but not NK cells. In future studies, it will be important to also measure NK cell activity if feasible, as the effects of soluble NKG2D ligands might not always necessarily be mediated via the downregulation of NKG2D at the cell surface, and NK cell activity may also be affected by other immunosuppressive mechanisms used by tumor cells as highlighted in a study of patients with myelodysplastic syndrome, where reduction of NK cell cytotoxicity did not correlate with the presence of soluble NKG2D ligands.67 The important role of NK cells and NKG2D in immunosurveillance of cancer suggests that the presence of soluble NKG2D ligands in the serum might serve as prognostic markers.90 Soluble MICA levels in the serum of 97 multiple myeloma patients correlated with progression-free survival.76 Strikingly, soluble MICA levels were associated with a poor progression-free survival in disease stage 1 patients. A study in Japan with 113 patients of oral squamous cell carcinoma and 20 healthy controls showed that patients with low serum MICA levels had significantly better overall survival rates than those with high soluble MICA levels.78 Low levels of soluble ULBP1 correlated with complete remission of acute myeloid leukemia patients receiving chemotherapy.83 Moreover, low levels of soluble MICB and soluble ULBP2 before chemotherapy were associated with a better survival after complete remission.83 Soluble ULBP2 levels also correlated with advanced disease stages in chronic lymphocytic leukemia.83 Similarly, in B-cell chronic lymphocytic leukemia patients, soluble ULBP2 was found to be an independent prognostic marker for disease progression, and levels of soluble ULBP2 allowed the identification of chronic lymphocytic leukemia patients with risk of early tumor progression.77 These data are supported by a different study on malignant melanoma, which also found a correlation between soluble ULBP2 and poor prognosis.91 CONCLUSION AND OUTLOOK Many signals and pathways regulate NKG2D ligand gene expression. Some of the pathways are linked and may offer the cells the means to distinguish between normal and disease-related events. Consistent with this hypothesis, mitogen-induced proliferation of cells triggers the transcription factors E2F1-3, but only briefly activates the DNA damage response and PI3K.31 In contrast, hyperproliferation in response to activated oncogenes leads to constitutive activation of E2Fs, the DNA damage response and PI3K, and the combination of all signals might be required for the induction of NKG2D ligands. Aberrant activation of PI3K due to mutations in many types of cancer could further enhance the expression of NKG2D ligands.31 Immunology and Cell Biology

Advances in NKG2D biology N Le Bert and S Gasser 234

The different levels of regulation of NKG2D ligands may also offer a back-up mechanism in case a signal or a pathway is impaired due to inhibition or loss. In the context of cancer, the role of p53 in regulation of NKG2D ligands supports such a hypothesis. Replication stress in response to activation of oncogenes was shown to induce DNA damage.92 The ensuing DNA damage response was suggested to represent a barrier against cancer by inducing cell cycle arrest or apoptosis.93 Consistent with this possibility, tumor progression is often accompanied by the loss of p53 function.94 Activation of p53 induces the expression of ULBP2, which may contribute to the recognition of precancerous cells by cytotoxic immune cells. However, many NKG2D ligands can also be upregulated in response to DNA damage in the absence of p53. Tumor cells that lack p53 often depend on the heat-shock factor 1, which is overexpressed in numerous cancer cell lines.95–97 Heat-shock factor 1 induces the expression of MICA and MICB, which may counter the loss of some ligands in cells that lack p53.98 Interestingly, a recent study suggested that p53 is critical for attracting NK cells to liver carcinoma cells, which might explain the loss of immunosurveillance in advanced tumor cells. It will be important to test whether p53 is also required for the recruitment of NK cells to other cancer and diseased cells.68 In light of the complex regulation of NKG2D ligands, the specificity of the different NKG2D ligands for particular diseases and disease stages remains to be investigated further before NKG2D ligands can be used as general diagnostic and prognostic markers in clinical settings. The current data suggest that in contrast to other NKG2D ligands, ULBP3 is mainly regulated by DNA damage. One could therefore assume that ULBP3 is a promising tumor-specific marker, a conclusion supported by the frequent expression of ULBP3 on glioma cells.58 However, despite the potentially straightforward regulation of ULBP3, B cell acute lymphoid leukemia and other tumor cells often do not express detectable levels of ULBP3.83 It is currently not clear what underlies these differences and whether they correlate with the presence of DNA damage or a functional DNA damage response in the different tumor cells. Furthermore, few studies have looked at ULBP3 expression in another disease context, especially diseases that might elicit a DNA damage response such as viral infections. Similar concerns will also apply when considering soluble NKG2D ligands as diagnostic or prognostic markers. Encouragingly, sera from most healthy controls were negative for soluble NKG2D ligands and a majority of studies found significant differences between healthy, benign and malignant tumor patients. However, there was generally a considerable overlap between the three different sample groups, and a number of cancer patients remain seronegative for soluble ligands. It is possible that the sensitivity of the assays is currently not adequate to detect low levels of NKG2D ligands in the serum of some patients, or that lack of distinction between cleaved, excreted and secreted NKG2D ligands confounds the results. Finally, a better understanding of the effects of soluble NKG2D ligands on immune cells in different diseases may also ameliorate the usefulness of soluble NKG2D ligands as prognostic markers. The NKG2D system could also offer the opportunity for therapeutic intervention. Several approaches are conceivable, some of which are currently being developed. Specific upregulation of NKG2D ligands on diseased cells by small molecules, such as genotoxic agents or protease inhibitors, or treatments, including radiation or heat shock, may render diseased cells more susceptible to NK cell-mediated lysis. Such clinical protocols might be particularly promising when combined with treatments that upregulate NKG2D on immune cells. Alternatively, in patients with soluble NKG2D ligands present in the serum, neutralization of soluble NKG2D ligands may enhance NK cell Immunology and Cell Biology

engagement with target cells and could restore expression levels of NKG2D receptor on NK cells. This might be achieved by the development of small molecules that specifically block the cleavage of NKG2D ligands on the cell surface of cancer cells. Such an approach could reinstate NKG2D expression on NK cells, whereas at the same time increasing the expression of NKG2D ligands on cancer cells, thereby enhancing their recognition and the activation of NK cells. Finally, NKG2D ligands might serve as clinically useful targets for antibody-directed therapies. Patients with low expression levels of NKG2D ligand on the cell surface, which was repeatedly found to correlate with a poor prognosis, may be good candidates for aggressive chemotherapy. In summary, much progress has been made in understanding NKG2D biology, but more disease-specific understanding of NKG2D ligand expression will be needed before NKG2D can be translated into clinical therapies. CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGEMENTS We thank all the current laboratory members and Prof David H Raulet at the University of California in Berkeley (USA) for useful discussions. This work was supported by the BMRC grant 07/1/21/19/513 and the NRF grant HUJ-CREATE—Cellular and Molecular Mechanisms of Inflammation.

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Advances in NKG2D ligand recognition and responses by NK cells.

The natural killer (NK) group 2 member D (NKG2D) is an activating immune receptor expressed on NK cells, cytotoxic T cells and a subset of other T cel...
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