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, activated NK cells sharing markers with dendritic cells member D) and its ligands play an important role in the (DCs), which are referred to as natural killer DCs NK, cd þ and CD8 þ T-cell-mediated immune response to or interferon (IFN)-producing killer DCs (Pillarisetty tumors. Ligands for NKG2D are rarely detectable on the et al., 2004; Chan et al., 2006; Taieb et al., 2006; surface of healthy cells and tissues, but are frequently Vosshenrich et al., 2007). In addition, NKG2D is expressed by tumor cell lines and in tumor tissues. It is present on the cell surface of all human CD8 þ T cells. evident that the expression levels of these ligands on target In contrast, in mice, expression of NKG2D is restricted cells have to be tightly regulated to allow immune cell to activated CD8 þ T cells (Ehrlich et al., 2005). In activation against tumors, but at the same time avoid tumor mouse models, NKG2D þ CD8 þ T cells prefer- destruction of healthy tissues. Importantly, it was recently entially accumulate in the tumor tissue (Gilfillan et al., discovered that another safeguard mechanism controlling 2002; Choi et al., 2007), suggesting that the activation via the receptor NKG2D exists. It was shown NKG2D þ CD8 þ T-cell population comprises T cells that NKG2D signaling is coupled to the IL-15 receptor involved in tumor cell recognition. Under non-patholo- pathway in a cell-specific manner suggesting that priming gical conditions, expression of NKG2D is not detectable of NKG2D-mediated activation depends on the cellular on a substantial proportion of human or mouse ab microenvironment and the distinct cellular context. This CD4 þ T cells. In contrast, NKG2D-expressing CD4 þ review will provide a broad overview of our up-to-date T cells accumulate in patients with MIC þ tumors (Groh knowledge of the NKG2D receptor and its ligands in the et al., 2006) as well as those suffering from autoimmune context of tumor immunology. Strategies to amplify diseases, including rheumatoid arthritis or Crohn’s NKG2D-mediated antitumor responses and counteract disease (Groh et al., 2003; Allez et al., 2007). tumor immune escape mechanisms will be discussed. Recently, klrk1À/À mice that are deficient in NKG2D Oncogene (2008) 27, 5944–5958; doi:10.1038/onc.2008.272 expression were generated (Guerra et al., 2008). These mice developed normal numbers of NK cells and other Keywords: NKG2D; RAE-1; MIC-A; ULBP; NK cells; lymphocyte subsets in all analysed organs. Moreover, no tumor immunology 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 dispen- sable for normal phenotypic development of NK cells. Expression of the NKG2D receptor NKG2D is a type II transmembrane protein (Garrity et al., 2005) and does not contain any known signaling The receptor NKG2D (natural-killer group 2, member motif within its intracellular domain (Houchins et al., D) belongs to the family of C-type lectin-like receptors. 1991). For signal transduction, NKG2D associates, like Therefore, NKG2D has been designated klrk1/KLRK1 many other activating receptors, with adaptor proteins (killer cell lectin-like receptor subfamily K, member 1). via charged residues in its transmembrane domain. As the gene encoding this receptor resides in the NKG2 Both human and mouse NKG2D form a complex with (natural killer group 2) complex, the gene product was DNAX-activating protein of 10 kDa (DAP10) (Wu originally designated as nkg2d/NKG2D. The NKG2 et al., 1999). In humans, NKG2D associates with complex is located on chromosome 6 in mice (Ho et al., DAP10 alone, and not with DAP12 (Rosen et al., 1998) and on chromosome 12 in humans (Glienke et al., 2004) (Figure 1). In addition, in mice, NKG2D binds to 1998). the adaptor DAP12, which is also known as killer cell- NKG2D is expressed by all NK cells, most NKT cells activating receptor-associated polypeptide or tyrosine and subsets of gd þ T cells (Wu et al., 1999; Jamieson kinase-binding protein (Paloneva et al., 2000; Diefenbach et al., 2002). NKG2D is also expressed by a subset of 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 Correspondence: Dr A Cerwenka, Division of Innate Immunity, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 structure (Garrity et al., 2005). Moreover, in mice, Heidelberg, Germany. NKG2D exists in two distinct splice variants. The long E-mail: [email protected] isoform (NKG2D-L) has 13 additional amino acids NKG2D in antitumor responses N Nausch and A Cerwenka 5945 Mouse Human

? NKG2D-L NKG2D-L NKG2D-S NKG2D-S NKG2D DAP

DAP10 DAP12 DAP DAP12 or or 10 YXXM YXXM P 10 YXXM I P I PP T PP P P

T Syk Grb2 Grb2 ZAP-70 Syk A Grb2

ZAP-70 PI3K A PI3K M P PI3K MP

Cytotoxicity Cytotoxicity Cytotoxicity IFN-γ IFN-γ IFN-γ

Expression on:

CD8+ T cells: Not present Activated/memory Not present Activated/memory Resting/activated

NK cells: 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 in T cells PI3K activation is a hallmark of co- the short variant (NKG2D-S) (Diefenbach et al., 2002). stimulatory receptors including CD28, NKG2D was Initially, it was reported that NKG2D-L does not bind originally described as a co-stimulatory molecule, to DAP12, whereas NKG2D-S associates with DAP10 augmenting T-cell receptor (TCR) signaling (Bauer and DAP12 (Diefenbach et al., 2002). In contrast, a et al., 1999; Diefenbach et al., 2002; Ehrlich et al., more recent study concluded that DAP10 and DAP12 2005). Under certain circumstances, however, NKG2D- compete equally for both variants of NKG2D mediated effector functions in T cells are independent of (Rabinovich et al., 2006). Since both studies relied on TCR engagement. For example, target cell killing of transfection of reporter cells in their association studies, long-term cultured or cloned CD8 þ T cells was shown a final proof in primary NK cells is still missing. It has to be mediated via NKG2D (Maccalli et al., 2003; been reported that NKG2D-L is expressed by resting Verneris et al., 2004). In addition, chronically activated NK cells, whereas the short isoform is induced upon intraepithelial CD8 þ T cells isolated from patients activation. Therefore, depending on the activation state suffering from celiac disease killed target via NKG2D of the NK cell, NKG2D signals via distinct intracellular without any contribution of TCR (Roberts et al., 2001; pathways to elicit effector function. DAP10 carries a Meresse et al., 2004). We conclude that cell-specific YXXM motif (Tyr-X-X-Meth) within the cytoplasmic features and the physiological context determine domain, which recruits phosphoinositide kinase-3 whether NKG2D acts as a stimulatory or a (PI3K) and growth factor receptor-bound protein 2 co-stimulatory molecule. 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 Priming of NKG2D-mediated effector functions protein kinase 70) and Syk (spleen tyrosine kinase). Therefore, in activated mouse NK cells, two different Exposure of purified NK cells to NKG2D ligand- pathways are triggered via NKG2D resulting in a expressing targets in vitro results only in low levels of PI3K- or Syk/ZAP70-dependent signaling cascade, target cell killing (Bryceson et al., 2006), indicating that respectively. for optimal triggering of NKG2D-mediated effector In CD8 þ T cells, which generally lack DAP12 functions, additional signals are required. In fact, expression, NKG2D binds to exclusively DAP10. As in vitro culture of NK cells in the presence of interleukin

