Immunobiology and Conflicting Roles of the Human NKG2D Lymphocyte Receptor and Its Ligands in

This information is current as Ahmed El-Gazzar, Veronika Groh and Thomas Spies of October 1, 2021. J Immunol 2013; 191:1509-1515; ; doi: 10.4049/jimmunol.1301071 http://www.jimmunol.org/content/191/4/1509 Downloaded from References This article cites 67 articles, 28 of which you can access for free at: http://www.jimmunol.org/content/191/4/1509.full#ref-list-1

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Immunobiology and Conflicting Roles of the Human NKG2D Lymphocyte Receptor and Its Ligands in Cancer Ahmed El-Gazzar, Veronika Groh, and Thomas Spies adopt diverse strategies to safeguard their survival, NKG2D forms homodimers that are assembled as hexameric which often involve blinding or incapacitating the im- complexes with four chains of the DAP10 signaling adaptor (6, mune response, thereby gaining battleground advantage 8). Distinct from mice, there is no shortened human NKG2D against the host. In immune responses against cancer, an isoform that can also bind DAP12. Upon ligation of NKG2D, important stimulatory lymphocyte receptor is NKG2D cytoplasmic tail tyrosine-phosphorylated DAP10 recruits PI3K because the tumor-associated expression of its ligands pro- or the growth factor receptor-bound 2/VAV1 signaling motes destruction of malignant cells. However, with ad- intermediate, thereby activating protein kinase B/AKT, as well as ERK and JNK in MAPK cascades (8). These branched sig-

vanced human cancers profound changes unfold wherein Downloaded from NKG2D and its ligands are targeted or exploited for im- naling pathways ultimately trigger lymphocyte cytotoxic gran- mune evasion and suppression. This negative imprinting ule polarization and degranulation, production, prolif- on the immune system may be accompanied by another eration, and survival. Stable surface expression of NKG2D is dependent on its association with DAP10, which is mediated by functional state wherein cancer cells coopt expression of oppositely charged amino acid residues in their transmem- NKG2D to complement the presence of its ligands for brane domains. self-stimulation of tumor growth and presumably malig- NKG2D expression can be induced by several http://www.jimmunol.org/ nant progression. This review emphasizes these conflicting (IL-2, IL-7, and IL-15), with IL-15 being biologically most functional dynamics at the immunity–cancer biology in- significant (7, 9). High-dose IL-15 can license NKG2D-DAP10 terface in humans, within an overview of the immunobi- for direct activation of NK cell and cytotoxic functions, ology of NKG2D and mechanisms underlying the regu- which play a detrimental role in certain T cell–mediated pa- lation of its ligands in cancer, with reference to instructive thologies such as in celiac disease (7). Moreover, IL-15 and 2 clinical observations and translational approaches. The TNF-a induce NKG2D receptors on CD28 CD4 T cells, Journal of Immunology, 2013, 191: 1509–1515. thus enabling costimulation of TCR-dependent autoreactivity

against rheumatoid arthritis synoviocytes displaying inappro- by guest on October 1, 2021 priately induced NKG2D ligands (5). Inhibitors of NKG2D he human NKG2D receptor is normally expressed expression include IL-21 and TGF-b, which regulates provision on NK cells, CD8 T cells, gd T cells, and some auto- of DAP10 (10, 11). migration inhibitory factor is reactive and immunosuppressive CD4 T cells (1, 2). Its T another negative regulator of NKG2D that is also produced by significance springs from the biology of its ligands, which are cancer cells (12). Finally, NKG2D is subject to ligand-induced absent or poorly expressed on most cells but are induced in downmodulation, which is especially significant in cancer set- and malignancies by viral transactivation, and mech- tings and is discussed further below. anisms associated with cellular stress responses and tumorigenic states, respectively (3, 4). These attributes have given rise to the concept of “induced self” serving to alert the immune system to Diversity and limited tissue distribution of NKG2D ligands pathological threats. Typically, engagement of NKG2D elicits NKG2D ligands are distant relatives of MHC class I poly- protective cytolytic and cytokine responses by activation, either peptides but without their attributes linked to Ag presentation. directly or via costimulation, of NK cells and CD8 T cells There is a multitude of structurally diverse ligands, including (5, 6). However, depending on cytokine environments together the closely related MHC class I–related chains A and B (MICA with conditions of anomalous ligand induction, NKG2D can and MICB) and the more distant family of six UL16-binding also initiate and/or exacerbate autoimmune disease inflamma- (ULBP1–6) (13). As with MHC class I, all NKG2D tory processes such as in rheumatoid arthritis and celiac disease ligands have a distal a1a2 receptor binding platform domain. (5, 7). MICA and MICB also have the membrane-proximal a3 do-

Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA Abbreviations used in this article: EGFR, epidermal growth factor receptor; ERp5, 98109 endoplasmic reticulum protein 5; MICA, MHC class I–related chain A; MICB, MHC class I–related chain B; ULBP, UL16-binding protein. Received for publication April 22, 2013. Accepted for publication June 11, 2013. A.E.-G. was supported by an Erwin Schro¨dinger Fellowship from the Austrian Science Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 Fund (Grant J3078). Work in the laboratory of V.G. and T.S. is supported by the National Institutes of Health. Address correspondence and reprint requests to Dr. Thomas Spies, Clinical Research Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, D1- 100, Seattle, WA 98109. E-mail address: [email protected]

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301071 1510 BRIEF REVIEWS: NKG2D AND ITS LIGANDS IN CANCER main and are transmembrane-anchored. All ULBP ligands folded protein responses, as well as to signaling intermediates lack the a3 domain. ULBP1–3 and ULBP6 have GPI instead and checkpoint anomalies associated with oncogenic states (3, of transmembrane anchors. In outer cell membranes, GPI- 4). Within this framework, detailed knowledge of ligand anchored ULBP proteins are clustered in lipid raft micro- regulation is fragmented. In addition to transcriptional acti- domains, which may increase avidity of lower affinity ligands vation, protein biosynthesis can modify ligand expression (4). for NKG2D (14). Reflecting the evolutionary divergence of Moreover, different mechanisms may activate transcription of NKG2D ligands, the crystal structure of MICA shows a pro- the same ligand gene, thus expanding the range of responses foundly altered MHC class I fold with only a remnant of a to cellular damage cues. shallow peptide binding groove and restructured a1a2 plat- The regulation of the genes for MICA and MICB shares form and a3 domain interfaces that preclude binding of similarities with heat shock protein 70 genes (21). These genes b2-microglobulin (5). MICA and MICB are represented by commonly have a promoter region heat shock response ele- numerous alleles, which differ in binding affinities for NKG2D ment for conditional binding of heat shock factor 1, a tran- (5, 15). scriptional activator that is responsive to homeostatic changes The ability of NKG2D to interact with diverse ligands seems caused by heat shock and . Promoter regions perplexing. In a complex crystal structure, the saddle-shaped of MICA/B also activate proliferation-induced transcription NKG2D homodimer sits astride the a1a2 platform domain (21). With epithelial tumor lines and presumably cancer cells of MICA, with each NKG2D monomer contacting either one as well, epigenetic derepression of promoter accessibility, ex- of the subdomains. Most of the interaction energy resides in two emplified by pharmacological histone deacetylase inhibition, NKG2D binding site tyrosines that make dominant contacts at plays an essential role in the transcriptional activation of Downloaded from each interface in the absence of conformational plasticity (16). NKG2D ligand genes (3). In accord with their regulation by Complementary information from an NKG2D–ULBP3 com- elementary cellular processes, relevant transcription factors in plex structure suggests that binding of different ligands involves addition to heat shock factor 1 are ubiquitous. They include common interfaces and overlapping sets of amino acid side-chain members of the SP zinc finger transcription factor family and interactions, thus permitting conservation of general shape the CCAAT box factor complex, which variably interact with complementarities and binding energies (17). 59-end flanking sequences at least of MICA/B and ULBP1 http://www.jimmunol.org/ The multiplicity and diversity of NKG2D ligands apparently (3). AP-1 (Fos/Jun), which has diverse roles in inflammatory reflect evolutionary pressure to maintain redundancy. One ex- responses as well as a tumor promoter or suppressor, has been planation is based on an evolutionary arms race with viruses en- implicated in the regulation of a mouse NKG2D ligand gene coding immunoevasins, which have the capacity to obstruct (22). In humans, AP-2a suppresses ULBP1 transcription by the functionality of different sets of NKG2D ligands (13, 18). interfering with SP complex binding site occupancy (3). Another concept emanates from the function of the ligands as These findings reflect the patchwork state of research with sensors of cellular damage cues. Raulet et al. (4) proposed that independent findings in humans and mice that are most often NKG2D ligands may convey composite specifications, analo- not mutually applicable. Along this line, E2F transcription by guest on October 1, 2021 gous to barcodes, on the surfaces of unhealthy cells, with var- factors involved in cell cycle entry have been implicated in iables including presence and relative abundancies of diverse mouse ligand transcriptional activation, but there is no cor- ligands and differences in posttranslational modifications. How- responding evidence in humans (4). Conversely, the tu- ever, NKG2D ligands are not absent from all normal cells mor suppressor has been implicated in transcriptional activation but presumably are insufficient, or ineffectively disposed, to of ULBP1 and ULBP2 but not of mouse NKG2D ligand genes stimulate an immune response when present. Some NKG2D (4, 23). Taken together, knowledge of promoter regions and ligand mRNAs are quite ubiquitous, which is inconsequential transcription factors relevant to tumor-associated expression presumably because of their negative regulation by microRNAs is limited for most NKG2D ligand genes. As of yet, repro- (19). Nevertheless, physiologically relevant examples for NKG2D gramming events associated with malignant transformation do ligand expression under normal conditions exist, including the not appear to have a principal role in their transcriptional ac- expression of MICA/B in intestinal mucosa, thymic epithelium, tivation. and the placental syncytiotrophoblast (5). However, NKG2D ligands can be posttranscriptionally reg- ulated by factors associated with oncogenic states. The BCR– Expression and regulation of NKG2D ligands in cancer ABL tyrosine kinase gene fusion is associated with MICA in- Expression of NKG2D ligands is pronounced in malignancies, duction in chronic myeloid leukemia (24). Moreover, chronic underscoring their role in promoting tumor immune surveil- activation of the RAS GTPase in response to extracellular lance. In the most comprehensive study of hematopoietic signals can lead to upregulation of ULBP1–3 and MICA via malignancies, Hilpert et al. (20) documented heterogeneous stimulation of MAPK and PI3K signaling cascades and, ulti- surface expression of at least one NKG2D ligand type in ∼75% mately, by eukaryotic translation initiation factor 4E–medi- of 205 leukemia patients. With solid tumors, a survey of widely ated increases of protein synthesis rates (4). An explanation for scattered data suggests that MICA/B predominate alongside at the near universal presence of NKG2D ligands in cancer cells least one ULBP family ligand in most epithelial cancers, in- was provided by Gasser et al. (25), who associated their ex- cluding head and neck, lung, breast, ovarian, cervical, prostate, pression with genotoxic stress and the activation of a DNA gastrointestinal, pancreatic, hepatocellular, and renal cell can- damage response owing to dsDNA breaks or stalled replication cer, as well as in malignant glioma, neuroblastoma, and mel- forks. This pathway involves the ataxia telangiectasia mutated anoma (1, 5). or ataxia telangiectasia mutated and Rad3-related protein Tumor expression of NKG2D ligands is linked to generic kinases, which activate checkpoint kinases that ultimately ef- mechanisms coupled to cellular stress, proliferation, and un- fect cell cycle arrest, DNA repair, or apoptosis. Accordingly, The Journal of Immunology 1511

