Engaging natural killer cells for cancer therapy via NKG2D, CD16A and other receptors.

The field of immuno-oncology has revolutionized cancer patient care and improved survival and quality of life for patients. Much of the focus in the field has been on exploiting the power of the adaptive immune response through therapeutic targeting of T cells. While these approaches have markedly advanced the field, some challenges remain, and the clinical benefit of T cell therapies does not extend to all patients or tumor indications. Alternative strategies, such as engaging the innate immune system, have become an intense area of focus in the field. In particular, the engagement of natural killer (NK) cells as potent effectors of the innate immune response has emerged as a promising modality in immunotherapy. Here, we review therapeutic approaches for selective engagement of NK cells for cancer therapy, with a particular focus on targeting the key activating receptors NK Group 2D (NKG2D) and cluster of differentiation 16A (CD16A).

The NKG2D ligand ULBP4 is not expressed by human monocytes.

The C-type lectin-like receptor NKG2D contributes to the immunosurveillance of virally infected and malignant cells by cytotoxic lymphocytes. A peculiar and puzzling feature of the NKG2D-based immunorecognition system is the high number of ligands for this single immunoreceptor. In humans, there are a total of eight NKG2D ligands (NKG2DL) comprising two members of the MIC (MICA, MICB) and six members of the ULBP family of glycoproteins (ULBP1 to ULBP6). While MICA has been extensively studied with regard to its biochemistry, cellular expression and function, very little is known about the NKG2DL ULBP4. This is, at least in part, due to its rather restricted expression by very few cell lines and tissues. Recently, constitutive ULBP4 expression by human monocytes was reported, questioning the view of tissue-restricted ULBP4 expression. Here, we scrutinized ULBP4 expression by human peripheral blood mononuclear cells and monocytes by analyzing ULBP4 transcripts and ULBP4 surface expression. In contrast to MICA, there was no ULBP4 expression detectable, neither by freshly isolated monocytes nor by PAMP-activated monocytes. However, a commercial antibody erroneously indicated surface ULBP4 on monocytes due to a non-ULBP4-specific binding activity, emphasizing the critical importance of validated reagents for life sciences. Collectively, our data show that ULBP4 is not expressed by monocytes, and likely also not by other peripheral blood immune cells, and therefore exhibits an expression pattern rather distinct from other human NKG2DL.

Targeting MICA/B with cytotoxic therapeutic antibodies leads to tumor control.

Background: MICA and MICB are tightly regulated stress-induced proteins that trigger the immune system by binding to the activating receptor NKG2D on cytotoxic lymphocytes. MICA and MICB are highly polymorphic molecules with prevalent expression on several types of solid tumors and limited expression in normal/healthy tissues, making them attractive targets for therapeutic intervention. Methods: We have generated a series of anti-MICA and MICB cross-reactive antibodies with the unique feature of binding to the most prevalent isoforms of both these molecules. Results: The anti-MICA and MICB antibody MICAB1, a human IgG1 Fc-engineered monoclonal antibody (mAb), displayed potent antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) of MICA/B-expressing tumor cells in vitro. However, it showed insufficient efficiency against solid tumors in vivo, which prompted the development of antibody-drug conjugates (ADC). Indeed, optimal tumor control was achieved with MICAB1-ADC format in several solid tumor models, including patient-derived xenografts (PDX) and carcinogen-induced tumors in immunocompetent MICAgen transgenic mice. Conclusions: These data indicate that MICA and MICB are promising targets for cytotoxic immunotherapy.

Arming cytotoxic lymphocytes for cancer immunotherapy by means of the NKG2D/NKG2D-ligand system.