Oncogene NKG2D in antitumor responses N Nausch and A Cerwenka 5946 (IL)-15 or high doses of IL-2 signiﬁcantly increases memory CD8 þ T cells isolated from celiac disease NKG2D-mediated killing of tumor targets. Recently, patients are activated via NKG2D in a TCR-indepen- Horng et al. (2007) demonstrated that the IL-15 receptor dent manner (Hue et al., 2004; Meresse et al., 2004). pathway couples to NKG2D signaling in NK cells There is evidence that this TCR-independent, NKG2D- (Figure 2). Triggering of the IL-15 receptor resulted in mediated activation is primed by a chronic exposure to phosphorylation of DAP10 by janus kinase 3, which IL-15 in vivo. In vitro, IL-15 not only upregulates the facilitated downstream signaling and NKG2D-mediated expression of NKG2D and DAP10 on CD8 þ T cells, effector function. Whether other cytokines activate the but also facilitates NKG2D-mediated signal transduc- NKG2D pathway in a similar fashion and whether tion via the induction of constitutive extracellular signal- other activating receptors are also controlled in a similar regulated kinase phosphorylation. Inhibitor studies manner by cytokines are currently unknown. One source revealed that phosphorylations of extracellular signal- of IL-15 is DCs, which produce IL-15 after exposure to regulated kinase and c-Jun N-terminal kinase are critical toll-like receptor ligands in vivo and subsequently in the NKG2D-induced cytotoxicity of CD8 þ T cells. present IL-15 on their cell surface to NK cells in Therefore, in CD8 þ T cells as well, IL-15 or high doses draining lymph nodes (Lucas et al., 2007). Whether this of IL-2 prime the NKG2D pathway and converts CD8 þ trans-presentation of IL-15 by DC is of importance T cells into NK-like lymphokine-activated killer cells during NK cell-mediated antitumor immune responses (Meresse et al., 2004). The exact mechanism of action of has not yet been addressed. IL-15-mediated priming of CD8 þ T cells is still elusive. Interestingly, naı¨ ve CD8 þ T cells from DAP10-Ub Taken together, there is increasing evidence that transgenic mice show no defects in IL-15 responsiveness, signaling initiated by activating receptors is not only suggesting that activation of NKG2D-mediated effector controlled by inhibitory receptors, as previously antici- function is controlled in a cell-speciﬁc manner depen- pated, but requires additional factors for optimal dent on the activation status. Nevertheless, effector/ activation.

IL - 15 P P PI P 3K Jak3

P P PI3K PI3K ERK P ERK P

Cytotoxicity Cytotoxicity

NKG2D ligand

NKG2D TLR

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 NKG2D in antitumor responses N Nausch and A Cerwenka 5947 NKG2D ligands ULBP3, bind to the UL16 glycoprotein of cytomegalo- virus (Cosman et al., 2001; Steinle et al., 2001; Chalupny NKG2D binds to a variety of ligands, which resemble et al., 2003). In total, 10 members of this gene family major histocompatibility complex (MHC) class I pro- were mapped, but only 6 RAET1 family members were teins (Figure 3) (Bauer et al., 1999; Cerwenka et al., shown to be expressed as functional proteins (Rado- 2000; Diefenbach et al., 2000). In mice, most ligands savljevic et al., 2002) (Figure 3). Two members of the belong to the family of retinoic acid early inducible-1 RAET1 family are anchored to the surface by a (RAE-1) proteins. So far, five family members, transmembrane region (RAET1E and G), whereas other RAE-1a–e, have been described. Whether all ligands can RAET1 molecules are linked to the cell membrane via be induced by RA is currently not known. It was glycosyl-phosphatidylinositol anchors (Bacon et al., suggested that the genes of the rae-1 family are 2004). In humans, an additional family of NKG2D differentially distributed in different mouse strains. There ligands, MHC class I-chain-related proteins A and B is evidence that rae-1d and -e exist in C57BL/6, whereas (MIC-A and -B), exists, which in addition to the a1and BALB/c and 129/J can express rae-1a, d and g (Ogasawara a2 domain has a third Ig-like domain (a3) (Bauer et al., and Lanier, 2005). It is not known whether this is a matter 1999; Bahram et al., 1994). MIC-A and -B are highly of different genes in different mouse strains or simply of polymorphic. So far, 61 MIC-A alleles and 30 MIC-B allelic variants. A documentation of the expression and alleles have been identified, which are listed on http:// genomic organization of rae-1 genes in different mouse www.anthonynolan.com/HIG/seq/nuc/text/. Some of strains should be the focus of future studies. these alleles differ in their ability to bind to the NKG2D The RAE-1 proteins consist of two Ig-like domains receptor (Steinle et al., 2001). Expression of certain (a1 and a2) and are attached to the cell membrane via a alleles might contribute to inter-individual differences in glycosyl-phosphatidylinositol anchor (Figure 3). In susceptibility to certain diseases (Bravo et al., 2007; contrast, MULT-1 (murine UL16-binding protein like Turkcapar et al., 2007). transcript-1) (Carayannopoulos et al., 2002; Diefenbach In humans and mice, a single receptor, NKG2D, et al., 2003) and the minor histocompatibility antigen binds to several distinct ligands. Why multiple ligands H60 (Malarkannan et al., 1998; Cerwenka et al., 2000; exist for this one, invariant, receptor is still not well Diefenbach et al., 2000), which bind to NKG2D in mice, understood; however, there are several possibilities that have a transmembrane region. Recently, two additional might account for this complexity (Cerwenka and variants of H60 were identified and named as H60b and Lanier, 2001; Eagle and Trowsdale, 2007). First, H60c (Takada et al., 2008). In contrast to H60(a), which different ligands bind to NKG2D with distinct affinities. is expressed in BALB/c but not in C57BL/6 mice, Therefore, engagement of NKG2D by different transcripts of H60b and H60c were found in both NKG2D ligands might fine-tune the extent of activa- C57BL/6 and BALB/c strains (Malarkannan et al., tion. The affinity of mouse ligands to NKG2D ranges 1998; Takada et al., 2008). H60c transcripts were between 2 and 700 nM (KD) and the affinity of the detected mainly in the skin, whereas H60a and H60b human ligands between 600 nM and 1.1 mM, respectively transcripts had a broader tissue distribution. In humans, (O’Callaghan et al., 2001; Strong, 2002; Mistry and RAET1 family members, which are orthologs of the O’Callaghan, 2007). Although striking differences in mouse RAE-1 proteins, have been described (Cosman binding affinities between different ligands exist, the et al., 2001; Radosavljevic et al., 2002). The RAET1 observed binding affinities in general are quite strong, as proteins were initially referred to as UL16-binding compared with other receptor/ligand interactions, in- proteins (ULBPs) and later renamed in accordance with cluding KIRs and MHC class I (KD ¼ 10 mM) (Strong, their mouse counterparts. In addition, the name ULBPs 2002). Second, there are hints that additional receptors is misleading, because only ULBP1 and ULBP2, but not for NKG2D ligands exist. In particular, Kriegeskorte