DNA damaging agents and replication inhibitors can induce surveillance in experimental spontaneous malignancies. Ab- expression of ULBP1–3 and mouse NKG2D ligands (4, 25). sence of NKG2D resulted in higher incidence and increased The mechanistic coupling of ligand induction to this cellular aggressiveness of early arising tumors. Reflecting the capacity of response is in essence not understood (4). NKG2D to trigger NK cell and CD8 T cell cytotoxicity, its Once induced in cancer cells, NKG2D ligands MICA/B and functionality was associated with negative selection of cancer various ULBPs are negatively regulated by cytokines, including cells bearing its ligands (33). TGF-b and IL-10 (1). Carcinoembryonic Ag-related adhesion In humans, evidence for a protective role of NKG2D against molecule 1 impedes glycosylation and hence intracellular cancer is limited and naturally indirect. Single-nucleotide poly- transport of NKG2D ligands (26). Of interest is a report of O- morphisms linked to NKG2D identified NK complex hap- glycan modification of MICA by core2 b-1,6-N-acetylgluco- lotypes that are associated with low or high NK cell cytotoxic saminyltransferase in bladder tumor cells. In that study, poly- activity and more or less risk of colorectal and aerodigestive N-acetyllactoseamine attached to MICA core2 O-glycans tract cancers in lifestyle-adjusted control and patient cohorts (N-acetylgalactosamine linked to branched N-acetylglucos- (34–36). A biallelic (threonine/alanine) variant at amino acid amine) binds galectin-3, which impairs ligand affinity for position 72 in its transmembrane segment may implicate NKG2D. This modification may explain correlations between NKG2D more directly. Threonine 72 may alter binding of high expression of core2 b-1,6-N-acetylglucosaminyltransfer- DAP10, thereby increasing signaling strength. This variant was ase and poor prognosis and highly metastatic behavior of blad- significantly less frequent in a study of patients with cervical der cancers (27). carcinoma than in the healthy controls (37). Perhaps more instructive are correlative studies of NKG2D ligand expression Downloaded from Regulation of NKG2D ligands by microRNAs in cancers and clinical outcomes. In a colorectal cancer study, As with many cellular processes, immune system functions and MICA was an independent marker of good prognosis for stage oncogenesis are regulated by microRNAs, which assemble with I and stage II but not later stage cancers (38). A similar rela- partially complementary sequences in 39-untranslated regions tionship was observed in pancreatic cancer (39). In a breast of target mRNAs, thus inhibiting translation or promoting cancer study, MICA/B and ULBP2 were associated with de- degradation. Stern-Ginossar et al. (19) and subsequent studies creased relapse in early stage cancer (40). These clinical data http://www.jimmunol.org/ identified cellular microRNAs of various families that sup- support a consensus that NKG2D and its ligands are protective press expression of MICA and/or MICB, or ULBP2 (28). at early but not advanced tumor stages, reflecting the progressive Most of those microRNAs are present in steady quantities in immune system failure that allows unchecked cancer growth. normal human cells, consistent with their proposed role in Consequently, tumor progression coupled to persistent expres- calibrating baseline expression of NKG2D ligand mRNAs sion of NKG2D ligands points to changes in functional dynamics such that no lymphocyte attack ensues. The current model that may increasingly favor host tumor susceptibility instead of hence posits that the repressive capacity of the microRNAs is resistance. exceeded in pathological conditions as in malignancies when by guest on October 1, 2021 transcriptional induction of NKG2D ligand genes leads to Exploitation of NKG2D ligands as tumor survival assets enabling consequential mRNA and protein expression. immune evasion Notwithstanding the attractiveness of this model, evidence NKG2D is subject to ligand-induced downmodulation by in support of the significance of microRNAs in suppressing endocytosis and partial degradation. This is most evident in NKG2D ligands in human cancer is scant because of insuf- patients with cancers positive for MICA/B, in which large ficient or contradictory correlative data. There are no larger proportions of CD8 T cells and NK cells among infiltrating studies correlating clusters of relevant microRNAs in ex vivo lymphocytes have diminished surface NKG2D. Moreover, cancer cells with absence or presence of surface NKG2D NKG2D is systemically reduced on peripheral blood CD8 ligands. Moreover, unrelated studies of microRNA expression T cells and NK cells. This deficiency is caused by circulating patterns in human cancers have yielded discordant results. A tumor-derived soluble ligands and is associated with impaired microRNA with regulatory activity for MICA/B (miR-106b) immune responses (41, 42). Shedding of soluble MICA and/or was found reduced in primary ovarian cancer and increased MICB and ULBP2 has been reported for most cancers and in colorectal cancer, gastric cancer, and hepatocellular carci- some hematopoietic malignancies. Tumor-derived exosomes noma (29–31). Among