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Review

Cancer exosomes and NKG2D receptor–ligand interactions: Impairing NKG2D-mediated cytotoxicity and anti-tumour immune surveillance Lucia Mincheva-Nilsson ∗ , Vladimir Baranov Department of Clinical Microbiology, Division of Clinical Immunology, Umeå University, S-90187 Umeå, Sweden

a r t i c l e

i n f o

a b s t r a c t

Keywords: Exosomes Cancer NKG2D MICA/B RAET1 ULBP NK cells Cytotoxicity Tumour surveillance Immune suppression

Human cancers constitutively produce and release endosome-derived nanometer-sized vesicles called exosomes that carry biologically active proteins, messenger and micro RNAs and serve as vehicles of intercellular communication. The tumour exosomes are present in the blood, urine and various malignant effusions such as peritoneal and pleural fluid of cancer patients and can modulate immune cells and responses thus deranging the immune system of cancer patients and giving advantage to the cancer to establish and spread itself. Here, the role of exosomes in the NKG2D receptor–ligand system’s interactions is discussed. The activating NK cell receptor NKG2D and its multiple ligands, the MHC class I-related chain (MIC) A/B and the retinoic acid transcript-1/UL-16 binding proteins (RAET1/ULBP) 1–6 comprise a powerful stress-inducible danger detector system that targets infected, inflamed and malignantly transformed cells and plays a decisive role in anti-tumour immune surveillance. Mounting evidence reveals that the MIC- and RAET1/ULBP ligand family members are enriched in the endosomal compartment of various tumour cells and expressed and released into the intercellular space and bodily fluids on exosomes thus preserving their entire molecule, three-dimensional protein structure and biologic activity. The NKG2D ligand-expressing exosomes serve as decoys with a powerful ability to down regulate the cognate receptor and impair the cytotoxic function of NK-, NKT-, gamma/delta- and cytotoxic T cells. This review summarizes recent findings concerning the role of NKG2D receptor–ligand system in cancer with emphasis on regulation of NKG2D ligand expression and the immunosuppressive role of exosomally expressed NKG2D ligands. © 2014 Published by Elsevier Ltd.

1. Introduction

microRNA in their lumen. Exosomes have been typically described as “cup-shaped”, a morphology that emanates from electron microscopy visualization of whole mount samples with negative contrast staining. This morphology is probably a result of a collapse of their spherical form due to the isolation procedure and the drying and fixation process during sample preparation. Although maybe an artefact the “cup-shaped” form description is frequently used and considered as a part of the definition of exosomes. Their protein and nucleic acid composition originates from the cells that produce them and the repertoire of expressed molecules at a certain time point reflects the current state of the parental cells. Certain proteins are generally enriched and common in exosomes; such are membranal targeting/adhesion molecules (e.g. tetraspanins, integrins and lactadherin), membrane trafficking molecules (e.g. annexins and Rab proteins), proteins involved in ESCRT complex (Alix, TSG101), cytoskeletal proteins (tubulin, actin), heat shock proteins (e.g. Hsp70 and 90), signal transduction proteins (protein kinases and heterotrimeric G proteins) and enzymes (e.g. GAPDH, pyrovate kinases, peroxidases) [2–4]. Besides the common molecules, molecules with specialized function like MHC class I and

1.1. Exosomes – vehicles for intercellular communication in health and disease Exosomes are specialized, nanometer-sized vesicles with cholesterol and sphingophospolipid-rich detergent-resistant membrane (DRM) produced within multivesicular bodies (MVB) of the late endosomal compartment and actively secreted into the extracellular space by a huge variety of cells in normal and pathologic conditions [1–3]. Exosomes are generated by inward budding of the MVBs’ limiting membrane and are characterized by a size of 30–100 nm, buoyant density of 1.13–1.19 g/ml on sucrose gradient and sedimentation at 100,000 × g [2]. They bear transmembrane (TM) and glycophosphatidylinositol (GPI)-anchored proteins on their membrane and cytosolic proteins together with mRNA and

∗ Corresponding author. Tel.: +46 90 7852237; fax: +46 90 7852843. E-mail address: [email protected] (L. Mincheva-Nilsson). http://dx.doi.org/10.1016/j.semcancer.2014.02.010 1044-579X/© 2014 Published by Elsevier Ltd.

