NKG2D ligands in tumor immunity - Nature

Oncogene (2008) 27, 5944–5958

& 2008 Macmillan Publishers Limited All rights reserved 0950-9232/08 $32.00 www.nature.com/onc

REVIEW

NKG2D ligands in tumor immunity N Nausch and A Cerwenka Division of Innate Immunity, German Cancer Research Center, Im Neuenheimer Feld 280, Heidelberg, Germany

The activating receptor NKG2D (natural-killer group 2, member D) and its ligands play an important role in the NK, cd þ and CD8 þ T-cell-mediated immune response to tumors. Ligands for NKG2D are rarely detectable on the surface of healthy cells and tissues, but are frequently expressed by tumor cell lines and in tumor tissues. It is evident that the expression levels of these ligands on target cells have to be tightly regulated to allow immune cell activation against tumors, but at the same time avoid destruction of healthy tissues. Importantly, it was recently discovered that another safeguard mechanism controlling activation via the receptor NKG2D exists. It was shown that NKG2D signaling is coupled to the IL-15 receptor pathway in a cell-specific manner suggesting that priming of NKG2D-mediated activation depends on the cellular microenvironment and the distinct cellular context. This review will provide a broad overview of our up-to-date knowledge of the NKG2D receptor and its ligands in the context of tumor immunology. Strategies to amplify NKG2D-mediated antitumor responses and counteract tumor immune escape mechanisms will be discussed. Oncogene (2008) 27, 5944–5958; doi:10.1038/onc.2008.272 Keywords: NKG2D; RAE-1; MIC-A; ULBP; NK cells; tumor immunology

Expression of the NKG2D receptor The receptor NKG2D (natural-killer group 2, member D) belongs to the family of C-type lectin-like receptors. Therefore, NKG2D has been designated klrk1/KLRK1 (killer cell lectin-like receptor subfamily K, member 1). As the gene encoding this receptor resides in the NKG2 (natural killer group 2) complex, the gene product was originally designated as nkg2d/NKG2D. The NKG2 complex is located on chromosome 6 in mice (Ho et al., 1998) and on chromosome 12 in humans (Glienke et al., 1998). NKG2D is expressed by all NK cells, most NKT cells and subsets of gd þ T cells (Wu et al., 1999; Jamieson et al., 2002). NKG2D is also expressed by a subset of Correspondence: Dr A Cerwenka, Division of Innate Immunity, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. E-mail: [email protected]

activated NK cells sharing markers with dendritic cells (DCs), which are referred to as natural killer DCs or interferon (IFN)-producing killer DCs (Pillarisetty et al., 2004; Chan et al., 2006; Taieb et al., 2006; Vosshenrich et al., 2007). In addition, NKG2D is present on the cell surface of all human CD8 þ T cells. In contrast, in mice, expression of NKG2D is restricted to activated CD8 þ T cells (Ehrlich et al., 2005). In tumor mouse models, NKG2D þ CD8 þ T cells preferentially accumulate in the tumor tissue (Gilfillan et al., 2002; Choi et al., 2007), suggesting that the NKG2D þ CD8 þ T-cell population comprises T cells involved in tumor cell recognition. Under non-pathological conditions, expression of NKG2D is not detectable on a substantial proportion of human or mouse ab CD4 þ T cells. In contrast, NKG2D-expressing CD4 þ T cells accumulate in patients with MIC þ tumors (Groh et al., 2006) as well as those suffering from autoimmune diseases, including rheumatoid arthritis or Crohn’s disease (Groh et al., 2003; Allez et al., 2007). Recently, klrk1/ mice that are deficient in NKG2D expression were generated (Guerra et al., 2008). These mice developed normal numbers of NK cells and other lymphocyte subsets in all analysed organs. Moreover, no significant differences were observed in the expression of NK cell maturation markers, including CD11b or CD43, or in the expression of activating or inhibitory receptors. These data suggest that NKG2D is dispensable for normal phenotypic development of NK cells. NKG2D is a type II transmembrane protein (Garrity et al., 2005) and does not contain any known signaling motif within its intracellular domain (Houchins et al., 1991). For signal transduction, NKG2D associates, like many other activating receptors, with adaptor proteins via charged residues in its transmembrane domain. Both human and mouse NKG2D form a complex with DNAX-activating protein of 10 kDa (DAP10) (Wu et al., 1999). In humans, NKG2D associates with DAP10 alone, and not with DAP12 (Rosen et al., 2004) (Figure 1). In addition, in mice, NKG2D binds to the adaptor DAP12, which is also known as killer cellactivating receptor-associated polypeptide or tyrosine kinase-binding protein (Paloneva et al., 2000; Diefenbach et al., 2002; Gilfillan et al., 2002). These adaptors couple to NKG2D as homodimers. As NKG2D is a homodimer, the adaptors and receptor form a hexameric structure (Garrity et al., 2005). Moreover, in mice, NKG2D exists in two distinct splice variants. The long isoform (NKG2D-L) has 13 additional amino acids

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P

P

PI3K

Grb2

or

Cytotoxicity IFN-γ

NKG2D

DAP12 I P T A M P

DA P1 YXXM 0

ZAP-70

10 YXXM

?

Syk

DA P

NKG2D-L

Human

NKG2D-L

NKG2D-S or Syk

I P T A M P

ZAP-70

NKG2D-S

DAP12

Mouse

P

P

PI3K

DA P1 0 YXXM

P

Grb2

Cytotoxicity IFN-γ

PI3K

P

Grb2

Cytotoxicity IFN-γ

Expression on: CD8+ T cells: NK cells:

Not present

Activated/memory

Not present

Activated/memory

Resting/activated

Resting/activated

Resting/activated

Resting/activated

Resting/activated

Resting/activated

Figure 1 Schematic illustration of NKG2D isoforms: expression patterns and transmembrane adaptors. Mouse NKG2D exists in two isoforms: NKG2D-short (S) and NKG2D-long (L). NKG2D-S couples to either DAP10 or DAP12. Whether NKG2D-L exclusively associates with DAP10 or binds to both adaptors DAP10 and DAP12 is controversial (indicated by ‘?’). NKG2D-L and -S are expressed by resting and activated natural killer cells. In mouse and human CD8 þ T cells, DAP12 is not detectable. Therefore, in T cells, NKG2D couples exclusively to DAP10. In mouse, NKG2D/DAP10 is expressed by activated/memory CD8 þ T cells. In humans, NKG2D/DAP10 is detectable on all CD8 þ T cells. The signaling cascade induced by DAP10 involves PI3K/Grb2. DAP12 mediates signaling via Syk/ZAP70 (Chang et al., 1999; Wu et al., 1999; Diefenbach et al., 2002; Rosen et al., 2004; Oppenheim et al., 2005; Rabinovich et al., 2006). DAP, DNAX-activating protein; Grb2, growth factor receptor-bound protein 2; PI3K, phosphoinositide kinase-3; Syk, spleen tyrosine kinase; ZAP70, zeta-chain-associated protein kinase 70.