other microRNAs specific for MICA/ may also contribute to effects initially attributed to soluble B, miR-17-5p and miR-373 were found highly expressed in ligands alone (43). Experimental support for a critical role of multiple cancers of diverse tissue origins; however, these micro- soluble ligands in promoting tumor immune evasion was RNAs have pleiotropic effects in tumor progression, which provided by a mouse model study comparing tumor formation may influence NKG2D ligand expression (29, 32). Lastly, by implants expressing either wild-type or a shedding-resistant there is the conundrum that most cancers comprise substantial form of MICB, or just the secreted soluble ligand. The results cell populations that are positive for NKG2D ligands, which confirmed the capacity of soluble ligand to foster tumor may in fact benefit tumor progression as discussed further growth, wherein impairment of NK cell responses was noted below. as the probable cause (44). This relationship is highlighted by beneficial effects of neutralizing anti-MICA autoantibodies that Protective role of NKG2D and its ligands in tumor immunity were induced as a result of anti–CTLA-4 immunotherapy in a Mouse model experiments substantiated NKG2D-dependent clinical trial with melanoma patients (45). By and large, the ob- rejection of ligand-bearing tumor lines by NK cells and/or served negative imprints on the immune response are corrobo- cytotoxic CD8 T cells in syngeneic mice (1). Most decisively, rated by clinical data, as tumor-associated or soluble NKG2D a study of NKG2D-deficient mice revealed defective immune ligands are associated with parameters of disease progression and 1512 BRIEF REVIEWS: NKG2D AND ITS LIGANDS IN CANCER poor prognosis in pancreatic and prostate cancer (MICA/B) Other immunosuppressive effects are associated with a small (39, 46), ovarian cancer (ULBP2 and ULBP4) (47), melanoma subset of normally occurring CD4 T cells with activation-in- (ULBP2) (48), and multiple myeloma (MICA) (49). dependent, constitutive expression of NKG2D. These T cells Biological precedent for tumor shedding of NKG2D li- lack proinflammatory cytokine and cytolytic signatures and gandsexistsinpregnancywhereimmunosuppressivefactors play no discernible role in antimicrobial recall responses. Instead, must prevent rejection of the semiallogeneic fetus. Placental they are autoreactive and biased toward an immunosuppressive syncytiotrophoblast cells express NKG2D ligands, including IL-10– and TGF-b–dominated cytokine profile (2, 58). For MICA/B and ULBP family members. Both shedding of sol- reasons unknown, the NKG2D+CD4+ T cells are not suscep- uble ligands and release of exosomes may contribute to tible to ligand-induced NKG2D downmodulation, thus main- NKG2D downmodulation on NK cells and CD8 T cells and taining NKG2D functionality under conditions of persistent thereby to maintenance of an immune tolerogenic environ- ligand exposure. Hence, in environments providing stimula- ment (5). tion of both TCR and NKG2D, the NKG2D+CD4+ T cells proliferate and may expand in numbers relative to other T cells, Regulation of NKG2D ligand shedding thereby causing imbalances in the lymphocyte pool and im- posing an immunosuppressive cytokine milieu. This homeo- Shedding of MICA and presumably MICB requires domain- static adjustment may normally serve to dampen chronic specific deconstruction by an accessory protein before proteo- immune activation. In advanced cancer patients, however, lytic cleavage can ensue. On the surface of tumor lines andex vivo tumor expression and shedding mainly of soluble MICA/B cancer cells, MICA interacts with endoplasmic reticulum pro- can drive substantial proliferative expansions of the NKG2D+ Downloaded from tein 5 (ERp5), a disulfide isomerase that typically assists in the CD4+ T cells. Casting on the physiological significance folding of nascent proteins inside cells. ERp5 and membrane- of these T cells, their frequencies are inversely correlated anchored MICA form transitory mixed-disulfide complexes with disease activity in systemic lupus erythematosus, sug- from which soluble MICA is released after proteolytic cleavage gesting that they participate in suppression of effector re- near the cell membrane (50). ERp5 reduces a deeply buried sponses (2). disulfide bond in the membrane-proximal MICA a3 domain, http://www.jimmunol.org/ thereby likely generating a conformational change that enables Coopting of NKG2D as a potential tumor growth factor receptor proteolytic cleavage of MICA. This physical relationship is re- flected by positive correlations between ERp5 and MICA/B In addition to promoting tumor immune evasion and sup- on the surface of leukemic cells and soluble ligands in multi- pression, NKG2D ligands may constitute yet another tumor ple myeloma and ERp5 (51, 52). Recruitment of MICA into survival asset. In a conceptual twist, variable proportions of ex cholesterol-enriched membrane lipid microdomains by palmi- vivo breast, ovarian, prostate, and colon cancer cells express toylation of cysteine residues in its cytoplasmic tail may pro- signaling-proficient NKG2D, thereby complementing the presence of its ligands for self-stimulation of tumor growth and