MHC I Human NKG2D-ligands Mouse NKG2D-ligands

MIC A, B RAET1I,H,N RAET1E,G RAE-1α–ε MULT-1, H60

α1 α2 α1 α2 α1 α2 α1 α2 α1 α2 α1 α2

β α3 2m α3

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 NKG2D in antitumor responses N Nausch and A Cerwenka 5948 et al. (2005) observed that soluble H60 and MIC-A lymph nodes (Cosman et al., 2001). In addition, ULBP4 inhibited anti-CD3-stimulated T-cell proliferation. This RAET1E is specifically transcribed in the skin (Cha- suppression of T-cell proliferation required IL-10, but lupny et al., 2003). Total tissue scans of MIC-A and -B was not blocked by the addition of a monoclonal of healthy individuals revealed that with the exception antibody (mAb) directed against NKG2D. So far, the of the central nervous system both genes are widely receptor responsible for this observation has not been transcribed (Schrambach et al., 2007). The extent to described. Third, it is feasible that an evolutionary which these ligands are expressed at protein levels is pressure to avoid escape mechanisms devised by certain unknown and has to be addressed in further studies with viruses to inhibit NKG2D ligand expression in virus- validated antibodies. However, it is likely that expres- infected cells may be driving the diversity of these sion of these proteins is tightly regulated at a post- ligands (Bahram et al., 2005). Moreover, the existence of transcriptional or possibly post-translational level. multiple ligands might also be of advantage to counter- The NKG2D ligands can be upregulated by multiple act escape mechanisms of transformed cells to NKG2D- stimuli, including infection by bacteria or viruses or by mediated antitumor responses. Finally, tissue-specific cellular transformation. In Table 1, stimuli leading to expression of NKG2D ligands and their differential the upregulation of human and mouse NKG2D ligands induction requirements might account for the necessity are summarized. RAE-1 is induced on mouse macro- of multiple ligands for one single receptor, as discussed phages after stimulation with toll-like receptor ligands in the following two sections. (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). NKG2D ligands: expression and regulation Surface expression of NKG2D on NKT cells and its ligand RAE-1 on hepatocytes are modulated upon The RAE-1 molecules were first described to be induced hepatitis B virus infection (Vilarinho et al., 2007). after treatment of the embryonic testicular teratoma cell Moreover, in certain cells and under distinct experi- line F9 with RA (Nomura et al., 1996; Zou et al., 1996). mental conditions, MIC-A and -B are inducible by heat Interestingly, similar to the mouse ligands, human shock (Groh et al., 1996). In addition, T cells activated MIC-A and -B proteins are also upregulated on by antigen-presenting cells, or T-cell blasts stimulated hepatocellular carcinoma cells upon treatment with with super antigen, anti-CD3/anti-CD28 in combination RA (Jinushi et al., 2003b). Indeed, expression of rae-1 with PMA, or infected with HIV, upregulate NKG2D mRNA was observed during embryogenesis from days 9 ligands (Diefenbach et al., 2000; Molinero et al., 2002; to 11, but was hardly detectable in healthy adult tissues. Rabinovich et al., 2003; Cerboni et al., 2007; Ward High levels of rae-1 are expressed in the brain of et al., 2007). The induction of MIC-A on TCR- embryos (Nomura et al., 1996). Furthermore, expression stimulated T cells involves activation of the transcrip- of RAE-1 is detected on the cell surface of embryonic tion factor nuclear factor-kB (Molinero et al., 2004). stem cells, which is further enhanced upon treatment In summary, these studies imply an important role of with RA (unpublished observation). Whether RAE-1 NKG2D and its ligands in infectious diseases, as proteins are expressed on the cell surface during reviewed elsewhere (Hamerman et al., 2005; Ogasawara embryogenesis and whether they are implicated in and Lanier, 2005). Since inflammatory diseases and developmental processes are currently unknown. infection with certain viruses are often linked to In BALB/c, but not in C57BL/6, mice, the expression malignant transformation, regulation of NKG2D li- of RAE-1 and, to a lesser extent, H60 was described on gands by infectious agents might increase the visibility Gr-1 þ and CD11b þ bone marrow cells that were of tumor cells to effector cells of the immune system. 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 NKG2D ligands: expression and regulation on irradiation-resistant NK cells in a C57BL/6 Â BALB/c transformed cells F1 recipient (Ogasawara et al., 2005). Similarly, in humans, NKG2D ligands are expressed at low levels on NKG2D ligands are expressed by a wide range of tumor CD34 þ hematopoietic stem cells and expression of the cell lines of different tissue origins. Mouse NKG2D RAET1 proteins is enhanced upon differentiation into ligands are detectable on T- and B-cell lymphoma cell myeloid progenitors (Guilloton et al., 2005; Nowbakht lines, including YAC-1, WEHI7.1 and A20, and on et al., 2005). multiple carcinoma cell lines, such as the lung carcinoma In healthy adult mice, NKG2D ligands are expressed lines LL/2 and MLg, rectum carcinoma line CMT, colon at low levels on the cell surface of hepatocytes carcinoma line MC38, prostate carcinoma line TRAMP (Vilarinho et al., 2007and our unpublished observa- and the Renca renal carcinoma cell line (Cerwenka tions) and on CD4 þ 8 þ double-positive thymocytes in et al., 2000; Diefenbach et al., 2000; Smyth et al., 2004). BALB/c mice, respectively (Li et al., 2005). In humans, Expression of the NKG2D ligands is not a ubiquitous RAET1 transcripts are detectable in different healthy feature of tumor cell lines, because the lymphoma cell tissues, including kidney, prostate, uterus, tonsil and lines RMA and RMA-S, the C57BL/6 melanoma cell