within the intracellular N-terminus as compared with the short variant (NKG2D-S) (Diefenbach et al., 2002). Initially, it was reported that NKG2D-L does not bind to DAP12, whereas NKG2D-S associates with DAP10 and DAP12 (Diefenbach et al., 2002). In contrast, a more recent study concluded that DAP10 and DAP12 compete equally for both variants of NKG2D (Rabinovich et al., 2006). Since both studies relied on transfection of reporter cells in their association studies, a final proof in primary NK cells is still missing. It has been reported that NKG2D-L is expressed by resting NK cells, whereas the short isoform is induced upon activation. Therefore, depending on the activation state of the NK cell, NKG2D signals via distinct intracellular pathways to elicit effector function. DAP10 carries a YXXM motif (Tyr-X-X-Meth) within the cytoplasmic domain, which recruits phosphoinositide kinase-3 (PI3K) and growth factor receptor-bound protein 2 upon activation (Chang et al., 1999; Wu et al., 1999). In contrast, DAP12 signals via an immunoreceptor tyrosine-based activation motif, which, after phosphorylation, recruits ZAP70 (zeta-chain-associated protein kinase 70) and Syk (spleen tyrosine kinase). Therefore, in activated mouse NK cells, two different pathways are triggered via NKG2D resulting in a PI3K- or Syk/ZAP70-dependent signaling cascade, respectively. In CD8 þ T cells, which generally lack DAP12 expression, NKG2D binds to exclusively DAP10. As

in T cells PI3K activation is a hallmark of costimulatory receptors including CD28, NKG2D was originally described as a co-stimulatory molecule, augmenting T-cell receptor (TCR) signaling (Bauer et al., 1999; Diefenbach et al., 2002; Ehrlich et al., 2005). Under certain circumstances, however, NKG2Dmediated effector functions in T cells are independent of TCR engagement. For example, target cell killing of long-term cultured or cloned CD8 þ T cells was shown to be mediated via NKG2D (Maccalli et al., 2003; Verneris et al., 2004). In addition, chronically activated intraepithelial CD8 þ T cells isolated from patients suffering from celiac disease killed target via NKG2D without any contribution of TCR (Roberts et al., 2001; Meresse et al., 2004). We conclude that cell-specific features and the physiological context determine whether NKG2D acts as a stimulatory or a co-stimulatory molecule.

Priming of NKG2D-mediated effector functions Exposure of purified NK cells to NKG2D ligandexpressing targets in vitro results only in low levels of target cell killing (Bryceson et al., 2006), indicating that for optimal triggering of NKG2D-mediated effector functions, additional signals are required. In fact, in vitro culture of NK cells in the presence of interleukin Oncogene

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(IL)-15 or high doses of IL-2 significantly increases NKG2D-mediated killing of tumor targets. Recently, Horng et al. (2007) demonstrated that the IL-15 receptor pathway couples to NKG2D signaling in NK cells (Figure 2). Triggering of the IL-15 receptor resulted in phosphorylation of DAP10 by janus kinase 3, which facilitated downstream signaling and NKG2D-mediated effector function. Whether other cytokines activate the NKG2D pathway in a similar fashion and whether other activating receptors are also controlled in a similar manner by cytokines are currently unknown. One source of IL-15 is DCs, which produce IL-15 after exposure to toll-like receptor ligands in vivo and subsequently present IL-15 on their cell surface to NK cells in draining lymph nodes (Lucas et al., 2007). Whether this trans-presentation of IL-15 by DC is of importance during NK cell-mediated antitumor immune responses has not yet been addressed. Interestingly, naı¨ ve CD8 þ T cells from DAP10-Ub transgenic mice show no defects in IL-15 responsiveness, suggesting that activation of NKG2D-mediated effector function is controlled in a cell-specific manner dependent on the activation status. Nevertheless, effector/

IL P

P

PI3 K

-1

memory CD8 þ T cells isolated from celiac disease patients are activated via NKG2D in a TCR-independent manner (Hue et al., 2004; Meresse et al., 2004). There is evidence that this TCR-independent, NKG2Dmediated activation is primed by a chronic exposure to IL-15 in vivo. In vitro, IL-15 not only upregulates the expression of NKG2D and DAP10 on CD8 þ T cells, but also facilitates NKG2D-mediated signal transduction via the induction of constitutive extracellular signalregulated kinase phosphorylation. Inhibitor studies revealed that phosphorylations of extracellular signalregulated kinase and c-Jun N-terminal kinase are critical in the NKG2D-induced cytotoxicity of CD8 þ T cells. Therefore, in CD8 þ T cells as well, IL-15 or high doses of IL-2 prime the NKG2D pathway and converts CD8 þ T cells into NK-like lymphokine-activated killer cells (Meresse et al., 2004). The exact mechanism of action of IL-15-mediated priming of CD8 þ T cells is still elusive. Taken together, there is increasing evidence that signaling initiated by activating receptors is not only controlled by inhibitory receptors, as previously anticipated, but requires additional factors for optimal activation.

P

5

Jak3 P

P

PI3K

PI3K

ERK

P

P

Cytotoxicity P

Cytotoxicity

ERK

IL-15

NKG2D ligand TLR

NKG2D DAP10/DAP12 DC

Figure 2 Priming of NKG2D-mediated effector function. (a) Stimulation of natural killer (NK) cells via NKG2D: engagement of NKG2D leads to phosphorylation of DAP10 and recruitment of PI3K. Subsequently, downstream signaling is triggered and NK cells are activated to kill NKG2D ligand-bearing targets at low levels. (b) Stimulation of NK cells via NKG2D and IL15R. IL-15 binding to its receptor results in phosphorylation of DAP10. DAP10 also associates with the IL-15R. Therefore, IL-15 primes NKG2D-mediated signaling. Upon binding of NKG2D ligands, NKG2D-mediated signaling is fully activated and high levels of target cell killing are observed. IL-15 is ‘trans-presented’ to NK cells by DCs after TLR engagement (Wu et al., 1999; Horng et al., 2007; Lucas et al., 2007). DAP, DNAX-activating protein; DC, dendritic cell; ERK, extracellular signal-regulated kinase; IL, interleukin; Jak3, janus kinase 3; PI3K, phosphoinositide kinase-3; TLR, toll-like receptor. Oncogene