INTRODUCTION: The activating NKG2D receptor plays a central role in the immune recognition and elimination of abnormal self-cells by cytotoxic lymphocytes. NKG2D binding to cell stress-inducible ligands (NKG2DL) up-regulated on cancer cells facilitates their immunorecognition. Yet tumor cells utilize various escape mechanisms to avert NKG2D-based immunosurveillance. Hence, therapeutic strategies targeting the potent NKG2D/NKG2DL axis and such immune escape mechanisms become increasingly attractive in cancer therapy. AREAS COVERED: This perspective provides a brief introduction into the NKG2D/NKG2DL axis and its relevance for cancer immune surveillance. Subsequently, the most advanced therapeutic approaches targeting the NKG2D system are presented focusing on NKG2D-CAR engineered immune cells and antibody-mediated strategies to inhibit NKG2DL shedding by tumors. EXPERT OPINION: Thus far, NKG2D-CAR engineered lymphocytes represent the most advanced therapeutic approach utilizing the NKG2D system. Similarly to other tumor-targeting CAR approaches, NKG2D-CAR cells demonstrate powerful on-target activity, but may also cause off-tumor toxicities or lose efficacy, if NKG2DL expression by tumors is reduced. However, NKG2D-CAR cells also act on the tumor microenvironment curtailing its immunosuppressive properties, thus providing an independent therapeutic benefit. The potency of tumoricidal NKG2D-expressing lymphocytes can be further boosted by enhancing NKG2DL expression through small molecules and therapeutic antibodies inhibiting tumor-associated shedding of NKG2DL.

MICAgen Mice Recapitulate the Highly Restricted but Activation-Inducible Expression of the Paradigmatic Human NKG2D Ligand MICA.

NKG2D is a potent activating immunoreceptor expressed on nearly all cytotoxic lymphocytes promoting their cytotoxicity against self-cells expressing NKG2D ligands (NKG2DLs). NKG2DLs are MHC class I-like glycoproteins that usually are not expressed on "healthy" cells. Rather, their surface expression is induced by various forms of cellular stress, viral infection, or malignant transformation. Hence, cell surface NKG2DLs mark "dangerous" cells for elimination by cytotoxic lymphocytes and therefore can be considered as "kill-me" signals. In addition, NKG2DLs are up-regulated on activated leukocytes, which facilitates containment of immune responses. While the NKG2D receptor is conserved among mammals, NKG2DL genes have rapidly diversified during mammalian speciation, likely due to strong selective pressures exerted by species-specific pathogens. Consequently, NKG2DL genes are not conserved in man and mice, although their NKG2D-binding domains maintained structural homology. Human NKG2DLs comprise two members of the MIC (MICA/MICB) and six members of the ULBP family of glycoproteins (ULBP1-6) with MICA representing the best-studied human NKG2DLs by far. Many of these studies implicate a role of MICA in various malignant, infectious, or autoimmune diseases. However, conclusions from these studies often were limited in default of supporting in vivo experiments. Here, we report a MICA transgenic (MICAgen) mouse model that replicates central features of human MICA expression and function and, therefore, constitutes a novel tool to critically assess and extend conclusions from previous in vitro studies on MICA. Similarly to humans, MICA transcripts are broadly present in organs of MICAgen mice, while MICA glycoproteins are barely detectable. Upon activation, hematopoietic cells up-regulate and proteolytically shed surface MICA. Shed soluble MICA (sMICA) is also present in plasma of MICAgen mice but affects neither surface NKG2D expression of circulating NK cells nor their functional recognition of MICA-expressing tumor cells. Accordingly, MICAgen mice also show a delayed growth of MICA-expressing B16F10 tumors, not accompanied by an emergence of MICA-specific antibodies. Such immunotolerance for the xenoantigen MICA ideally suits MICAgen mice for anti-MICA-based immunotherapies. Altogether, MICAgen mice represent a valuable model to study regulation, function, disease relevance, and therapeutic targeting of MICA in vivo.

Impairment of NKG2D-Mediated Tumor Immunity by TGF-ß.

Transforming growth factor-ß (TGF-ß) suppresses innate and adaptive immune responses via multiple mechanisms. TGF-ß also importantly contributes to the formation of an immunosuppressive tumor microenvironment thereby promoting tumor growth. Amongst others, TGF-ß impairs tumor recognition by cytotoxic lymphocytes via NKG2D. NKG2D is a homodimeric C-type lectin-like receptor expressed on virtually all human NK cells and cytotoxic T cells, and stimulates their effector functions upon engagement by NKG2D ligands (NKG2DL). While NKG2DL are mostly absent from healthy cells, their expression is induced by cellular stress and malignant transformation, and, accordingly, frequently detected on various tumor cells. Hence, the NKG2D axis is thought to play a decisive role in cancer immunosurveillance and, obviously, often is compromised in clinically apparent tumors. There is mounting evidence that TGF-ß, produced by tumor cells and immune cells in the tumor microenvironment, plays a key role in blunting the NKG2D-mediated tumor surveillance. Here, we review the current knowledge on the impairment of NKG2D-mediated cancer immunity through TGF-ß and discuss therapeutic approaches aiming at counteracting this major immune escape pathway. By reducing tumor-associated expression of NKG2DL and blinding cytotoxic lymphocytes through down-regulation of NKG2D, TGF-ß is acting upon both sides of the NKG2D axis severely compromising NKG2D-mediated tumor rejection. Consequently, novel therapies targeting the TGF-ß pathway are expected to reinvigorate NKG2D-mediated tumor elimination and thereby to improve the survival of cancer patients.