Oncogene 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 rae-1 Nomura et al. (1996) RAE-1a Unpublished observations RA Hepatocellular carcinoma tissue MIC-A/B Jinushi et al. (2003b) and cell lines TLR ligands Macrophages RAE-1a–e Hamerman et al. (2004) LPS Macrophages ULBP1/2/3 Nedvetzki et al. (2007) LPS B cells (BALB/c) NKG2D mouse ligands Diefenbach et al. 2000) Mycobacterium tuberculosis Macrophages RAE-1 Rausch et al. (2006) Pseudomonas aeruginosa Airway epithelial cell, macrophages RAE-1, ULBP2 Borchers et al. (2006) Influenza A, Sendai infection Macrophages MIC-A Siren et al. (2004) Escherichia coli Epithelial cell lines MIC-A Tieng et al. (2002) Super antigen, antigens T cells MIC-A, ULBP1/2/3 Cerboni et al. (2007) HIV T cells ULBP1/2/3 Ward et al. (2007) aCD3/CD28/PMA T cells MIC-A Molinero et al. (2002) ConA, APC, PMA T cells NKG2D mouse ligands Diefenbach et al. (2000); Rabinovich et al. (2003) EBV infection B cells in lytic cycle ULBP1 Pappworth et al. (2007) IFN-a DC MIC-A/B Jinushi et al. (2003a) Differentiation Myeloid progenitors ULBP1 Nowbakht et al. (2005) ULBP2/3 Guilloton et al. (2005) Heat shock Epithelial cells MIC-A/B Groh et al. (1996) E1A (serotype 5) Human, mouse tumor cells, primary RAE-1, MIC and ULBPs Routes et al. (2005) baby mouse kidney fibroblasts BCR/ABL Leukemic, hematopoietic CD34+ MIC Boissel et al. (2006) DNA damage Human and mouse fibroblasts RAE-1, ULBP1/2/3, MIC-A Gasser et al. (2005) JunB/AP-1 Mouse embryonic fibroblasts, RAE-1e, MULT-1 Nausch et al. (2006) endothelioma cells HDAC AML hepatoma cells MIC-A/B, ULBP1 MIC-A/B Armeanu et al. (2005); Diermayr et al. (2007)

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 tumor entity studied. This observation was further transgenic MT-ret mice lack expression of NKG2D extended by a study analysing NKG2D ligand expres- ligands (Cerwenka et al., 2000, 2001; Diefenbach et al., sion in ovarian carcinomas. Of six samples of freshly 2000; unpublished observation). Furthermore, differ- isolated ovarian carcinoma cells, one expressed MIC-A, ences in expression levels of NKG2D ligands are two ULBP2 and two ULBP3. In one isolate, no observed on tumor cell lines derived from progressing NKG2D ligands were detectable. Furthermore, tumors isolated from different mice. In this context, MIC-A and ULBP3 were expressed at low densities, Smyth et al. (2005) reported that only one-third of whereas expression levels of ULBP2 were relatively high MCA-induced sarcoma cell lines derived from wild-type (Carlsten et al., 2007). In addition, it was reported that mice expressed RAE-1 molecules on the cell surface and myeloma cell lines express NKG2D ligands and that the levels of expression varied. These data suggest that NKG2D was one of the key NK-activating receptors for tumor editing by the immune system might occur. In bone marrow-derived myeloma cell recognition (Car- fact, in mice lacking perforin, all generated tumor lines bone et al., 2005; El-Sherbiny et al., 2007). expressed RAE-1, indicating that elimination via perfor- It is important to understand the factors leading to in was responsible for the observed variable expression NKG2D ligand expression in tumor cells. As NKG2D of NKG2D ligands in situ (Smyth et al., 2005). ligands are rarely expressed on the cell surface of adult The human RAET1 proteins were reported to be healthy cells, their expression is likely to be associated expressed by tumors of different origin, including with malignant transformation and the process of leukemia, gliomas and melanomas (Pende et al., 2002; tumorigenesis. Cell transformation by certain oncogenes Friese et al., 2003; Salih et al., 2003). MICs are including K-ras, c-myc, Akt, E1A or Ras V12, or their expressed by an even broader range of tumors and were combinations, does not directly force expression of detected in leukemia, various carcinomas (breast, lung, NKG2D ligands. These data suggest that additional colon, kidney, ovary and prostate), gliomas, neuroblas- events are required for NKG2D ligand expression tomas and melanomas (Groh et al., 1999; Pende et al., (Gasser et al., 2005). To mimic tumorigenesis, trans- 2002; Vetter et al., 2002; Friese et al., 2003; Salih et al., formed ovarian cell lines were implanted in the ovary of 2003; Watson et al., 2006; Castriconi et al., 2007). In nude mice, which resulted in the development of tumors general, as in mice, the proportions of ligand-positive (Gasser et al., 2005). Cell lines derived from these tumors, the levels of ligand expression and the types of tumors expressed signiﬁcant amounts of NKG2D ligands expressed are heterogeneous between different ligands. Whether primary tumors directly isolated from tumor types and between individual samples of one mice expressed NKG2D ligands in situ was not