mote shedding, although there is no confirmatory evidence in by guest on October 1, 2021 cancer cells (53). NKG2D ligands are cleaved near the cell possibly malignant progression (59). Triggering of cancer cell membrane by ADAM family of cell surface disintegrins and NKG2D by Ab crosslinking or engagement of ligands on metalloproteases, among which members 9, 10, 14, and 17 adjacent cancer cells activates the oncogenic PI3K–AKT– have been variously implicated (54). mammalian target of rapamycin signaling axis, which is commonly hyperactive in cancer, and downstream effectors controlling protein synthesis and cell growth. Additionally, as Persistent NKG2D ligand expression may cause systemic immune in lymphocytes, engagement of NKG2D stimulates phos- suppression phorylation of ERK and JNK in MAPK cascades (59). These Chronic expression of NKG2D ligands, as in advanced can- kinases are activation targets of receptor tyrosine kinases such cer, may have broader immunosuppressive effects. Peripheral as the epidermal growth factor receptor (EGFR), which, be- blood NK cells from patients with colorectal or cervical cancer cause of mutation or aberrant expression, frequently cause lack functional natural cytotoxicity receptors NKp30 and/or excessive cancer cell proliferation and increased motility and NKp46, which has been associated with soluble MICA or survival (60). diminished NKG2D (42, 55). Moreover, loss or diminished Consistent with these signaling activities, above-threshold expression of the CD3z signaling adaptor is well documented expression of NKG2D-DAP10 in ligand-bearing tumor lines in T cells and NK cells from patients with cancer (56). CD3z increases their bioenergetic metabolism and proliferation, thus is associated with CD16 (FcgRIII) and is essential for NKp30/ implying similarities with oncogenic growth factor receptors 46 signaling and TCR complex expression and function. Ex- such as the EGFR and the insulin-like growth factor-1 receptor. perimentally, sustained ligand stimulation of signaling-com- Thus, cancers may coopt expression of NKG2D-DAP10 for petent NKG2D on activated human CD8 T cells or NK cells their own benefit to promote tumorigenesis. In a preliminary results in loss of CD3z concomitant with the gradual decrease assessment, this role is supported by significant correlations of surface NKG2D (57). Mechanistically, these seemingly between percentages of ex vivo cancer cells that are positive for unrelated events are linked in a causal chain wherein NKG2D surface NKG2D and criteria of tumor progression (59). Hence, signaling initiates /–mediated activa- NKG2D itself might in fact represent the main factor un- tion of caspases-3/7, resulting in CD3z cleavage and subse- derlying the typically poor clinical outcomes that have been quent degradation (57). Hence, cancer expression of NKG2D associated with cancer cell expression of its ligands (39, 46, 47, ligands may lead to impairment of multiple CD3z-dependent 49). Notably, tumor cell expression of NKG2D was not de- receptor functions, thus promoting far-reaching lymphocyte tol- tected in several experimental models of spontaneous cancers erization. in mice (59). The Journal of Immunology 1513