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NKG2D ligands NKG2D binds to a variety of ligands, which resemble major histocompatibility complex (MHC) class I proteins (Figure 3) (Bauer et al., 1999; Cerwenka et al., 2000; Diefenbach et al., 2000). In mice, most ligands belong to the family of retinoic acid early inducible-1 (RAE-1) proteins. So far, five family members, RAE-1a–e, have been described. Whether all ligands can be induced by RA is currently not known. It was suggested that the genes of the rae-1 family are differentially distributed in different mouse strains. There is evidence that rae-1d and -e exist in C57BL/6, whereas BALB/c and 129/J can express rae-1a, d and g (Ogasawara and Lanier, 2005). It is not known whether this is a matter of different genes in different mouse strains or simply of allelic variants. A documentation of the expression and genomic organization of rae-1 genes in different mouse strains should be the focus of future studies. The RAE-1 proteins consist of two Ig-like domains (a1 and a2) and are attached to the cell membrane via a glycosyl-phosphatidylinositol anchor (Figure 3). In contrast, MULT-1 (murine UL16-binding protein like transcript-1) (Carayannopoulos et al., 2002; Diefenbach et al., 2003) and the minor histocompatibility antigen H60 (Malarkannan et al., 1998; Cerwenka et al., 2000; Diefenbach et al., 2000), which bind to NKG2D in mice, have a transmembrane region. Recently, two additional variants of H60 were identified and named as H60b and H60c (Takada et al., 2008). In contrast to H60(a), which is expressed in BALB/c but not in C57BL/6 mice, transcripts of H60b and H60c were found in both C57BL/6 and BALB/c strains (Malarkannan et al., 1998; Takada et al., 2008). H60c transcripts were detected mainly in the skin, whereas H60a and H60b transcripts had a broader tissue distribution. In humans, RAET1 family members, which are orthologs of the mouse RAE-1 proteins, have been described (Cosman et al., 2001; Radosavljevic et al., 2002). The RAET1 proteins were initially referred to as UL16-binding proteins (ULBPs) and later renamed in accordance with their mouse counterparts. In addition, the name ULBPs is misleading, because only ULBP1 and ULBP2, but not

ULBP3, bind to the UL16 glycoprotein of cytomegalovirus (Cosman et al., 2001; Steinle et al., 2001; Chalupny et al., 2003). In total, 10 members of this gene family were mapped, but only 6 RAET1 family members were shown to be expressed as functional proteins (Radosavljevic et al., 2002) (Figure 3). Two members of the RAET1 family are anchored to the surface by a transmembrane region (RAET1E and G), whereas other RAET1 molecules are linked to the cell membrane via glycosyl-phosphatidylinositol anchors (Bacon et al., 2004). In humans, an additional family of NKG2D ligands, MHC class I-chain-related proteins A and B (MIC-A and -B), exists, which in addition to the a1 and a2 domain has a third Ig-like domain (a3) (Bauer et al., 1999; Bahram et al., 1994). MIC-A and -B are highly polymorphic. So far, 61 MIC-A alleles and 30 MIC-B alleles have been identified, which are listed on http:// www.anthonynolan.com/HIG/seq/nuc/text/. Some of these alleles differ in their ability to bind to the NKG2D receptor (Steinle et al., 2001). Expression of certain alleles might contribute to inter-individual differences in susceptibility to certain diseases (Bravo et al., 2007; Turkcapar et al., 2007). In humans and mice, a single receptor, NKG2D, binds to several distinct ligands. Why multiple ligands exist for this one, invariant, receptor is still not well understood; however, there are several possibilities that might account for this complexity (Cerwenka and Lanier, 2001; Eagle and Trowsdale, 2007). First, different ligands bind to NKG2D with distinct affinities. Therefore, engagement of NKG2D by different NKG2D ligands might fine-tune the extent of activation. The affinity of mouse ligands to NKG2D ranges between 2 and 700 nM (KD) and the affinity of the human ligands between 600 nM and 1.1 mM, respectively (O’Callaghan et al., 2001; Strong, 2002; Mistry and O’Callaghan, 2007). Although striking differences in binding affinities between different ligands exist, the observed binding affinities in general are quite strong, as compared with other receptor/ligand interactions, including KIRs and MHC class I (KD ¼ 10 mM) (Strong, 2002). Second, there are hints that additional receptors for NKG2D ligands exist. In particular, Kriegeskorte

Human NKG2D-ligands

MHC I MIC A, B

α1

α2

α1

α3

β2m

α3

α2

RAET1I,H,N

α1

α2

Mouse NKG2D-ligands

RAET1E,G

α1

α2

RAE-1α–ε

α1

α2

MULT-1, H60

α1

α2

Figure 3 Schematic illustration of human and mouse ligands for NKG2D in comparison with the MHC class I molecule. Synonyms for the RAET1 molecules are RAET1I ¼ ULBP1, RAET1H ¼ ULBP2, RAET1N ¼ ULBP3, RAET1E ¼ ULBP4 ¼ LETAL (Radosavljevic et al., 2002). a1–a3, Ig-like domains; b2m, b2 microglobulin light chain; MHC I, major histocompatibility complex class I; MIC, MHC class I-chain-related protein; MULT-1, murine UL16-binding protein like transcript 1. Oncogene

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et al. (2005) observed that soluble H60 and MIC-A inhibited anti-CD3-stimulated T-cell proliferation. This suppression of T-cell proliferation required IL-10, but was not blocked by the addition of a monoclonal antibody (mAb) directed against NKG2D. So far, the receptor responsible for this observation has not been described. Third, it is feasible that an evolutionary pressure to avoid escape mechanisms devised by certain viruses to inhibit NKG2D ligand expression in virusinfected cells may be driving the diversity of these ligands (Bahram et al., 2005). Moreover, the existence of multiple ligands might also be of advantage to counteract escape mechanisms of transformed cells to NKG2Dmediated antitumor responses. Finally, tissue-specific expression of NKG2D ligands and their differential induction requirements might account for the necessity of multiple ligands for one single receptor, as discussed in the following two sections.

NKG2D ligands: expression and regulation The RAE-1 molecules were first described to be induced after treatment of the embryonic testicular teratoma cell line F9 with RA (Nomura et al., 1996; Zou et al., 1996). Interestingly, similar to the mouse ligands, human MIC-A and -B proteins are also upregulated on hepatocellular carcinoma cells upon treatment with RA (Jinushi et al., 2003b). Indeed, expression of rae-1 mRNA was observed during embryogenesis from days 9 to 11, but was hardly detectable in healthy adult tissues. High levels of rae-1 are expressed in the brain of embryos (Nomura et al., 1996). Furthermore, expression of RAE-1 is detected on the cell surface of embryonic stem cells, which is further enhanced upon treatment with RA (unpublished observation). Whether RAE-1 proteins are expressed on the cell surface during embryogenesis and whether they are implicated in developmental processes are currently unknown. In BALB/c, but not in C57BL/6, mice, the expression of RAE-1 and, to a lesser extent, H60 was described on Gr-1 þ and CD11b þ bone marrow cells that were repopulating lethally irradiated mice reconstituted with hematopoietic stem cells. Moreover, expression of these NKG2D ligands on the repopulating BALB/c bone marrow cells targeted these cells for rejection by irradiation-resistant NK cells in a C57BL/6  BALB/c F1 recipient (Ogasawara et al., 2005). Similarly, in humans, NKG2D ligands are expressed at low levels on CD34 þ hematopoietic stem cells and expression of the RAET1 proteins is enhanced upon differentiation into myeloid progenitors (Guilloton et al., 2005; Nowbakht et al., 2005). In healthy adult mice, NKG2D ligands are expressed at low levels on the cell surface of hepatocytes (Vilarinho et al., 2007 and our unpublished observations) and on CD4 þ 8 þ double-positive thymocytes in BALB/c mice, respectively (Li et al., 2005). In humans, RAET1 transcripts are detectable in different healthy tissues, including kidney, prostate, uterus, tonsil and Oncogene