Oncogene NKG2D in antitumor responses N Nausch and A Cerwenka 5950 investigated in this study. In contrast, other studies The relevance of NKG2D and its ligands in cancer showed that overexpression of the oncogenes adenovirus E1A or BCR/ABL by itself resulted in expression of Initially, Bauer et al. (1999) reported that target cells RAE-1 or MIC-A, respectively (Routes et al., 2005; ectopically expressing the NKG2D ligand MIC-A are Boissel et al., 2006). Therefore, the role of oncogenes in efficiently killed via NKG2D despite the expression of the induction of NKG2D ligands is likely to be MHC class I molecules (Groh et al., 1999). Mouse NK influenced by additional factors, such as the origin of cells also efficiently eliminated MHC class I-positive the transformed cell line. The detailed molecular RMA cells that were transduced with the NKG2D mechanisms leading to the upregulation of NKG2D ligands H60 or RAE-1 (Cerwenka et al., 2000; ligands by different oncogenes are not completely Diefenbach et al., 2000). These data suggested that understood. NKG2D ligands would also play an important role in The activation of NKG2D ligand expression during tumor rejection in vivo. Indeed, ectopic expression of transformation is a multifaceted process, which is NKG2D ligands on tumor cells overcomes MHC class controlled at several levels. Indeed, we have shown that I-dependent inhibition of NK cells and enables NK cells expression of RAE-1e and MULT-1 are enhanced in to reject tumors in vivo (Cerwenka et al., 2001; mouse embryonic fibroblasts deficient in JunB, a subunit Diefenbach et al., 2001). of the transcription factor AP-1 (Nausch et al., 2006). Subsequently, Girardi et al. (2001) reported that the Notably, recently an AP-1-binding site was also exposure of skin to carcinogens rapidly induced expres- reported in the promoter of MIC-A (Venkataraman sion of RAE-1, whereas in healthy skin, NKG2D et al., 2007). AP-1 is involved in the regulation of ligands were not detectable. Interestingly, skin-asso- complex biological processes, for example, proliferation, ciated NKG2D þ gd þ T cells killed skin carcinoma cells differentiation and apoptosis, which are often dysregu- in vitro, which was blocked by a mAb directed against lated during tumorigenesis (Angel et al., 2001). There- NKG2D. These data suggested an important role of fore, AP-1 plays a pivotal role in the development of NKG2D/NKG2D ligands during the early immune tumors. It appears reasonable that the expression of response against spontaneously arising tumors. It was RAE-1 is linked to such a central ‘sensor’ of transfor- reported that the neutralization of NKG2D by using a mation to make transformed cells visible to the immune mAb increased the frequency of mice developing MCA- system. Furthermore, it is well established that the DNA induced sarcomas (Smyth et al., 2005), confirming the damage pathway is activated in many tumors. Key impact of the NKG2D/NKG2D ligand interaction players in the DNA damage response are the ATM during antitumor immune responses in vivo. In addition, (ataxia telangiectasia, mutated) and ATR (ATM- and these data suggest that the outgrowth of transformed Rad3-related) kinases. Gasser et al. (2005) have recently cells can be prevented by an NKG2D-dependent reported that DNA damage induces surface expression clearance mechanism early during tumor development. of NKG2D ligands, which was dependent on both ATM NKG2D-deficient mice crossed to TRAMP mice that and ATR kinases. Also, activation of the DNA damage spontaneously develop prostate adenocarcinoma pathway can be interpreted as a ‘sensor’ for transformed showed a higher incidence of aggressive early-arising and stressed cells, which are marked for destruction by carcinomas, as compared with control mice (Guerra immune cells via NKG2D. et al., 2008). Moreover, an earlier onset of c-myc- It is possible that multiple mechanisms cooperate in expressing lymphoma was observed in NKG2D-defi- the upregulation of NKG2D ligands. DNA damage cient Em-myc mice. These results confirm that NKG2D indirectly activates c-Jun N-terminal kinase leading to plays a critical role in tumor surveillance in vivo. Tumors phosphorylation of c-Jun (Roos and Kaina, 2006). We expressing NKG2D ligands can develop in fully have evidence that c-Jun antagonizes JunB and up- immune-competent animals. Therefore, we suspect that regulates RAE-1 expression (unpublished observations). mechanisms exist in tumor-bearing mice and cancer Furthermore, it was reported that AP-1 acts down- patients that impair NKG2D-dependent effector func- stream of the ATM kinase, which is involved in the tions of NK cells, or, alternatively, tumors are likely to DNA damage response (Weizman et al., 2003). There- exploit mechanisms to evade NK cells by targeting fore, AP-1 and the DNA damage pathway might pathways other than NKG2D. The different aspects of cooperate in the upregulation of NKG2D ligands. NKG2D-mediated responses in cancer are illustrated in Furthermore, there is evidence that histone deacetylase Figure 4. 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. Immune evasion from NKG2D-dependent immune Taken together, our understanding of the mechanisms response leading to the expression of NKG2D ligands is in its infancy. It is possible that the existence of multiple Immune evasion by interference with the NKG2D receptor NKG2D ligands (as discussed in the section ‘NKG2D In transgenic mice that constitutively expressed RAE-1 ligands’) reflects their ability to become induced by or MIC-A in all organs, or which specifically express certain stimuli and pathways ensuring visibility of stressed RAE-1 in the skin, systemically downregulated NKG2D cells to immune cells under different circumstances. expression on NK cells was observed and NKG2D

Oncogene NKG2D in antitumor responses N Nausch and A Cerwenka 5951 (i) Immune response

NK +

CD8 NK IFN-γ, Perforin (ii) Immune escape (iii) Immune therapy

TGF-β ATRA γ IFN- HDAC inhibitor

NK ERp5 Tumor IL-15, IL-2, NK MMPs IL-12, IL-18 ? IL-21 ?