CD56 as a marker of an ILC1-like population with NK cell properties that is functionally impaired in AML.

An understanding of natural killer (NK) cell physiology in acute myeloid leukemia (AML) has led to the use of NK cell transfer in patients, demonstrating promising clinical results. However, AML is still characterized by a high relapse rate and poor overall survival. In addition to conventional NKs that can be considered the innate counterparts of CD8 T cells, another family of innate lymphocytes has been recently described with phenotypes and functions mirroring those of helper CD4 T cells. Here, in blood and tissues, we identified a CD56+ innate cell population harboring mixed transcriptional and phenotypic attributes of conventional helper innate lymphoid cells (ILCs) and lytic NK cells. These CD56+ ILC1-like cells possess strong cytotoxic capacities that are impaired in AML patients at diagnosis but are restored upon remission. Their cytotoxicity is KIR independent and relies on the expression of TRAIL, NKp30, NKp80, and NKG2A. However, the presence of leukemic blasts, HLA-E-positive cells, and/or transforming growth factor-ß1 (TGF-ß1) strongly affect their cytotoxic potential, at least partially by reducing the expression of cytotoxic-related molecules. Notably, CD56+ ILC1-like cells are also present in the NK cell preparations used in NK transfer-based clinical trials. Overall, we identified an NK cell-related CD56+ ILC population involved in tumor immunosurveillance in humans, and we propose that restoring their functions with anti-NKG2A antibodies and/or small molecules inhibiting TGF-ß1 might represent a novel strategy for improving current immunotherapies.

Publisher Correction: Absence of NKG2D ligands defines leukaemia stem cells and mediates their immune evasion.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Absence of NKG2D ligands defines leukaemia stem cells and mediates their immune evasion.

Patients with acute myeloid leukaemia (AML) often achieve remission after therapy, but subsequently die of relapse1 that is driven by chemotherapy-resistant leukaemic stem cells (LSCs)2,3. LSCs are defined by their capacity to initiate leukaemia in immunocompromised mice4. However, this precludes analyses of their interaction with lymphocytes as components of anti-tumour immunity5, which LSCs must escape to induce cancer. Here we demonstrate that stemness and immune evasion are closely intertwined in AML. Using xenografts of human AML as well as syngeneic mouse models of leukaemia, we show that ligands of the danger detector NKG2D-a critical mediator of anti-tumour immunity by cytotoxic lymphocytes, such as NK cells6-9-are generally expressed on bulk AML cells but not on LSCs. AML cells with LSC properties can be isolated by their lack of expression of NKG2D ligands (NKG2DLs) in both CD34-expressing and non-CD34-expressing cases of AML. AML cells that express NKG2DLs are cleared by NK cells, whereas NKG2DL-negative leukaemic cells isolated from the same individual escape cell killing by NK cells. These NKG2DL-negative AML cells show an immature morphology, display molecular and functional stemness characteristics, and can initiate serially re-transplantable leukaemia and survive chemotherapy in patient-derived xenotransplant models. Mechanistically, poly-ADP-ribose polymerase 1 (PARP1) represses expression of NKG2DLs. Genetic or pharmacologic inhibition of PARP1 induces NKG2DLs on the LSC surface but not on healthy or pre-leukaemic cells. Treatment with PARP1 inhibitors, followed by transfer of polyclonal NK cells, suppresses leukaemogenesis in patient-derived xenotransplant models. In summary, our data link the LSC concept to immune escape and provide a strong rationale for targeting therapy-resistant LSCs by PARP1 inhibition, which renders them amenable to control by NK cells in vivo.

The NKG2D axis: an emerging target in cancer immunotherapy.