Targeting of NKG2D or its ligands for cancer therapy Transduction of chimeric activating lymphocyte receptors Therapy approaches are tailored to enhance or redirect NK consisting of human NKG2D, CD3z, and either costimulatory cell– and/or T cell–mediated cytotoxicity by utilizing agents CD28 or 4-1BB (CD137), a TNFR family member, repre- that may increase NKG2D ligand expression or bifunctional sents a strategy for adoptive T cell therapy (66, 67). Finally, fusion proteins including an NKG2D ligand and a single- another approach aims at induction or administration of Abs chain Ab fragment (scFv) targeting a specific tumor cell sur- for neutralization of soluble NKG2D ligands. It remains to be face marker. Among pharmaceuticals tested for upregulation seen how these or other therapeutic approaches impact the of NKG2D ligands on tumor lines in vitro are demethylating dynamic functionality of NKG2D and its ligands at the in- agents, proteasomal inhibitors, and genotoxic drugs used for terface between the immune system and cancer, as there may chemotherapy such as histone deacetylase inhibitors (61). be unintended consequences. Objectives for the bifunctional fusion proteins are to target NK cells and T cells onto tumor cells and engage NKG2D. Conclusions Examples are ULBP2/anti–prostate-specific membrane Ag scFv Although best known for their protective role in tumor im- and ULBP2/anti–syndecan-1 (CD138) scFv (62). CD138 is mune surveillance, NKG2D and its ligands are also exploited as highly expressed in some hematopoietic malignancies such as tumor survival assets, enabling immune evasion and sup- multiple myeloma. Other ligand fusion proteins incorporat- pression, and quite possibly stimulation of tumor growth and ing anti-HER2 (EGFR-2) Ab fragments have been developed malignant progression (Fig. 1). The tentative role of NKG2D