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II complexes, costimulatory molecules, various receptor ligands (such as FasL, TRAIL, PD-L1, NKG2D ligands), proteins emanating from their cellular origin serving as “address tags”, cytokines and functional mRNA and microRNA have been identified in the cargo of various exosomes [5–7]. Exosomes are important intercellular communicators and can be viewed as parcels or mail, mediating cell–cell contact by proxy between the paternal cells producing them and the target cells receiving their message. Several types of exosome–target cell interactions are proposed; among them binding to the cellular surface by receptor–ligand interactions, exosomal adhesion molecules, phosphatidylserine, direct fusion of vesicles with the recipient plasma membrane after adhesion or internalization into endocytic compartments through receptor-mediated endocytosis and phagocytosis [2]. The interaction between exosomes and target cells can result in direct stimulation or inhibition of the target cell, transfer of membrane receptors and adhesion molecules and/or delivery of proteins. Furthermore, genetic material exchange through exosomal transport of mRNA and microRNA is an additional level of exosome-mediated intercellular communication that can reprogram the recipient cells [7]. Exosome secretion is observed in many cell types including hematopoietic cells (reticulocytes, platelets, dendritic cells [DC], macrophages, T-and B lymphocytes), mast cells, various epithelial cells, syncytiotrophoblast, fibroblasts, neurons, glial cells and many tumour-transformed cells [6,8–17]. The degree of exosome secretion can be upregulated by parental cell exposure to biological signals or stress conditions such as radiation, oxidative and thermal stress, inflammation, infection or DNA transformation or damage [17,18]. 1.2. Cancer exosomes modulate immune responses in a dual way As mentioned above production and secretion of high amounts of exosomes by various carcinomas is regularly reported [6,16–29]. It has been shown that aberrant signalling pathways involving activation of p53 and its subsequent response pathways such as TsAP6/Steap3 up-regulate exosome secretion in tumour cells [18,20]. Thus, exosome secretion seems to be a general characteristic of malignant transformation. Consistently, tumour-derived exosomes are enriched in the blood, urine and various malignant effusions such as peritoneal ascites and pleural fluid of cancer patients [6,16]. Tumour exosomes are closely reflecting the original cancer cells and usually carry tumour antigens, specific for the tumours that produce and release them such as Melan A, HER2, Silv, CEA, mesothelin, CD24 and EpCAM [16,21–24]. Exosomes can be divided generally into two major groups – exosomes with immunoactivating properties and those that are tolerogenic or immunosuppressive. In general, exosomes originating from immune cells such as antigen presenting cells (APC: macrophages, dendritic cells and B cells) enhance immune responses and are immune activating either in a direct way functioning as antigen presenters by proxy, or indirectly by influencing APC and various immune mechanisms. In contrast, normal epithelium-derived exosomes in physiological conditions, e.g. enterocyte-derived exosomes, so called tolerosomes, and placental exosomes exert immunosuppression promoting homeostasis and immune tolerance [11,25,26]. Tumour-derived exosomes have a dual effect on the immune system. From one side, they can enhance tumour antigen recognition indirectly by providing tumour antigens for antigen processing and presentation when uptaken by professional APC. It has been shown in murine in vivo and human ex vivo model systems that dendritic cells pulsed with cancer cellderived exosomes can prime cytotoxic T cells to induce protective antitumor immune responses [16,21]. Additionally, exosomes from heat-shocked tumour cells contained high levels of Hsp 70 and were reported to elicit a direct Th1-polarized immune response in