INTRODUCTION: The immunoreceptor NKG2D belongs to the best-characterized activating receptors of cytotoxic lymphocytes. NKG2D binds to a variety of cell surface glycoproteins distantly related to MHC class I molecules, termed NKG2D ligands (NKG2DL). NKG2DL are inducibly expressed upon cellular stress, viral infection or malignant transformation thus marking 'stressed' or 'harmful' cells for clearance through NKG2D+ lymphocytes. However, certain viruses and many tumors employ various strategies to escape from NKG2D-mediated immunosurveillance. Areas covered: Expression and regulation of both NKG2D and NKG2DL, especially at sites of immune responses or in the tumor microenvironment, as well as mechanisms of NKG2D escape strategies, as their understanding is key for harnessing the NKG2D/NKG2DL axis for immunotherapy. Studies documenting the importance of the NKG2D/NKG2DL axis for cancer immunosurveillance. Therapeutic approaches targeting the NKG2D/NKG2DL axis in cancer. Expert opinion: The selective expression of NKG2DL on malignant cells together with the strong activating potency of NKG2D renders the NKG2D/NKG2DL axis a prime target for immunotherapies. Based on a thorough understanding of the NKG2D/NKG2DL system as well as of the most relevant escape strategies of tumors, the diligent and thoughtful design of novel treatment modalities harnessing the NKG2D/NKG2DL axis holds great promise for the future therapy of cancer.

A point mutation in the Ncr1 signal peptide impairs the development of innate lymphoid cell subsets.

NKp46 (CD335) is a surface receptor shared by both human and mouse natural killer (NK) cells and innate lymphoid cells (ILCs) that transduces activating signals necessary to eliminate virus-infected cells and tumors. Here, we describe a spontaneous point mutation of cysteine to arginine (C14R) in the signal peptide of the NKp46 protein in congenic Ly5.1 mice and the newly generated NCRB6C14R strain. Ly5.1C14R NK cells expressed similar levels of Ncr1 mRNA as C57BL/6, but showed impaired surface NKp46 and reduced ability to control melanoma tumors in vivo. Expression of the mutant NKp46C14R in 293T cells showed that NKp46 protein trafficking to the cell surface was compromised. Although Ly5.1C14R mice had normal number of NK cells, they showed an increased number of early maturation stage NK cells. CD49a+ILC1s were also increased but these cells lacked the expression of TRAIL. ILC3s that expressed NKp46 were not detectable and were not apparent when examined by T-bet expression. Thus, the C14R mutation reveals that NKp46 is important for NK cell and ILC differentiation, maturation and function. Significance Innate lymphoid cells (ILCs) play important roles in immune protection. Various subsets of ILCs express the activating receptor NKp46 which is capable of recognizing pathogen derived and tumor ligands and is necessary for immune protection. Here, we describe a spontaneous point mutation in the signal peptide of the NKp46 protein in congenic Ly5.1 mice which are widely used for tracking cells in vivo. This Ncr1 C14R mutation impairs NKp46 surface expression resulting in destabilization of Ncr1 and accumulation of NKp46 in the endoplasmic reticulum. Loss of stable NKp46 expression impaired the maturation of NKp46+ ILCs and altered the expression of TRAIL and T-bet in ILC1 and ILC3, respectively.

Cutting an NKG2D Ligand Short: Cellular Processing of the Peculiar Human NKG2D Ligand ULBP4.

Stress-induced cell surface expression of MHC class I-related glycoproteins of the MIC and ULBP families allows for immune recognition of dangerous "self cells" by human cytotoxic lymphocytes via the NKG2D receptor. With two MIC molecules (MICA and MICB) and six ULBP molecules (ULBP1-6), there are a total of eight human NKG2D ligands (NKG2DL). Since the discovery of the NKG2D-NKG2DL system, the cause for both redundancy and diversity of NKG2DL has been a major and ongoing matter of debate. NKG2DL diversity has been attributed, among others, to the selective pressure by viral immunoevasins, to diverse regulation of expression, to differential tissue expression as well as to variations in receptor interactions. Here, we critically review the current state of knowledge on the poorly studied human NKG2DL ULBP4. Summarizing available facts and previous studies, we picture ULBP4 as a peculiar ULBP family member distinct from other ULBP family members by various aspects. In addition, we provide novel experimental evidence suggesting that cellular processing gives rise to mature ULBP4 glycoproteins different to previous reports. Finally, we report on the proteolytic release of soluble ULBP4 and discuss these results in the light of known mechanisms for generation of soluble NKG2DL.