for preliminary testing in mice (63). as an oncoprotein still requires in vivo experimental substan- Downloaded from Another type of bispecific T cell and NK cell engager, thus tiation in an animal model. Cancer biology questions of fur- far tested in mice only, combines the extracellular domain of ther relevance are whether NKG2D imparts cellular changes NKG2D with either CD3« or the Fc portion of IgG2a for in cancer environments that are distinct or merely synergistic binding to CD16 (64, 65). In an in vivo tumor model, ad- to those conferred by prototypic growth factor receptors, and ministration of the NKG2D–CD3« fusion protein reduced whether its activities affect other aspects of tumorigenesis.

growth of tumors bearing NKG2D ligands and promoted These issues broadly reflect an increasing appreciation of a http://www.jimmunol.org/ T cell infiltration. Likewise, the NKG2D–Fc region fusion need of an integrated approach that considers tumor and host protein triggered lysis and reduced growth of ligand-positive factors together in the evaluation and targeting of tumor lymphomas (64, 65). progression. by guest on October 1, 2021

FIGURE 1. Schematic representation of NKG2D/ligand-mediated effects in cancer settings. (A) Tumor-associated NKG2D ligands trigger effector func- tions of NK cells and, via costimulation, of CD8 T cells. NKG2D-mediated CD8 T cell stimulation triggers Fas ligand release. At this stage, the TCR, CD16, and NKp30 and NKp46 nat- ural cytotoxicity receptors are function- ally expressed in complex with CD3z. (B) Persistence of tumor cell mem- brane-bound and soluble NKG2D ligands cause NKG2D downmodulation and functional impairment. Still, func- tional NKG2D [see (A)and(C)] initiates Fas ligand/Fas–mediated caspase activa- tion and resultant CD3z loss. As a con- sequence, the functional capacities of the TCR, CD16, and NKp30 and NKp46, whichallsignalthroughCD3z,areim- paired. (C) NKG2D costimulates pro- liferation and IL-10, TGF-b,andFas ligand release by immunosuppressive NKG2D+CD4+ Tcells.(D)NKG2D+ cancer cells proliferate in response to autocrine NKG2D-mediated stimula- tion. See text for further explanations. 1514 BRIEF REVIEWS: NKG2D AND ITS LIGANDS IN CANCER

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