lymph nodes (Cosman et al., 2001). In addition, ULBP4 RAET1E is specifically transcribed in the skin (Chalupny et al., 2003). Total tissue scans of MIC-A and -B of healthy individuals revealed that with the exception of the central nervous system both genes are widely transcribed (Schrambach et al., 2007). The extent to which these ligands are expressed at protein levels is unknown and has to be addressed in further studies with validated antibodies. However, it is likely that expression of these proteins is tightly regulated at a posttranscriptional or possibly post-translational level. The NKG2D ligands can be upregulated by multiple stimuli, including infection by bacteria or viruses or by cellular transformation. In Table 1, stimuli leading to the upregulation of human and mouse NKG2D ligands are summarized. RAE-1 is induced on mouse macrophages after stimulation with toll-like receptor ligands (Hamerman et al., 2004) and after infection with Mycobacterium tuberculosis (Rausch et al., 2006). MIC-B is upregulated on human macrophages infected with Influenza A or with Sendai virus (Siren et al., 2004). Surface expression of NKG2D on NKT cells and its ligand RAE-1 on hepatocytes are modulated upon hepatitis B virus infection (Vilarinho et al., 2007). Moreover, in certain cells and under distinct experimental conditions, MIC-A and -B are inducible by heat shock (Groh et al., 1996). In addition, T cells activated by antigen-presenting cells, or T-cell blasts stimulated with super antigen, anti-CD3/anti-CD28 in combination with PMA, or infected with HIV, upregulate NKG2D ligands (Diefenbach et al., 2000; Molinero et al., 2002; Rabinovich et al., 2003; Cerboni et al., 2007; Ward et al., 2007). The induction of MIC-A on TCRstimulated T cells involves activation of the transcription factor nuclear factor-kB (Molinero et al., 2004). In summary, these studies imply an important role of NKG2D and its ligands in infectious diseases, as reviewed elsewhere (Hamerman et al., 2005; Ogasawara and Lanier, 2005). Since inflammatory diseases and infection with certain viruses are often linked to malignant transformation, regulation of NKG2D ligands by infectious agents might increase the visibility of tumor cells to effector cells of the immune system.

NKG2D ligands: expression and regulation on transformed cells NKG2D ligands are expressed by a wide range of tumor cell lines of different tissue origins. Mouse NKG2D ligands are detectable on T- and B-cell lymphoma cell lines, including YAC-1, WEHI7.1 and A20, and on multiple carcinoma cell lines, such as the lung carcinoma lines LL/2 and MLg, rectum carcinoma line CMT, colon carcinoma line MC38, prostate carcinoma line TRAMP and the Renca renal carcinoma cell line (Cerwenka et al., 2000; Diefenbach et al., 2000; Smyth et al., 2004). Expression of the NKG2D ligands is not a ubiquitous feature of tumor cell lines, because the lymphoma cell lines RMA and RMA-S, the C57BL/6 melanoma cell

NKG2D in antitumor responses N Nausch and A Cerwenka

5949 Table 1 Regulation of ligands for the activating receptor NKG2D Stimuli/treatment

Cells/cell line

Ligands

Reference

RA

F9 embryonic carcinoma

RA

Nomura et al. (1996) Unpublished observations Jinushi et al. (2003b)

TLR ligands LPS LPS Mycobacterium tuberculosis Pseudomonas aeruginosa Influenza A, Sendai infection Escherichia coli Super antigen, antigens HIV aCD3/CD28/PMA ConA, APC, PMA

Hepatocellular carcinoma tissue and cell lines Macrophages Macrophages B cells (BALB/c) Macrophages Airway epithelial cell, macrophages Macrophages Epithelial cell lines T cells T cells T cells T cells

rae-1 RAE-1a MIC-A/B RAE-1a–e ULBP1/2/3 NKG2D mouse ligands RAE-1 RAE-1, ULBP2 MIC-A MIC-A MIC-A, ULBP1/2/3 ULBP1/2/3 MIC-A NKG2D mouse ligands

EBV infection IFN-a Differentiation

B cells in lytic cycle DC Myeloid progenitors

Heat shock E1A (serotype 5)

Epithelial cells Human, mouse tumor cells, primary baby mouse kidney fibroblasts Leukemic, hematopoietic CD34+ Human and mouse fibroblasts Mouse embryonic fibroblasts, endothelioma cells AML hepatoma cells

ULBP1 MIC-A/B ULBP1 ULBP2/3 MIC-A/B RAE-1, MIC and ULBPs

Hamerman et al. (2004) Nedvetzki et al. (2007) Diefenbach et al. 2000) Rausch et al. (2006) Borchers et al. (2006) Siren et al. (2004) Tieng et al. (2002) Cerboni et al. (2007) Ward et al. (2007) Molinero et al. (2002) Diefenbach et al. (2000); Rabinovich et al. (2003) Pappworth et al. (2007) Jinushi et al. (2003a) Nowbakht et al. (2005) Guilloton et al. (2005) Groh et al. (1996) Routes et al. (2005)

MIC RAE-1, ULBP1/2/3, MIC-A RAE-1e, MULT-1

Boissel et al. (2006) Gasser et al. (2005) Nausch et al. (2006)

MIC-A/B, ULBP1 MIC-A/B

Armeanu et al. (2005); Diermayr et al. (2007)

BCR/ABL DNA damage JunB/AP-1 HDAC

Abbreviations: AML, acute myeloid leukemia; APC, antigen-presenting cell; ConA, concavalin A; EBV, Epstein–Barr virus; HDAC, histone deacetylase; LPS, lipopolysaccharide; MIC, MHC class I-chain-related protein; RAE-1, retinoic acid early inducible-1 protein; TLR, toll-like receptor; ULBP, UL16-binding protein.

line B16, and melanoma cell lines established from transgenic MT-ret mice lack expression of NKG2D ligands (Cerwenka et al., 2000, 2001; Diefenbach et al., 2000; unpublished observation). Furthermore, differences in expression levels of NKG2D ligands are observed on tumor cell lines derived from progressing tumors isolated from different mice. In this context, Smyth et al. (2005) reported that only one-third of MCA-induced sarcoma cell lines derived from wild-type mice expressed RAE-1 molecules on the cell surface and the levels of expression varied. These data suggest that tumor editing by the immune system might occur. In fact, in mice lacking perforin, all generated tumor lines expressed RAE-1, indicating that elimination via perforin was responsible for the observed variable expression of NKG2D ligands in situ (Smyth et al., 2005). The human RAET1 proteins were reported to be expressed by tumors of different origin, including leukemia, gliomas and melanomas (Pende et al., 2002; Friese et al., 2003; Salih et al., 2003). MICs are expressed by an even broader range of tumors and were detected in leukemia, various carcinomas (breast, lung, colon, kidney, ovary and prostate), gliomas, neuroblastomas and melanomas (Groh et al., 1999; Pende et al., 2002; Vetter et al., 2002; Friese et al., 2003; Salih et al., 2003; Watson et al., 2006; Castriconi et al., 2007). In general, as in mice, the proportions of ligand-positive tumors, the levels of ligand expression and the types of ligands expressed are heterogeneous between different tumor types and between individual samples of one