a MHC-independent manner in autologous and allogeneic murine models [27,28]. The boostering effect of anti-tumour immunity conveyed by tumour exosomes comprised the basis for clinical trials of antitumor immune priming based on vaccination with tumour exosomes or tumour exosome-pulsed DCs [29–32]. On the other side, as much as this induction and maintenance of antitumour immunity is desirable and wanted, the major direct effect of tumour exosomes on the immune system of cancer patients is that of immune suppression. Tumour exosomes not only carry tumour markers but also proteins with detrimental effect on the immune system. They express key immune molecules and signalling substances that promote apoptosis such as FasL, TRAIL, PD-L1 [33,34]. They are able to selectively impair lymphocyte IL-2 response while supporting Treg cells by inducing and up regulating their suppressive function through TGF␤ and IL10-dependent pathways [23,35]. By expressing immune receptor ligands on their surface, tumour exosomes can serve as decoys down modulating important immune responses, e.g. the NKG2D-mediated NKand CTL cytotoxicity [17,36–38]. It seems that tumours escape immune attack mimicking the behaviour of the immune system in an adverse way by using exosomes that dysregulate normal protective immune function and enhance immune suppression by T regulatory cells. Thus, despite the documented indirect immune priming function of tumour exosomes, convincing evidence shows that the net effect of tumour exosomes originating from mammary, lung, colon, prostate and ovarian cancer [39] is a powerful immune suppression, promoting the establishment and metastatic spreading of the primary tumour. In this review the influence of tumour-derived exosomes on the major cytotoxic system in the body–the NKG2D receptor–ligand system operating as an antitumour immune surveillance mechanism will be discussed. An illustration of cancer-derived exosomes and their expression of NKG2D ligands by negative contrast staining and immunoelectron microscopy is shown in Fig. 1. As a background, a short description of the NKG2D receptor and its ligands, the MIC- and ULBP/RAET1 family members, is given below.

2. The NKG2D receptor–ligand system and its role in anti-tumour immune surveillance 2.1. NK cells, their activation and receptors Natural killer (NK) cells are cytotoxic effector cells of the innate immunity engaged in the defence against viral infections and tumours. They differ in their activation and reactivity compared to T and B cells in that they lack clonally distributed antigenspecific receptors and do not recognize and react to antigens in a MHC-restricted manner. Instead, their reactivity is decided by an intricate interplay of a set of inhibitory and activating receptors. In humans these receptors are roughly divided into three groups depending on their structure: (1) the killer immunoglobulin receptors (KIRs), (2) the natural cytotoxicity receptors (NCRs), and (3) the c-type lectin-like receptors (Table 1). Most of the inhibitory receptors are the Ig superfamily members KIRs and Ig-like transcripts ILT binding classical and non-classical MHC I molecules; and a member of the C-type lectin family – NKG2A/B binding the non-classical MHC-E. These inhibitory receptors react on the presence of “healthy” MHC class I molecules and transmit their inhibitory signals through immunoreceptor tyrosine-based inhibition motifs (ITIMs) present in the intracellular tail of the receptors. The phosphorylated ITIMs recruit SHP1/2 phosphatases that dephosphorylate and inactivate adaptors involved in NK cell activation [reviewed in 40]. Thus, normal cells are protected from NK cell attack due to adequate expression of MHC class I molecules. In addition to altered and/or missing self [41] NK-cell

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Fig. 1. Electron micrographs illustrating that tumours constitutively secrete exosomes expressing NKG2D ligands on their surface. (A) Tumour exosomes isolated from supernatants of epithelial ovarian cancer explant cultures, T-cell leukaemia cell line Jurkat and B-cell lymphoma cell line Raji. Negative contrast staining showing the vesicle size of 30–100 nanometers and the cup-shaped form typical for exosomes. Immunoelectron microscopy (IEM) of CD63 marker stained by gold particles, proving the exosomal nature of the vesicles. (B) IEM of B-cell lymphoma (Raji) secreted exosomes stained by immunogold for the NKG2D ligands MICA/B, ULBP1 and 2. Note that the stained vesicles have the size and cup-shape typical for exosomes. Similar staining was obtained with Jurkat- and ovarian cancer-derived exosomes. Bars in each micrograph represent 100 nm.