CD8 IFN-γ, Perforin Treg + CD4 ζζ ζ ζ + ζ NKG2D ζ NK ericR+ Chim CD8

? CD4+ MICs T reg NKG2D- RAE-1 / RAET-1 MDSC NKG2D

DAP10/DAP12

TCR

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. NKG2D- þ þ mediated activation of NK cells is suppressed by Treg. NKG2D CD4 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 þ of Treg counteracts inhibition of NK cell activity. CD8 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; NKG2D downregulation in mice have not been Oppenheim et al., 2005; Wiemann et al., 2005). In determined. addition, transgenic mice expressing RAE-1 specifically It is possible that reduced expression of the adaptors in the skin were more susceptible to chemically induced DAP10 or DAP12, which are necessary for cell surface tumors (Oppenheim et al., 2005). Whether local surface- expression of NKG2D, might also lead to impaired bound expression of NKG2D ligands on tumors is activation of NK cells in response to NKG2D ligand- indeed sufficient to mediate systemic downregulation of expressing targets. In this context, it was reported that NKG2D receptor expression on infiltrating NK cells or upon chronic exposure of NK cells to NKG2D ligand- T cells is currently unknown. expressing tumors, expressions of NKG2D and its High levels of soluble MIC-A are detected in the sera adaptors DAP10 and DAP12 were reduced (Coudert of transgenic mice overexpressing MIC-A (Wiemann et al., 2005). Whether in MIC-A and RAE-1 transgenic et al., 2005). Soluble MIC was reported to effectively mice the observed reduction of cell surface NKG2D downregulate NKG2D expression (Groh et al., 2002). expression is caused by an enhanced receptor inter- Interestingly, NKG2D expression of non-transgenic nalization or owing to lower protein levels of the mice was not affected by soluble MIC-A from sera of adaptors DAP10 or DAP12 was not addressed in these transgenic mice, suggesting that the observed down- studies (Oppenheim et al., 2005; Wiemann et al., 2005). regulation 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 Immune evasion by interference with NKG2D ligands produced in RAE-1 transgenic mice as well (Oppenheim Initially, soluble MIC-A was detected in the sera of et al., 2005) and whether it might result in a systemic patients suffering from different types of cancer,

Oncogene NKG2D in antitumor responses N Nausch and A Cerwenka 5952 including breast and lung cancer (Groh et al., 2002). The (iii) blocking of the NKG2D-binding site for surface- appearance of soluble MIC-A was subsequently de- expressed NKG2D ligands. Moreover, it was reported scribed in patients suffering from cancers of different that after co-culture of NK cells with MIC-B þ target origins, including leukemia (Salih et al., 2003), pancrea- cells, an exchange of MIC-B and NKG2D occurs at the tic carcinomas (Marten et al., 2006), hepatocellular immune synapse, resulting in diminished NK-cell- cancer (Jinushi et al., 2005) and colon carcinoma mediated cytotoxicity (Roda-Navarro et al., 2006). (Doubrovina et al., 2003). Since soluble MIC-A and Whether this in vitro observation is of physiological -B are found in substantial amounts in the sera of relevance is debatable (Roda-Navarro and Reyburn, patients suffering from different cancers (Raffaghello 2007). et al., 2004; Marten et al., 2006; Salih et al., 2006; Waldhauer and Steinle, 2006), these proteins were Cytokines impairing the function of NKG2D receptor and suggested as diagnostic markers for cancer progression NKG2D ligands (Yokoyama, 2002). First studies confirmed that soluble During antitumor immune responses, numerous cyto- MIC-A can be used as a diagnostic marker for cancer at kines are produced by immune and non-immune cells, as early stages, whereas MIC-B serum levels are correlated well as by the tumor cells, in the tumor microenviron- with advanced cancer stages and metastasis (Holdenrie- ment. Interestingly, IFN-g and -a downregulate the der et al., 2006a, b). The shedding of MIC-A and -B was expression of the NKG2D ligand H60 on MCA reported to be mediated via metalloproteases owing to sarcomas (Bui et al., 2006). In addition, there is evidence proteolytic cleavage of the extracellular parts (Salih that MIC-A is also downregulated by IFN-g on human et al., 2002, 2006). In addition, a recent report melanoma cell lines (Schwinn et al., manuscript demonstrated that cell surface endoplasmic reticulum submitted). Downmodulation of the NKG2D receptor protein 5 (ERp5) is required for MIC-A shedding was observed upon treatment of human NK cell lines (Kaiser et al., 2007) by the formation of mixed disulfide with IFN-g (Zhang et al., 2005a). Therefore, high levels complexes that release MIC-A. A recent study revealed of IFN-g impair NK cell activation by several mechan- that enhanced levels of circulating sMIC-A in patients isms. First, IFN-g downregulates the expression of suffering from multiple myeloma correlated with en- NKG2D receptor and NKG2D ligands, thereby coun- hanced levels of ERp5 surface expression on CD138 þ teracting the activation of NK cells. Furthermore, plasma cells (Jinushi et al., 2008). Notably, ERp5 was IFN-g upregulates MHC class I on tumor cells, leading identified as metastasis-promoting factor in a mouse to inhibition of NK cells through their inhibitory MHC breast cancer model, and enhanced levels of ERp5 class I receptors. It will be important to define the exact expression were found in human surgical samples of sources and kinetics of IFN-g production in tumor- invasive breast cancer (Gumireddy et al., 2008). In this bearing mice in vivo. study, the levels of MIC-A on tumor cells and sMIC-A Transforming growth factor (TGF)-b downregulates in the sera of patients were not determined. Never- the expression of the NKG2D receptor in vitro theless, it is tempting to speculate that enhanced levels of (Castriconi et al., 2003; Song et al., 2006b) in mouse ERp5 on tumor cells might lead to high levels of sMIC- models (Dasgupta et al., 2005) and in cancer patients A and thereby impair NK-cell-mediated elimination of (Lee et al., 2004). Similarly, TGF-b decreases the primary tumors and metastases. expression of NKG2D ligands (Friese et al., 2004; Furthermore, soluble forms of ULBP2 and ULBP4 Eisele et al., 2006). Substantial amounts of TGF-b are (also named as RAET1E2 or LETAL (lymphocyte found in the sera of cancer patients, and cancer cells effector cell toxicity activating ligand)) have been often produce TGF-b. In addition, several studies have described (Salih et al., 2006; Waldhauer and Steinle, demonstrated that regulatory T cells (T ) are a source 2006; Cao et al., 2007). However, engagement of the reg of TGF-b, which mediates their immunosuppressive NKG2D receptor by soluble ligands does not necessa- function. rily 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 cell- Cells inhibiting NKG2D-mediated immune responses bound ligands to the NKG2D receptor. Most ULBPs It is evident that NK cell activation is not only are inserted in the cell membrane by glycosyl-phospha- suppressed by signaling via inhibitory receptors but also tidylinositol anchors and are therefore sensitive to by encountering suppressive cell populations. Suppres- treatment with phosphatidylinositol-specific phospholi- sion of cells of the adaptive immune system has pase C. In this context, Song et al. (2006a) demonstrated attracted lots of attention. Comparably little is known that shedding of ULBPs can be prevented by inhibition about the cross-talk of NK cells with immunosuppres- of phosphatidylinositol-specific phospholipase C. In sive cell populations. In this context, Ghiringhelli et al. contrast, Waldhauer et al. reported that ULBP2 is (2005) reported that human Treg inhibited the cytolytic cleaved by metalloproteases and not by phospholipases function and IFN-g secretion of NK cells after stimula- (Waldhauer and Steinle, 2006). tion with IL-12. This inhibition was mediated by Thus, shedding of NKG2D ligands has multiple membrane-bound TGF-b. Importantly, Treg also caused consequences on NKG2D-mediated responses: (i) re- a TGF-b-mediated downregulation of NKG2D on duction of ligand density on the tumor cell surface, (ii) human and mouse NK cells, suggesting an inhibitory downmodulation of the receptor on effector cells and role for Treg in the NKG2D-mediated activation of NK