tumor entity studied. This observation was further extended by a study analysing NKG2D ligand expression in ovarian carcinomas. Of six samples of freshly isolated ovarian carcinoma cells, one expressed MIC-A, two ULBP2 and two ULBP3. In one isolate, no NKG2D ligands were detectable. Furthermore, MIC-A and ULBP3 were expressed at low densities, whereas expression levels of ULBP2 were relatively high (Carlsten et al., 2007). In addition, it was reported that myeloma cell lines express NKG2D ligands and that NKG2D was one of the key NK-activating receptors for bone marrow-derived myeloma cell recognition (Carbone et al., 2005; El-Sherbiny et al., 2007). It is important to understand the factors leading to NKG2D ligand expression in tumor cells. As NKG2D ligands are rarely expressed on the cell surface of adult healthy cells, their expression is likely to be associated with malignant transformation and the process of tumorigenesis. Cell transformation by certain oncogenes including K-ras, c-myc, Akt, E1A or Ras V12, or their combinations, does not directly force expression of NKG2D ligands. These data suggest that additional events are required for NKG2D ligand expression (Gasser et al., 2005). To mimic tumorigenesis, transformed ovarian cell lines were implanted in the ovary of nude mice, which resulted in the development of tumors (Gasser et al., 2005). Cell lines derived from these tumors expressed significant amounts of NKG2D ligands. Whether primary tumors directly isolated from mice expressed NKG2D ligands in situ was not Oncogene

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investigated in this study. In contrast, other studies showed that overexpression of the oncogenes adenovirus E1A or BCR/ABL by itself resulted in expression of RAE-1 or MIC-A, respectively (Routes et al., 2005; Boissel et al., 2006). Therefore, the role of oncogenes in the induction of NKG2D ligands is likely to be influenced by additional factors, such as the origin of the transformed cell line. The detailed molecular mechanisms leading to the upregulation of NKG2D ligands by different oncogenes are not completely understood. The activation of NKG2D ligand expression during transformation is a multifaceted process, which is controlled at several levels. Indeed, we have shown that expression of RAE-1e and MULT-1 are enhanced in mouse embryonic fibroblasts deficient in JunB, a subunit of the transcription factor AP-1 (Nausch et al., 2006). Notably, recently an AP-1-binding site was also reported in the promoter of MIC-A (Venkataraman et al., 2007). AP-1 is involved in the regulation of complex biological processes, for example, proliferation, differentiation and apoptosis, which are often dysregulated during tumorigenesis (Angel et al., 2001). Therefore, AP-1 plays a pivotal role in the development of tumors. It appears reasonable that the expression of RAE-1 is linked to such a central ‘sensor’ of transformation to make transformed cells visible to the immune system. Furthermore, it is well established that the DNA damage pathway is activated in many tumors. Key players in the DNA damage response are the ATM (ataxia telangiectasia, mutated) and ATR (ATM- and Rad3-related) kinases. Gasser et al. (2005) have recently reported that DNA damage induces surface expression of NKG2D ligands, which was dependent on both ATM and ATR kinases. Also, activation of the DNA damage pathway can be interpreted as a ‘sensor’ for transformed and stressed cells, which are marked for destruction by immune cells via NKG2D. It is possible that multiple mechanisms cooperate in the upregulation of NKG2D ligands. DNA damage indirectly activates c-Jun N-terminal kinase leading to phosphorylation of c-Jun (Roos and Kaina, 2006). We have evidence that c-Jun antagonizes JunB and upregulates RAE-1 expression (unpublished observations). Furthermore, it was reported that AP-1 acts downstream of the ATM kinase, which is involved in the DNA damage response (Weizman et al., 2003). Therefore, AP-1 and the DNA damage pathway might cooperate in the upregulation of NKG2D ligands. Furthermore, there is evidence that histone deacetylase inhibitors also lead to an upregulation of NKG2D ligands (Armeanu et al., 2005; Diermayr et al., 2007), suggesting that the expression of NKG2D ligands is also regulated by epigenetic changes. Taken together, our understanding of the mechanisms leading to the expression of NKG2D ligands is in its infancy. It is possible that the existence of multiple NKG2D ligands (as discussed in the section ‘NKG2D ligands’) reflects their ability to become induced by certain stimuli and pathways ensuring visibility of stressed cells to immune cells under different circumstances. Oncogene

The relevance of NKG2D and its ligands in cancer Initially, Bauer et al. (1999) reported that target cells ectopically expressing the NKG2D ligand MIC-A are efficiently killed via NKG2D despite the expression of MHC class I molecules (Groh et al., 1999). Mouse NK cells also efficiently eliminated MHC class I-positive RMA cells that were transduced with the NKG2D ligands H60 or RAE-1 (Cerwenka et al., 2000; Diefenbach et al., 2000). These data suggested that NKG2D ligands would also play an important role in tumor rejection in vivo. Indeed, ectopic expression of NKG2D ligands on tumor cells overcomes MHC class I-dependent inhibition of NK cells and enables NK cells to reject tumors in vivo (Cerwenka et al., 2001; Diefenbach et al., 2001). Subsequently, Girardi et al. (2001) reported that the exposure of skin to carcinogens rapidly induced expression of RAE-1, whereas in healthy skin, NKG2D ligands were not detectable. Interestingly, skin-associated NKG2D þ gd þ T cells killed skin carcinoma cells in vitro, which was blocked by a mAb directed against NKG2D. These data suggested an important role of NKG2D/NKG2D ligands during the early immune response against spontaneously arising tumors. It was reported that the neutralization of NKG2D by using a mAb increased the frequency of mice developing MCAinduced sarcomas (Smyth et al., 2005), confirming the impact of the NKG2D/NKG2D ligand interaction during antitumor immune responses in vivo. In addition, these data suggest that the outgrowth of transformed cells can be prevented by an NKG2D-dependent clearance mechanism early during tumor development. NKG2D-deficient mice crossed to TRAMP mice that spontaneously develop prostate adenocarcinoma showed a higher incidence of aggressive early-arising carcinomas, as compared with control mice (Guerra et al., 2008). Moreover, an earlier onset of c-mycexpressing lymphoma was observed in NKG2D-deficient Em-myc mice. These results confirm that NKG2D plays a critical role in tumor surveillance in vivo. Tumors expressing NKG2D ligands can develop in fully immune-competent animals. Therefore, we suspect that mechanisms exist in tumor-bearing mice and cancer patients that impair NKG2D-dependent effector functions of NK cells, or, alternatively, tumors are likely to exploit mechanisms to evade NK cells by targeting pathways other than NKG2D. The different aspects of NKG2D-mediated responses in cancer are illustrated in Figure 4.