activation requires a positive signal delivered by the engagement of activating receptors such as NCRs – NKp30, NKp44 and NKp46, NKG2D and co-receptors such as 2B4(CD244) or NTB-A (=NK, T and B lymphocytes-activated). The activating NCRs, NKG2C/E and NKG2D receptors are transducing signals through tyrosine-based activation molecules (ITAM) to NK cells resulting in up-regulation of their killing ability and/or cytokine production [reviewed in 40]. 2.2. The NKG2D immunoreceptor Among the activating NK cell receptors NKG2D holds a central position as a major activating immune receptor expressed on most cytotoxic lymphocytes in humans such as all NK cells, all CD8+ ␣␤T cells, subsets of ␥␦T – and NKT cells and a small subset of CD4+ ␣␤ T cells [42–44]. NKG2D is a type 2 transmembrane homodimer that belongs to the C lectin-like family. NKG2D lacks classical signalling

sequences in its cytoplasmic tale. Instead, ligation of NKG2D transduces activation signals through the adaptor molecule DAP10 in humans and DAP10 and 12 in mice and induces degranulation and/or cytokine production in cytotoxic effector cells. In NK cells, NKG2D ligation is sufficient to trigger NK cell cytotoxicity while it has a co-stimulatory function in CD8+ T cells, where it enhances TCR-driven cytotoxicity but cannot activate target cell lysis without TCR engagement [45–47]. NKG2D membrane expression is regulated by cytokine expression in the tissue microenvironment as well as by soluble NKG2D ligands. IL-2, IL-15 can rapidly increase NKG2D and DAP10 expression [47]. In inflammatory settings, such as rheumatoid arthritis, it was shown that TNF␣ and IL-15 can up-regulate NKG2D expression in a subset of CD4+ NKG2D+ cells [44]. In contrast, exposure to IL-21 in combination with IL12 or TGF-␤1 down modulates NKG2D expression thus interfering with immunosurveillance by impairing the initiation of the cytotoxic

Table 1 Some of the human NK cell receptors and their ligands. Group by molecular structure of the receptors

Receptors

Signal

Ligand

Ig superfamily – killer immunoglobulin-like receptors (KIRs), Ig-like transcripts (ILT)

KIR3DL1 KIR3DL2 KIR2DL1, 2, 3 KIR2DL4 KIR2DS1, 2 ILT 2 NKp44 NKp30 NKp46 NKp80 NKG2A/B NKG2C/E NKG2D

−a − − − +b − + + + + − + +

MHC-B MHC-A MHC-C MHC-A, -B, -G MHC-C MHC-A, -B, -G Unknown, influenza virus Unknown Unknown, influenza virus Unknown MHC-E MHC-E MICA/B; ULBPs

Natural cytotoxicity receptors (NCRs)

C-type lectin like receptors

a b

−, inhibitory signals transduced by immunoreceptor tyrosine-based inhibition motif (ITIM). +, activating signals transduced by immunoreceptor tyrosine-based activating molecule (ITAM).

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ARTICLE IN PRESS RAE1 ␣ − ␧, H60a-c, MULT 1 RAE1 ␣ − ␧, H60a-c, MULT 1 RAE1 ␣ − ␧, H60a-c, MULT 1 ND = not determined.