The novel deubiquitinase inhibitor b-AP15 induces direct and NK cell-mediated antitumor effects in human mantle cell lymphoma.

The first therapeutic proteasome inhibitor bortezomib has clinical efficacy in mantle cell lymphoma (MCL) which resulted in its incorporation in treatment algorithms for this disease. Impairment of proteasomal function by bortezomib is mediated via inhibition of the 20S core particle. However, proteasome function can also be modified by targeting upstream components of the ubiquitin-proteasome system. Recently, b-AP15 has been identified as a small molecule achieving proteasome inhibition by targeting the deubiquitinase (DUB) activity of the 19S regulatory subunit and was found to inhibit cancer cell growth in preclinical analyses. In the present study, both direct antitumor effects and the possibility to induce natural killer group 2 member D ligands (NKG2DL) to reinforce NK cell immunity with b-AP15 were investigated to provide a rational basis for clinical evaluation of this novel DUB inhibitor in MCL. Treatment with b-AP15 resulted in reduced viability as well as induction of apoptosis in a time- and dose-dependent manner, which could be attributed to caspase activation in MCL cells. In addition, treatment with b-AP15 differentially induced NKG2DL expression and subsequent NK cell lysis of MCL cells. These results indicate that the DUB inhibitor b-AP15 displays substantial antitumor activity in human MCL and suggest that b-AP15 might be a novel therapeutic option in the treatment of MCL that warrants clinical investigation.

Platelet-mediated shedding of NKG2D ligands impairs NK cell immune-surveillance of tumor cells.

Platelets promote metastasis, among others by coating cancer cells traveling through the blood, which results in protection from NK cell immune-surveillance. The underlying mechanisms, however, remain to be fully elucidated. Here we report that platelet-coating reduces surface expression of NKG2D ligands, in particular MICA and MICB, on tumor cells, which was mirrored by enhanced release of their soluble ectodomains. Similar results were obtained upon exposure of tumor cells to platelet-releasate and can be attributed to the sheddases ADAM10 and ADAM17 that are detectable on the platelet surface and in releasate following activation and at higher levels on platelets of patients with metastasized lung cancer compared with healthy controls. Platelet-mediated NKG2DL-shedding in turn resulted in impaired "induced self" recognition by NK cells as revealed by diminished NKG2D-dependent lysis of tumor cells. Our results indicate that platelet-mediated NKG2DL-shedding may be involved in immune-evasion of (metastasizing) tumor cells from NK cell reactivity.

NKG2D-Dependent Antitumor Effects of Chemotherapy and Radiotherapy against Glioblastoma.

Purpose: NKG2D is a potent activating immune cell receptor, and glioma cells express the cognate ligands (NKG2DL). These ligands are inducible by cellular stress and temozolomide (TMZ) or irradiation (IR), the standard treatment of glioblastoma, could affect their expression. However, a role of NKG2DL for the efficacy of TMZ and IR has never been addressed.Experimental Design: We assessed the effect of TMZ and IR on NKG2DL in vitro and in vivo in a variety of murine and human glioblastoma models, including glioma-initiating cells, and a cohort of paired glioblastoma samples from patients before and after therapy. Functional effects were studied with immune cell assays. The relevance of the NKG2D system for the efficacy of TMZ and IR was assessed in vivo in syngeneic orthotopic glioblastoma models with blocking antibodies and NKG2D knockout mice.Results: TMZ or IR induced NKG2DL in vitro and in vivo in all glioblastoma models, and glioblastoma patient samples had increased levels of NKG2DL after therapy with TMZ and IR. This enhanced the immunogenicity of glioma cells in a NGK2D-dependent manner, was independent from cytotoxic or growth inhibitory effects, attenuated by O6-methylguanine-DNA-methyltransferase (MGMT), and required the DNA damage response. The survival benefit afforded by TMZ or IR relied on an intact NKG2D system and was decreased upon inhibition of the NKG2D pathway.Conclusions: The immune system may influence the activity of convential cancer treatments with particular importance of the NKG2D pathway in glioblastoma. Our data provide a rationale to combine NKG2D-based immunotherapies with TMZ and IR. Clin Cancer Res; 24(4); 882-95. ©2017 AACR.

Select Clr-g Expression on Activated Dendritic Cells Facilitates Cognate Interaction with a Minor Subset of Splenic NK Cells Expressing the Inhibitory Nkrp1g Receptor.