Immune evasion from NKG2D-dependent immune response Immune evasion by interference with the NKG2D receptor In transgenic mice that constitutively expressed RAE-1 or MIC-A in all organs, or which specifically express RAE-1 in the skin, systemically downregulated NKG2D expression on NK cells was observed and NKG2D

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Figure 4 Schematic illustration of three different aspects of NKG2D/NKG2D ligands in cancer. (i) NKG2D-mediated immune response: NK and CD8 þ effector cells are activated after triggering with ligands, produce IFN-g and exert cytotoxic activity against the tumor. (ii) Immune escape: MIC-A interacts with ERp5 and is shed by MMPs from the cell surface, which subsequently leads to a downmodulation and/or blocking of NKG2D. Expression of NKG2D and its ligands is reduced by TGF-b or IFN-g. NKG2Dmediated activation of NK cells is suppressed by Treg. NKG2D þ CD4 þ T cells expand upon NKG2D ligand engagement and inhibit NKG2D þ CD4 þ T-cell proliferation via Fas/FasL. (iii) Immune therapy: ATRA or HDAC inhibitors upregulate the expression of NKG2D ligands on certain types of tumors. Cytokines enhance the expression of NKG2D and/or the activity of NK cells. Depletion of Treg counteracts inhibition of NK cell activity. CD8 þ T cells carrying a chimeric receptor of NKG2D-CD3z kill NKG2D ligand positive targets and elicit a Th1 immune response. ATRA, all-trans retinoic acid; DAP, DNAX-activating protein; ERp5, endoplasmic reticulum protein 5; HDAC, histone deacetylase; IFN, interferon; MIC, MHC class I-chain-related protein; MDSC, myeloid-derived suppressor cell; MMP, matrix metalloproteinase; NK, natural killer; TCR, T-cell receptor; TGF, transforming growth factor; Treg, regulatory T cell.

functions were impaired (Ogasawara et al., 2005; Oppenheim et al., 2005; Wiemann et al., 2005). In addition, transgenic mice expressing RAE-1 specifically in the skin were more susceptible to chemically induced tumors (Oppenheim et al., 2005). Whether local surfacebound expression of NKG2D ligands on tumors is indeed sufficient to mediate systemic downregulation of NKG2D receptor expression on infiltrating NK cells or T cells is currently unknown. High levels of soluble MIC-A are detected in the sera of transgenic mice overexpressing MIC-A (Wiemann et al., 2005). Soluble MIC was reported to effectively downregulate NKG2D expression (Groh et al., 2002). Interestingly, NKG2D expression of non-transgenic mice was not affected by soluble MIC-A from sera of transgenic mice, suggesting that the observed downregulation of the NKG2D receptor in transgenic mice might be mainly caused by the persistent exposure to cell-bound expressed MIC-A. Whether soluble RAE-1 is produced in RAE-1 transgenic mice as well (Oppenheim et al., 2005) and whether it might result in a systemic

NKG2D downregulation in mice have not been determined. It is possible that reduced expression of the adaptors DAP10 or DAP12, which are necessary for cell surface expression of NKG2D, might also lead to impaired activation of NK cells in response to NKG2D ligandexpressing targets. In this context, it was reported that upon chronic exposure of NK cells to NKG2D ligandexpressing tumors, expressions of NKG2D and its adaptors DAP10 and DAP12 were reduced (Coudert et al., 2005). Whether in MIC-A and RAE-1 transgenic mice the observed reduction of cell surface NKG2D expression is caused by an enhanced receptor internalization or owing to lower protein levels of the adaptors DAP10 or DAP12 was not addressed in these studies (Oppenheim et al., 2005; Wiemann et al., 2005). Immune evasion by interference with NKG2D ligands Initially, soluble MIC-A was detected in the sera of patients suffering from different types of cancer, Oncogene

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including breast and lung cancer (Groh et al., 2002). The appearance of soluble MIC-A was subsequently described in patients suffering from cancers of different origins, including leukemia (Salih et al., 2003), pancreatic carcinomas (Marten et al., 2006), hepatocellular cancer (Jinushi et al., 2005) and colon carcinoma (Doubrovina et al., 2003). Since soluble MIC-A and -B are found in substantial amounts in the sera of patients suffering from different cancers (Raffaghello et al., 2004; Marten et al., 2006; Salih et al., 2006; Waldhauer and Steinle, 2006), these proteins were suggested as diagnostic markers for cancer progression (Yokoyama, 2002). First studies confirmed that soluble MIC-A can be used as a diagnostic marker for cancer at early stages, whereas MIC-B serum levels are correlated with advanced cancer stages and metastasis (Holdenrieder et al., 2006a, b). The shedding of MIC-A and -B was reported to be mediated via metalloproteases owing to proteolytic cleavage of the extracellular parts (Salih et al., 2002, 2006). In addition, a recent report demonstrated that cell surface endoplasmic reticulum protein 5 (ERp5) is required for MIC-A shedding (Kaiser et al., 2007) by the formation of mixed disulfide complexes that release MIC-A. A recent study revealed that enhanced levels of circulating sMIC-A in patients suffering from multiple myeloma correlated with enhanced levels of ERp5 surface expression on CD138 þ plasma cells (Jinushi et al., 2008). Notably, ERp5 was identified as metastasis-promoting factor in a mouse breast cancer model, and enhanced levels of ERp5 expression were found in human surgical samples of invasive breast cancer (Gumireddy et al., 2008). In this study, the levels of MIC-A on tumor cells and sMIC-A in the sera of patients were not determined. Nevertheless, it is tempting to speculate that enhanced levels of ERp5 on tumor cells might lead to high levels of sMICA and thereby impair NK-cell-mediated elimination of primary tumors and metastases. Furthermore, soluble forms of ULBP2 and ULBP4 (also named as RAET1E2 or LETAL (lymphocyte effector cell toxicity activating ligand)) have been described (Salih et al., 2006; Waldhauer and Steinle, 2006; Cao et al., 2007). However, engagement of the NKG2D receptor by soluble ligands does not necessarily lead to the downregulation of cell surface expression of NKG2D (Waldhauer and Steinle, 2006). It is possible that soluble ligands might simply inhibit binding of cellbound ligands to the NKG2D receptor. Most ULBPs are inserted in the cell membrane by glycosyl-phosphatidylinositol anchors and are therefore sensitive to treatment with phosphatidylinositol-specific phospholipase C. In this context, Song et al. (2006a) demonstrated that shedding of ULBPs can be prevented by inhibition of phosphatidylinositol-specific phospholipase C. In contrast, Waldhauer et al. reported that ULBP2 is cleaved by metalloproteases and not by phospholipases (Waldhauer and Steinle, 2006). Thus, shedding of NKG2D ligands has multiple consequences on NKG2D-mediated responses: (i) reduction of ligand density on the tumor cell surface, (ii) downmodulation of the receptor on effector cells and Oncogene