RAE1 ␣ − ␧, H60a-c, MULT 1 None None

RAE1 ␣ − ␧, H60a-c, MULT 1

RAE1 ␣ − ␧, H60a-c, MULT 1

Low/none Yes Low/none Yes Low/none Yes Low/none Yes Low/none Yes Low/none Yes

Low/none Yes

1.1 × 10−6 None None 8 × 10−7 None None

Receptor affinity KD (M) Peptide presentation Beta 2 microglobulin association Expression in normal cells Up-regulation upon infection, inflammation, transformation/genotoxic stress Murine orthologues

2 ␣1/␣2 domains Glycophosphatidylinositol (GPI) linked >30 ␣1/␣2/␣3 domains TM/cyt

>70 ␣1/␣2/␣3 domains Transmembrane and cyto-plasmic region (TM/cyt) 0.9-1 × 10−6 None None

Low/none Yes

ND None None ND None None ND None None ND None None

ULBP6

Polymorphism Molecular structure Membrane anchor

ND None None

RAET1G Chromosome 6, outside the MHC locus 6p25.1 3 ␣1/␣2 domains TM/cyt

ULBP5 ULBP4

RAET1E Chromosome 6, outside the MHC locus 6p25.1 6 ␣1/␣2 domains TM/cyt RAET1N Chromosome 6, outside the MHC locus 6p25.1 4 ␣1/␣2 domains GPI- linked

ULBP3 ULBP2

PERB11.1 Chromosome 6, MHC locus 6p21.33 Alternative name Gene location

MICB

ULBP1

PERB11.2 Chromosome 6, MHC locus 6p21.33

MICA Member name

Retinoic early transcript-1/UL-16 binding proteins (RAET1/ULBP) MHC class I chain-related proteins (MIC) Family

Table 2 Some of the major features of the human NKG2D ligand families MIC and RAET1/ULBP.

To date two families of human NKG2D ligands are known both encoded on chromosome 6: the MHC class I chain related antigens A and B (MICA and MICB) located within the MHC locus [52] and the six UL-16 binding proteins (ULBPs) also known as retinoic acid early transcript-1 proteins (RAET1) located outside the MHC locus. The ULBPs were named by their discovery as ligands to the human cytomegalovirus (CMV) – three of the members – ULBP1, 2 and 6 bind to the human CMV glycoprotein UL16 [53]. The molecules of the both family members have structural similarity to the MHC members but in comparison of coding sequences they are distantly related to the MHC proteins and to each other (about 20% sequence similarity). Similar to the classical MHC class I molecules MICA and B comprise of three domains ␣1, ␣2, ␣3, transmembrane region and a cytoplasmic tail, however, they do not present antigen peptides and are not associated with ␤2-microglobulin, functional features shared with the ULBP family of ligands. But in contrast to MICA/B, ULBPs lack ␣3 domain in their molecule and the majority of their members are linked to the membrane via a GPI-anchor. Some of the main characteristics of the NKG2D ligands are summarized in Table 2. The cellular expression of NKG2D ligands is complex and its control is not completely understood. The protein expression of these ligands on normal healthy cells is limited or absent although RNA message for both MIC and ULBPs has been found in various cell types and tissues. As mentioned before, these ligands act as inducible self antigens up-regulated by different types of pathological conditions that lead to cellular stress, such as tumour transformation, bacterial and viral infections, inflammation and autoimmunity. Heat shock, oxidative stress, proteasome inhibition and DNA damage by various agents like chemicals, irradiation and UV-light have all been described as stimuli leading to an induction and/or increase in the NKG2D ligand expression [42,54]. The exact molecular mechanisms controlling induction/up-regulation of the NKG2D ligands are not clear, however, numerous reports suggest that their expression is differentially regulated depending on the cell type, its metabolic status and the surrounding microenvironment. There are several reports on differential stress-induced MIC and ULBP regulation at many cellular levels – e.g. transcriptional and post-transcriptional level, as well as post-translational regulation including protein modifications, shedding and endosomal trafficking and release [reviewed in 54]. In addition to the mechanisms described above, diverse biochemical properties due to molecular structure, membranal anchor and MICA/B polymorphism are further contributing to the complexity of these ligands and their biological role that spans from participation in immune surveillance and protection of homeostasis by direct killing of infected, transformed or damaged cells to immune evasion by impairment of the cytotoxic response.