Oncogene NKG2D in antitumor responses N Nausch and A Cerwenka 5953 cells. Indeed, after adoptive transfer of activated Treg during immune responses to NK-cell-sensitive tumors into Rag1À/À mice, development of lung metastasis was in vivo will be addressed in future studies. less efficiently controlled in a B16-RAE-1 þ model. In In summary, multiple mechanisms to escape contrast, transfer of Treg did not influence the number of NKG2D-dependent antitumor immune responses exist. metastasis caused by the parental B16 tumor cells. Therefore, it is not surprising that the immune system Inhibition of NKG2D-dependent NK cell activation often fails to control the outgrowth of NKG2D ligand- was also observed by Smyth et al. (2006). In this study, positive tumors. Nevertheless, the expression of depletion of Treg resulted in tumor rejection of NK- NKG2D ligands marks transformed cells and alerts sensitive tumors, such as 3LL and RMA-S-RAE-1b. the immune system. Amplification of NKG2D-mediated In contrast, no downregulation of NKG2D was antitumor immune responses and simultaneous circum- observed, suggesting that Treg might act in several ways vention of immune evasion should be objectives for the on NKG2D-dependent activation. Treg modulates design of strategies for immune therapy of cancer. 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 Perspectives of immunotherapy exploiting immune cell melanoma after application of an anti-4-1BB mAb and activation via NKG2D an anti-CD4 mAb, which deplete Treg and conventional CD4 þ T cells (Choi et al., 2007). In NK-depleted mice, To elicit successful antitumor immune responses, it is of the observed tumor regression after mAb treatment overall importance to guide highly active cytolytic was reduced by the application of a neutralizing effector cells in high numbers in close proximity to the anti-NKG2D mAb, suggesting that Treg also suppress tumor cells. At the same time, tumors have to be visible NKG2D-mediated effector functions of to immune cells and rendered highly susceptible to their NKG2D þ CD8 þ CD11c þ T cells. attack, which facilitate their rapid elimination. A distinct population of potentially immunosuppres- It was observed that tumor cells expressing high levels sive NKG2D þ CD4 þ T cells was recently described in of NKG2D ligands were efficiently rejected by NK cells, cancer patients (Groh et al., 2006). Interestingly, this whereas tumor cells expressing intermediate levels of population was expanded in patients bearing MIC þ , but NKG2D ligands were less immunogenic (Diefenbach not MICÀ, epithelial tumors. Also, in vitro expansion of et al., 2001). Therefore, it is a promising strategy to NKG2D þ CD4 þ T cells required stimulation via TCR increase the density of NKG2D ligands on tumor cells in the presence of soluble MIC-A. Importantly, to efficiently activate antitumor immune responses. NKG2D þ CD4 þ T cells inhibited proliferation of Several pathways to upregulate NKG2D ligand expres- NKG2DÀCD4 þ T cells in vitro via FasL. Similar results sion exist as discussed in the sections ‘NKG2D ligands: were obtained with NKG2D þ CD8 þ T cells. Therefore, expression and regulation’ and ‘NKG2D ligands: soluble MIC-A not only directly impairs NKG2D- expression and regulation on transformed cells’. RA mediated effector function of NK and CD8 þ T cells, but induces both human and mouse ligands. All-trans also facilitates the expansion of immunosuppressive retinoic acid (ATRA) has been used in therapeutic NKG2D þ CD4 þ T cells to dampen antitumor immune protocols in the treatment of acute myeloid leukemic responses. Whether NKG2D þ CD4 þ T cells also exist in patients with promising results, in particular in patients mice bearing NKG2D ligand-positive tumors and with the acute promyelocytic leukemia subtype (Tall- whether they are of physiological importance in vivo man, 2006). ATRA potently induces the differentiation are currently not known. Notably, there is also evidence of malignant leukemia cells, which is considered to be that anti-CD3-stimulated T-cell proliferation is inhib- the main mode of action of ATRA. In addition, it was ited by soluble MIC-A or H60 owing to the production discovered recently that ATRA increases ULBP levels of IL-10. However, in this study, no involvement of the on the acute myeloid leukemia cell line HL-60 (Rohner NKG2D receptor was observed (Kriegeskorte et al., et al., 2007). Furthermore, ULBP3 and MIC-A were 2005). upregulated by ATRA on leukemic B cells of patients Finally, myeloid-derived suppressor cells (MDSCs), suffering from chronic lymphocytic leukemia of B-cell which expand in tumor patients and tumor-bearing type (Poggi et al., 2004). High expression of ULBP in mice, are implicated in potent suppression of T-cell leukemic B cells was correlated with a better prognosis responses against tumors. Recently, Liu et al. (2007) in combination with high levels of IL-4 and circulating reported that MDSCs inhibited the cytotoxicity of IL-2- Vdelta1 T cells (Catellani et al., 2007). Apart from activated NK cells in vitro and repressed NK cell ATRA, trichostatin A and vitamin D3, which promote function in vivo. In contrast, we have evidence that myeloid cell differentiation, also upregulate NKG2D MDSCs can activate NK cells to produce high amounts ligands on acute myeloid leukemia cells (Rohner et al., of IFN-g. We further observed that a MDSC subset 2007). expressed RAE-1 and that induction of IFN-g by Several agents used in classical chemotherapies or MDSC required the interaction of NKG2D with radiotherapies cause DNA damage, which is one of the its ligands (Nausch et al., manuscript submitted). pathways for the induction of NKG2D ligands (Gasser Therefore, we conclude that MDSCs also activate et al., 2005). It is tempting to speculate that the curative certain effector functions of NK cells. Their exact role effect of these therapies is at least partially caused by an