(iii) blocking of the NKG2D-binding site for surfaceexpressed NKG2D ligands. Moreover, it was reported that after co-culture of NK cells with MIC-B þ target cells, an exchange of MIC-B and NKG2D occurs at the immune synapse, resulting in diminished NK-cellmediated cytotoxicity (Roda-Navarro et al., 2006). Whether this in vitro observation is of physiological relevance is debatable (Roda-Navarro and Reyburn, 2007). Cytokines impairing the function of NKG2D receptor and NKG2D ligands During antitumor immune responses, numerous cytokines are produced by immune and non-immune cells, as well as by the tumor cells, in the tumor microenvironment. Interestingly, IFN-g and -a downregulate the expression of the NKG2D ligand H60 on MCA sarcomas (Bui et al., 2006). In addition, there is evidence that MIC-A is also downregulated by IFN-g on human melanoma cell lines (Schwinn et al., manuscript submitted). Downmodulation of the NKG2D receptor was observed upon treatment of human NK cell lines with IFN-g (Zhang et al., 2005a). Therefore, high levels of IFN-g impair NK cell activation by several mechanisms. First, IFN-g downregulates the expression of NKG2D receptor and NKG2D ligands, thereby counteracting the activation of NK cells. Furthermore, IFN-g upregulates MHC class I on tumor cells, leading to inhibition of NK cells through their inhibitory MHC class I receptors. It will be important to define the exact sources and kinetics of IFN-g production in tumorbearing mice in vivo. Transforming growth factor (TGF)-b downregulates the expression of the NKG2D receptor in vitro (Castriconi et al., 2003; Song et al., 2006b) in mouse models (Dasgupta et al., 2005) and in cancer patients (Lee et al., 2004). Similarly, TGF-b decreases the expression of NKG2D ligands (Friese et al., 2004; Eisele et al., 2006). Substantial amounts of TGF-b are found in the sera of cancer patients, and cancer cells often produce TGF-b. In addition, several studies have demonstrated that regulatory T cells (Treg) are a source of TGF-b, which mediates their immunosuppressive function. Cells inhibiting NKG2D-mediated immune responses It is evident that NK cell activation is not only suppressed by signaling via inhibitory receptors but also by encountering suppressive cell populations. Suppression of cells of the adaptive immune system has attracted lots of attention. Comparably little is known about the cross-talk of NK cells with immunosuppressive cell populations. In this context, Ghiringhelli et al. (2005) reported that human Treg inhibited the cytolytic function and IFN-g secretion of NK cells after stimulation with IL-12. This inhibition was mediated by membrane-bound TGF-b. Importantly, Treg also caused a TGF-b-mediated downregulation of NKG2D on human and mouse NK cells, suggesting an inhibitory role for Treg in the NKG2D-mediated activation of NK

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cells. Indeed, after adoptive transfer of activated Treg into Rag1/ mice, development of lung metastasis was less efficiently controlled in a B16-RAE-1 þ model. In contrast, transfer of Treg did not influence the number of metastasis caused by the parental B16 tumor cells. Inhibition of NKG2D-dependent NK cell activation was also observed by Smyth et al. (2006). In this study, depletion of Treg resulted in tumor rejection of NKsensitive tumors, such as 3LL and RMA-S-RAE-1b. In contrast, no downregulation of NKG2D was observed, suggesting that Treg might act in several ways on NKG2D-dependent activation. Treg modulates NKG2D-mediated immune responses of not only NK cells but also CD8 þ T cells. In this context, it was reported that NKG2D on NKG2D þ CD8 þ CD11c þ T cells was involved in the antitumor response against B16 melanoma after application of an anti-4-1BB mAb and an anti-CD4 mAb, which deplete Treg and conventional CD4 þ T cells (Choi et al., 2007). In NK-depleted mice, the observed tumor regression after mAb treatment was reduced by the application of a neutralizing anti-NKG2D mAb, suggesting that Treg also suppress NKG2D-mediated effector functions of NKG2D þ CD8 þ CD11c þ T cells. A distinct population of potentially immunosuppressive NKG2D þ CD4 þ T cells was recently described in cancer patients (Groh et al., 2006). Interestingly, this population was expanded in patients bearing MIC þ , but not MIC, epithelial tumors. Also, in vitro expansion of NKG2D þ CD4 þ T cells required stimulation via TCR in the presence of soluble MIC-A. Importantly, NKG2D þ CD4 þ T cells inhibited proliferation of NKG2DCD4 þ T cells in vitro via FasL. Similar results were obtained with NKG2D þ CD8 þ T cells. Therefore, soluble MIC-A not only directly impairs NKG2Dmediated effector function of NK and CD8 þ T cells, but also facilitates the expansion of immunosuppressive NKG2D þ CD4 þ T cells to dampen antitumor immune responses. Whether NKG2D þ CD4 þ T cells also exist in mice bearing NKG2D ligand-positive tumors and whether they are of physiological importance in vivo are currently not known. Notably, there is also evidence that anti-CD3-stimulated T-cell proliferation is inhibited by soluble MIC-A or H60 owing to the production of IL-10. However, in this study, no involvement of the NKG2D receptor was observed (Kriegeskorte et al., 2005). Finally, myeloid-derived suppressor cells (MDSCs), which expand in tumor patients and tumor-bearing mice, are implicated in potent suppression of T-cell responses against tumors. Recently, Liu et al. (2007) reported that MDSCs inhibited the cytotoxicity of IL-2activated NK cells in vitro and repressed NK cell function in vivo. In contrast, we have evidence that MDSCs can activate NK cells to produce high amounts of IFN-g. We further observed that a MDSC subset expressed RAE-1 and that induction of IFN-g by MDSC required the interaction of NKG2D with its ligands (Nausch et al., manuscript submitted). Therefore, we conclude that MDSCs also activate certain effector functions of NK cells. Their exact role

during immune responses to NK-cell-sensitive tumors in vivo will be addressed in future studies. In summary, multiple mechanisms to escape NKG2D-dependent antitumor immune responses exist. Therefore, it is not surprising that the immune system often fails to control the outgrowth of NKG2D ligandpositive tumors. Nevertheless, the expression of NKG2D ligands marks transformed cells and alerts the immune system. Amplification of NKG2D-mediated antitumor immune responses and simultaneous circumvention of immune evasion should be objectives for the design of strategies for immune therapy of cancer.

Perspectives of immunotherapy exploiting immune cell activation via NKG2D To elicit successful antitumor immune responses, it is of overall importance to guide highly active cytolytic effector cells in high numbers in close proximity to the tumor cells. At the same time, tumors have to be visible to immune cells and rendered highly susceptible to their attack, which facilitate their rapid elimination. It was observed that tumor cells expressing high levels of NKG2D ligands were efficiently rejected by NK cells, whereas tumor cells expressing intermediate levels of NKG2D ligands were less immunogenic (Diefenbach et al., 2001). Therefore, it is a promising strategy to increase the density of NKG2D ligands on tumor cells to efficiently activate antitumor immune responses. Several pathways to upregulate NKG2D ligand expression exist as discussed in the sections ‘NKG2D ligands: expression and regulation’ and ‘NKG2D ligands: expression and regulation on transformed cells’. RA induces both human and mouse ligands. All-trans retinoic acid (ATRA) has been used in therapeutic protocols in the treatment of acute myeloid leukemic patients with promising results, in particular in patients with the acute promyelocytic leukemia subtype (Tallman, 2006). ATRA potently induces the differentiation of malignant leukemia cells, which is considered to be the main mode of action of ATRA. In addition, it was discovered recently that ATRA increases ULBP levels on the acute myeloid leukemia cell line HL-60 (Rohner et al., 2007). Furthermore, ULBP3 and MIC-A were upregulated by ATRA on leukemic B cells of patients suffering from chronic lymphocytic leukemia of B-cell type (Poggi et al., 2004). High expression of ULBP in leukemic B cells was correlated with a better prognosis in combination with high levels of IL-4 and circulating Vdelta1 T cells (Catellani et al., 2007). Apart from ATRA, trichostatin A and vitamin D3, which promote myeloid cell differentiation, also upregulate NKG2D ligands on acute myeloid leukemia cells (Rohner et al., 2007). Several agents used in classical chemotherapies or radiotherapies cause DNA damage, which is one of the pathways for the induction of NKG2D ligands (Gasser et al., 2005). It is tempting to speculate that the curative effect of these therapies is at least partially caused by an Oncogene