RAET1H Chromosome 6, outside the MHC locus 6p25.1 1 ␣1/␣2 domains GPI- linked

2.3. NKG2D ligands

RAET1I Chromosome 6, outside the MHC locus 6p25.1

hit of effector cells [48,49]. NKG2D is one of the most important tumour recognition receptors on NK cells although NCRs and other receptors like DNAM1 have also been associated with anti-tumour defense [50,51]. The role of NKG2D as a key tumour recognition receptor lies in its specificity for induced self-proteins as compared to other receptors, e.g. T cell and B cell receptors that recognize foreign antigens. The ligands recognized by NKG2D, further described below, are poorly or not at all expressed on healthy cells. Instead their expression is induced as a result of cellular stress caused by infection, inflammation, tumorogenesis or damage by cellular or genotoxic agents. Thus, the activating cytotoxic NKG2D receptor participates in immune surveillance by induced-self recognition and elimination of damaged and transformed cells.

RAET1L Chromosome 6, outside the MHC locus 6p25.1 1 ␣1/␣2 domains GPI-linked

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2.4. NKG2D-ligand expression in tumours and the role of NKG2D in tumour surveillance There are several studies reporting expression of human ULBP/RAET1 ligands by tumours of different origin including leukemias, lymphomas, gliomas, melanomas, and ovarian carcinomas. The MIC molecules were detected in even broader range of tumours – haematological malignances and various adenocarcinomas such as breast, lung, colon, kidney, ovary and prostate tumours, gliomas, neuroblastomas and melanomas [17,37, 55, reviewed in 56]. The NKG2D ligand expression is not a constitutive feature of all tumour-transformed cells, not even within the same cell type. The ligand expression is heterogeneous between different tumour types and between individual cells within a tumour entity – e.g. ovarian carcinomas display a great difference in NKG2D ligand expression both regarding the ligands expressed and the level of expression [51]. Similarly to humans, murine NKG2D ligands are found on haematological malignancies and murine carcinomas, including lung, rectum, colon and prostate tumour-derived cell lines. Differences in NKG2D ligand expression in murine tumours homologous to those in humans were also observed. For example, only one-third of MCA-induced murine sarcoma cell lines were found to express RAE-1 molecules [50]. Thus, several factors and additional events besides malignant transformation and tumorogenesis are required for NKG2D ligand expression [57]. However, there are studies with conflicting results and the exact regulation of these ligands in cancer is still poorly understood and remains to be elucidated [reviewed in 56]. The finding that tumour cells express NKG2D ligands that can activate NKG2D dependent killing in vitro and in vivo brought a revival of the anti-tumour immune surveillance concept. Tumour cells expressing NKG2D ligands can be rejected in an NKG2D receptor depending fashion. Moreover, it was shown in vivo that neutralizing the NKG2D receptor by administration of antiNKG2D antibodies in mice increased the incidence of MCA-induced fibrosarcoma [50] and that NKG2D-deficient mice in models of spontaneous malignancy were prone to develop severe prostate adenocarcinoma and B cell lymphoma, underlying that the NKG2D receptor-mediated cytotoxicity is critical in anti-tumour immune surveillance [58]. Cancers undermine NKG2D-mediated anti-tumour immune surveillance by release of NKG2D-ligand-expressing exosomes that are more potent NKG2D receptor inhibitors than truncated NKG2D ligands, shed by protease-cleavage. The NKG2D cytotoxicity destroying NKG2D ligand-expressing tumour cells shown in vitro could not be completely translated in vivo. While NKG2D-dependent tumour rejection was effective at the early stages of tumour development, sustained expression of NKG2D ligands in late stages of malignancy were negatively associated with the immune response thus facilitating the malignant process. What could be the reason for that besides downregulation of the NKG2D receptor due to sustained NIG2D engagement by its ligands? There is mounting evidence demonstrating that tumour cells avoid becoming NK cell targets by shedding NKG2D ligands from the cell surface through cleavage by matrix metalloproteases (MMPs), a finding supported by presence of soluble MICA and B in serum of cancer patients but not in healthy controls. The effect is not only decreasing the cell surface ligand expression in tumours but also down modulating expression of the NKG2D receptor. However, there is equally strong and abundant evidence that besides MMP-mediated shedding, the NKG2D ligands are expressed and carried on the surface of tumour exosomes, constitutively released in the blood and other body fluids and tumoral effusions such as pleural and ascitic fluid. At present the mechanisms that decide the mode of NKG2D ligand expression, on the cellular surface or on