Oncogene NKG2D in antitumor responses N Nausch and A Cerwenka 5954 immune response triggered by enhanced levels of RMA lymphoma cells were injected intravenously NKG2D ligand expression. NKG2D ligands were (Zhang et al., 2007). In this model, IFN-g production also applied as DNA-based vaccines. Vaccination with by chimeric T cells was an absolute requirement for H60-containing vectors, in combination with the tumor rejection. In addition, GM-CSF and cytotoxicity tumor-associated antigen survivin, enhanced innate mediated via FasL or perforin were involved in the and adaptive immune cell activation in comparison with observed antitumor activity of chimeric receptor-bear- survivin alone. In this study, addition of the H60 vaccine ing T cells. A recent clinical trial showed that adoptive enhanced the cross-talk between DC, CD8 þ T cells transfer of genetically engineered T cells with tumor- and NK cells, whereas CD4 þ T cells were suppressive specific TCR led to cancer regression in some patients (Zhou et al., 2005, 2006). (Morgan et al., 2006). However, as only little is known Furthermore, it is desirable to enhance the expression about tumor-associated antigens, the use of TCR- levels of the NKG2D receptor and increase its engineered T cells in immunotherapy is limited. There- functionality. Several cytokines effectively enhance fore, NKG2D-engineered T cells represent a promising NKG2D-mediated immune response. IFN-a, which is alternative tool for the treatment of NKG2D ligand- frequently used in cancer therapies including therapies expressing tumors. against melanoma (reviewed in Jack et al. (2006)), NKG2D-mediated activation of NK and CD8 þ T upregulated NKG2D (Zhang et al., 2005a). The cells can also be beneficial in response to tumors, which same group reported that NKG2D expression was express no or low levels of ligands. In these studies, Fab enhanced in an NK cell line stably transfected with fragments of antibodies specific for tumor antigens, IL-15 (Zhang et al., 2004). Moreover, IL-2 and -12 including carcinoembryonic antigen or CD20, were were reported to amplify NKG2D-mediated tumor conjugated to recombinant soluble MIC-A proteins. suppression of MCA-induced sarcomas (Smyth et al., These antibodies targeted tumor cells by recognizing the 2004, 2005). tumor-associated antigens and passively coated the IL-21, another member of the IL-2 cytokine family, tumor cells with MIC-A. Indeed, antibody-mediated enhances NKG2D-dependent tumor rejection, although coating of tumor cells with MIC-A sensitized tumor expression levels of NKG2D in mice remain unaffected targets to NKG2D-dependent NK cell lysis. These data (Takaki et al., 2005). imply that NKG2D-mediated immune cell activation is Interestingly, in human NK cells, downregulated applicable in response to a broad number of tumors expression of NKG2D was observed upon IL-21 (Germain et al., 2005). treatment (Burgess et al., 2006). It will be interesting It is possible that strategies enhancing NKG2D- to assess the effect of therapies exploiting cytokines mediated immune activation benefit enormously by the with regard to enhancing NKG2D-mediated effector simultaneous prevention of immune evasion. Currently, functions. ONTAK, a fusion protein of IL-2 and diphtheria toxin, Recently, several studies investigated whether T cells which reduces the numbers of Treg, is being evaluated in can be manipulated to eliminate NKG2D ligand- clinical trials in tumor patients (Barnett et al., 2005). It positive tumor targets in vitro and in vivo. Teng et al. will be interesting to determine whether the elimination (2005) generated DAP12-expressing T cells by retroviral of Treg in patients enhances NK cell activity in response transduction of primary T cells. These DAP12 þ T cells to NKG2D ligand-positive tumor targets. specifically lysed RAE-1b-positive tumor cell targets. Adoptive transfer of DAP12 þ T cells into Rag-1À/À mice bearing the RAE-1b-transfected RMA-S lymphoma enhanced survival of these mice. These data indicate Conclusion that T cells can be converted to acquire NK-like function by overexpression of a single adaptor. As During the last 10 years, the activating receptor human NKG2D does not bind to DAP12 (Rosen et al., NKG2D and its ligands were extensively studied with 2004), beneficial effects of DAP12 overexpression in regards to expression, regulation and function. It is T cells in response to NKG2D ligand-expressing targets commonly accepted that the interaction between this are likely to be restricted to mouse tumor models. Using receptor and its multiple ligands, which are induced in a similar approach, chimeric receptors of NKG2D or ‘inflammation and cancer’, plays a pivotal role in the DAP10 linked to the cytoplasmic domain of CD3z were recognition of tumor cells. So far, most studies generated (Zhang et al., 2005b). Primary T cells establishing the involvement of NKG2D in antitumor transduced with these chimeric receptors efficiently responses were performed with neutralizing anti- killed NKG2D ligand-positive tumor cell targets and NKG2D mAbs. Studies with NKG2D-deficient mice produced high amounts of Th1 cytokines and proin- will reveal the exact role of NKG2D in tumor flammatory chemokines (Zhang et al., 2005b). In surveillance in response to spontaneously arising addition, upon adoptive transfer of NKG2D-CD3z T tumors. In addition, recent evidence emerged stating that cells mixed with tumor cells or intravenously injected 1 the IL-15R couples to the NKG2D pathway, providing day before tumor cell inoculation, RMA-RAE-1b tumor novel insight into the priming of NKG2D-mediated growth was inhibited and immunological memory responses. Future studies are required to further elucidate against RMA cells was generated. Furthermore, the cross-talk of NKG2D and other activating receptors NKG2D-CD3z T cells promoted long-term survival, if with cytokine receptors in different cell types. Together,

Oncogene NKG2D in antitumor responses N Nausch and A Cerwenka 5955 efﬁcient strategies of NKG2D-mediated immune therapy Acknowledgements against cancer should aim at optimal activation of NKG2D, induction of the highest levels of NKG2D We thank Professor Lewis Lanier and Ioanna Galani for critically reading the paper. The work is supported by a Marie ligands on tumor cells and simultaneous counteraction of Curie Excellent Grant, Deutsche Jose´ Carreras Leuka¨ mie immune evasion. Recent studies mainly performed in Stiftung, German-Israel DKFZ/MOST cooperation and tumor mouse models suggest that exploitation of the Boehringer Ingelheim Fonds. We want to apologize to all NKG2D/NKG2D ligand pathway could be a promising authors whose work could not be cited in this review because strategy for antitumor immune therapy. of space limitations.

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