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immune response triggered by enhanced levels of NKG2D ligand expression. NKG2D ligands were also applied as DNA-based vaccines. Vaccination with H60-containing vectors, in combination with the tumor-associated antigen survivin, enhanced innate and adaptive immune cell activation in comparison with survivin alone. In this study, addition of the H60 vaccine enhanced the cross-talk between DC, CD8 þ T cells and NK cells, whereas CD4 þ T cells were suppressive (Zhou et al., 2005, 2006). Furthermore, it is desirable to enhance the expression levels of the NKG2D receptor and increase its functionality. Several cytokines effectively enhance NKG2D-mediated immune response. IFN-a, which is frequently used in cancer therapies including therapies against melanoma (reviewed in Jack et al. (2006)), upregulated NKG2D (Zhang et al., 2005a). The same group reported that NKG2D expression was enhanced in an NK cell line stably transfected with IL-15 (Zhang et al., 2004). Moreover, IL-2 and -12 were reported to amplify NKG2D-mediated tumor suppression of MCA-induced sarcomas (Smyth et al., 2004, 2005). IL-21, another member of the IL-2 cytokine family, enhances NKG2D-dependent tumor rejection, although expression levels of NKG2D in mice remain unaffected (Takaki et al., 2005). Interestingly, in human NK cells, downregulated expression of NKG2D was observed upon IL-21 treatment (Burgess et al., 2006). It will be interesting to assess the effect of therapies exploiting cytokines with regard to enhancing NKG2D-mediated effector functions. Recently, several studies investigated whether T cells can be manipulated to eliminate NKG2D ligandpositive tumor targets in vitro and in vivo. Teng et al. (2005) generated DAP12-expressing T cells by retroviral transduction of primary T cells. These DAP12 þ T cells specifically lysed RAE-1b-positive tumor cell targets. Adoptive transfer of DAP12 þ T cells into Rag-1/ mice bearing the RAE-1b-transfected RMA-S lymphoma enhanced survival of these mice. These data indicate that T cells can be converted to acquire NK-like function by overexpression of a single adaptor. As human NKG2D does not bind to DAP12 (Rosen et al., 2004), beneficial effects of DAP12 overexpression in T cells in response to NKG2D ligand-expressing targets are likely to be restricted to mouse tumor models. Using a similar approach, chimeric receptors of NKG2D or DAP10 linked to the cytoplasmic domain of CD3z were generated (Zhang et al., 2005b). Primary T cells transduced with these chimeric receptors efficiently killed NKG2D ligand-positive tumor cell targets and produced high amounts of Th1 cytokines and proinflammatory chemokines (Zhang et al., 2005b). In addition, upon adoptive transfer of NKG2D-CD3z T cells mixed with tumor cells or intravenously injected 1 day before tumor cell inoculation, RMA-RAE-1b tumor growth was inhibited and immunological memory against RMA cells was generated. Furthermore, NKG2D-CD3z T cells promoted long-term survival, if Oncogene

RMA lymphoma cells were injected intravenously (Zhang et al., 2007). In this model, IFN-g production by chimeric T cells was an absolute requirement for tumor rejection. In addition, GM-CSF and cytotoxicity mediated via FasL or perforin were involved in the observed antitumor activity of chimeric receptor-bearing T cells. A recent clinical trial showed that adoptive transfer of genetically engineered T cells with tumorspecific TCR led to cancer regression in some patients (Morgan et al., 2006). However, as only little is known about tumor-associated antigens, the use of TCRengineered T cells in immunotherapy is limited. Therefore, NKG2D-engineered T cells represent a promising alternative tool for the treatment of NKG2D ligandexpressing tumors. NKG2D-mediated activation of NK and CD8 þ T cells can also be beneficial in response to tumors, which express no or low levels of ligands. In these studies, Fab fragments of antibodies specific for tumor antigens, including carcinoembryonic antigen or CD20, were conjugated to recombinant soluble MIC-A proteins. These antibodies targeted tumor cells by recognizing the tumor-associated antigens and passively coated the tumor cells with MIC-A. Indeed, antibody-mediated coating of tumor cells with MIC-A sensitized tumor targets to NKG2D-dependent NK cell lysis. These data imply that NKG2D-mediated immune cell activation is applicable in response to a broad number of tumors (Germain et al., 2005). It is possible that strategies enhancing NKG2Dmediated immune activation benefit enormously by the simultaneous prevention of immune evasion. Currently, ONTAK, a fusion protein of IL-2 and diphtheria toxin, which reduces the numbers of Treg, is being evaluated in clinical trials in tumor patients (Barnett et al., 2005). It will be interesting to determine whether the elimination of Treg in patients enhances NK cell activity in response to NKG2D ligand-positive tumor targets.

Conclusion During the last 10 years, the activating receptor NKG2D and its ligands were extensively studied with regards to expression, regulation and function. It is commonly accepted that the interaction between this receptor and its multiple ligands, which are induced in ‘inflammation and cancer’, plays a pivotal role in the recognition of tumor cells. So far, most studies establishing the involvement of NKG2D in antitumor responses were performed with neutralizing antiNKG2D mAbs. Studies with NKG2D-deficient mice will reveal the exact role of NKG2D in tumor surveillance in response to spontaneously arising tumors. In addition, recent evidence emerged stating that the IL-15R couples to the NKG2D pathway, providing novel insight into the priming of NKG2D-mediated responses. Future studies are required to further elucidate the cross-talk of NKG2D and other activating receptors with cytokine receptors in different cell types. Together,

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efficient strategies of NKG2D-mediated immune therapy against cancer should aim at optimal activation of NKG2D, induction of the highest levels of NKG2D ligands on tumor cells and simultaneous counteraction of immune evasion. Recent studies mainly performed in tumor mouse models suggest that exploitation of the NKG2D/NKG2D ligand pathway could be a promising strategy for antitumor immune therapy.

Acknowledgements We thank Professor Lewis Lanier and Ioanna Galani for critically reading the paper. The work is supported by a Marie Curie Excellent Grant, Deutsche Jose´ Carreras Leuka¨mie Stiftung, German-Israel DKFZ/MOST cooperation and Boehringer Ingelheim Fonds. We want to apologize to all authors whose work could not be cited in this review because of space limitations.

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