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exosomes in MVBs, are not known. As shown in Table 2 the NKG2D ligands are expressed either as transmembranally-attached or GPI-linked molecules. It has been proposed that difference in membranal attachment might play a role on how NKG2D ligands are expressed and how they function. It was reported that murine NKG2D ligands with transmembranal attachment have a higher receptor affinity compared to GPI-linked [59], however, these differences were not observed in humans [60]. Another suggestion was that the GPI-linked ligands are preferably expressed on exosomes since GPI-anchored proteins are preferably enriched in DRM and thus they will be internalized to early and late endosomes and eventually end up on exosomes that are released in the intercellular space. In line with this suggestion it was shown that the GPI-linked ULBP1-3 were recruited to DRM regions [61] while only a low proportion of the transmembranally attached MICA/B molecules were present in DRM [62]. Although this observation was experimentally substantiated it is not a general rule for all or for a particular NKG2D ligand(s). There are several reports contradicting this “rule” where differences in NKG2D ligand expression were found not only between different family members but also within one and the same molecule expressed in different settings. Thus, TM-attached MICA/B family members can be enriched in DRM, since a high proportion of the very common MICA*008 proteins were recruited to DRM and exosomally expressed as full length proteins [63]. Furthermore, it has been postulated that ULBP3 is often expressed on exosomes while ULBP1 and 2 are MMP-cleaved. Treatment with MMP-inhibitors could up regulate and shift the ULBP2 expression to exosomes and enrich MICA*019 proteins from the cellular to the exosomal membrane [61,65]. In contrast, Viaud et al. found ULBP1 on dendritic cell-derived exosomes [64]. We found MIC and ULBP1 and 2 but not 3 expressed on exosomes secreted by leukaemia/lymphoma T and B cells that could upregulate their exosome secretion when subjected to thermal and oxidative cellular stress [17]. Moreover, in our biogenetic studies of NKG2D ligand-expressing human placental exosomes we found that all NKG2D ligands, regardless of their membrane anchoring were enriched on exosomes in the late endosomes (MVBs) of the syncytiotrophoblast and released by membrane fusion of the MVB membrane and the apical syncytiotrophoblastic membrane [25,26]. Thus, there are intrinsic and extrinsic differences in the expression of NKG2D ligands and the exact mechanisms regulating their expression remain unclear. One can, though, conclude based on mounting evidence that the NKG2D ligands are differentially expressed and the “soluble” NKG2D ligand moiety in blood, urine and other bodily effusions of cancer patients originates from MMP-cleaved truncated ligand proteins and full length NKG2D ligand proteins that are TM- or GPI-anchored on the membrane of released exosomes thus preserving their three-dimensional protein structure and orientation. The differences in these two moieties of soluble NKG2D ligands are important in regard to their function and ability to modulate the immune system. Ligation of the NKG2D receptor by soluble NKG2DL produced by MMP-mediated shedding of cell surface-bound ligands or by membrane-bound exosomal ligands can result in down modulation of cognate receptor leading to impairment of the cytotoxic response, a strategy for tumour escape from immune attack [17,26,27,38,55]. However, the NKG2D ligands expressed on exosomes seem to be far more potent as receptor down modulators compared to the truncated MMP-cleaved ligands [61,63]. One explanation of the greater potency of exosome-mediated down modulation might be that besides preservation of the molecular structure and biologic activity of the ligand(s), the exosome can enrich several molecules of the same ligand and probably express other NKG2D ligands as well and thus be a multipotent carrier of NKG2D ligands impairing the cytotoxic response to a greater extent. The presence of soluble MIC molecules in the serum of cancer patients was documented

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and proposed to be MMP-cleaved molecules [66]. However, the method of detection does not distinguish between MMP-cleaved and exosomally-carried MIC molecules and the soluble MIC in serum of cancer patients must be a mixture of both. In this regard it is important to note that the most abundantly expressed MIC protein MICA*008 is entirely released on exosomes [63]. A careful examination of the mechanisms behind the tumour exosome-mediated NKG2D receptor down modulation shows that the NKG2D ligand-expressing tumour exosomes down regulate the cognate receptor and impair the cytotoxic response but preserve the constitutive levels of granzyme B and perforin in cytotoxic T cells and NK cells leaving their lytic machinery intact [37]. We found a similar immunosuppressive action mediated by human placental exosomes [25,26]. Another interesting observation was that exosomally carried TGF␤1, delivered to CD8+ T and NK cells, had a powerful down-modulatory effect to NKG2D receptor, which was exosome-dependent and could not be further increased by addition of exogenous TGF␤1 [37]. The powerful down modulation of the NKG2D receptor and the subsequent impairment of cytotoxicity in the presence of tumour exosomes could not be restored by IL-15, a cytokine well known as a positive regulator of NKG2Ddependent NK cell responses. Thus, it seems that NKG2D ligands and TGF␤1 delivered by exosomes cooperate to decrease NKG2D expression and NKG2D-mediated cytotoxic responses by a mechanism(s) so far unclarified [37]. In addition, cancer exosomes exert negative effect on NK and CTL function by other direct or indirect mechanisms such as inhibiting lymphocyte responses to IL-2 and inducing T regulatory cells [23]. 3. Concluding remarks The NKG2D receptor–ligand system is a multifunctional danger detecting system alerting the immune system to respond to diverse situations of cellular stress such as infections, inflammation, DNA damage and malignant transformation. Ligand recognition by the cognate receptor mediates cytotoxic responses of innate and adaptive immune effector cells resulting in elimination of infected, damaged or tumour transformed cells. However, exosomal release and/or MMP-shedding of these same ligands in the sera of cancer patients down regulates the receptor on NK and cytotoxic T cells and weakens the immune response. A convincing body of evidence indicates that NKG2D ligands released on tumour exosomes in the blood and bodily fluids of cancer patients are far more potent NKG2D inhibitors than their MMP-cleaved truncated soluble counterparts. Tumour exosomes deliver multiple NKG2D ligands in combination with other signals that derange the NKG2D-mediated anti-tumour immune surveillance thus promoting cancer establishment and its metastatic spreading. Much is known about the nature and biochemical properties of the NKG2D ligands, however, more remains to be elucidated in order to gain a comprehensive understanding of how the cellular and exosomal expression of different ligands is regulated. Revealing their precise regulation of expression and elucidating the biochemical composition and biological role of their carriers, the immunosuppressive exosomes, that are constitutively released by most cancers, is a prerequisite for finding new strategies to counteract their inhibitory effect and assist the immune system in regaining its ability to combat tumour through immune surveillance. Acknowledgements This work was supported by the Swedish National Research (Vetenskapsrådet, K2013-54X-22341-01-05); Foundation Swedish National Cancer Research Foundation (Cancerfonden, CAN2013/439); Central ALF funding and Spjutspetsanslag, County

of Västerbotten; and the Insamlingsstiftelsen at the Medical Faculty, Umeå University, Sweden.

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Please cite this article in press as: Mincheva-Nilsson L, Baranov V. Cancer exosomes and NKG2D receptor–ligand interactions: Impairing NKG2D-mediated cytotoxicity and anti-tumour immune surveillance. Semin Cancer Biol (2014), http://dx.doi.org/10.1016/j.semcancer.2014.02.010