Natural killer gene complex-encoded immunomodulatory C-type lectin-like receptors include members of the NKRP1 and C-type lectin-like 2 (CLEC2) gene families, which constitute genetically linked receptor-ligand pairs and are thought to allow for NK cell-mediated immunosurveillance of stressed or infected tissues. The mouse C-type lectin-like receptor Nkrp1g was previously shown to form several receptor-ligand pairs with the CLEC2 proteins Clr-d, Clr-f, and Clr-g, respectively. However, the physiological expression of Nkrp1g and its CLEC2 ligands as well as their functional relevance remained poorly understood. Recently, we demonstrated a gut-restricted expression of Clr-f on intestinal epithelial cells that is spatially matched by Nkrp1g on subsets of intraepithelial lymphocytes. In this study, we investigated expression and ligand interaction of Nkrp1g in the splenic compartment, and found an exclusive expression on a small subset of NK cells that upregulates Nkrp1g after cytokine exposure. Whereas transcripts of Clr-d and Clr-f are virtually absent from the spleen, Clr-g transcripts were abundantly detected throughout different leukocyte populations and hematopoietic cell lines. However, a newly generated anti-Clr-g mAb detected only residual Clr-g surface expression on splenic monocytes, whereas many hematopoietic cell lines brightly display Clr-g. Clr-g surface expression was strongly upregulated on splenic CD8α+ conventional dendritic cells (DCs) and plasmacytoid DCs upon TLR-mediated activation and detectable by Nkrp1g, which dampens NK cell effector functions upon Clr-g engagement. Hence, different to the intestinal tract, in the spleen, Nkrp1g is selectively expressed by a subset of NK cells, thereby potentially allowing for an inhibitory engagement with Clr-g-expressing activated DCs during immune responses.

HemITAM: A single tyrosine motif that packs a punch.

Innate immune cells sense danger through a plethora of germline-encoded receptors that recognize pathogen-associated molecular patterns (PAMPs) or cellular molecules that are exposed only by stressed, infected, malignant, or dead cells. Many of these danger-sensing receptors belong to the C-type lectin-like superfamily (CLSF) and therefore are called C-type lectin-like receptors (CTLRs). Certain activating CTLRs, namely, CLEC-2, Dectin-1, DNGR-1, NKp80, and NKp65, which are encoded by genes that are clustered together in a subregion of the mammalian natural killer gene complex (NKC), use a single copy tyrosine signaling module termed the hemi-immunoreceptor tyrosine-based activation motif (hemITAM). These hemITAM-bearing CTLRs are present on myeloid cells and innate lymphocytes and stimulate various functions, such as phagocytosis, cytokine production, and cytotoxicity. Proximal signaling mechanisms involve the tyrosine phosphorylation of the hemITAM and the subsequent activation of the kinase Syk. Signaling and Syk recruitment by the hemITAM appear to be tuned by variable amino acids within or near the hemITAM, which give rise to differences in downstream signaling events and diverging functional outcomes among hemITAM-bearing receptors.

Chronic NKG2D Engagement In Vivo Differentially Impacts NK Cell Responsiveness by Activating NK Receptors.

Immunosuppression is a typical hallmark of cancer and frequently includes perturbations of the NKG2D tumor recognition system as well as impaired signaling by other activating NK cell receptors. Several in vitro studies suggested that sustained engagement of the NKG2D receptor, as it is occurring in the tumor microenvironment, not only impairs expression and function of NKG2D but also impacts signaling by other activating NK receptors. Here, we made use of a transgenic mouse model of ubiquitous NKG2D ligand expression (H2-Kb-MICA mice) to investigate consequences of chronic NKG2D engagement in vivo for functional responsiveness by other activating NK receptors such as NKp46 and Ly49D. Unexpectedly, we found no evidence for an impairment of NKp46 expression and function in H2-Kb-MICA mice, as anticipated from previous in vitro experiments. However, we observed a marked downregulation and dysfunction of the activating receptor Ly49D in activated NK cells from H2-Kb-MICA mice. Ly49D shares the adaptor proteins DAP10 and DAP12 with NKG2D possibly explaining the collateral impairment of Ly49D function in situations of chronic NKG2D engagement. Altogether, our results demonstrate that persistent engagement of NKG2D in vivo, as often observed in tumors, can selectively impair functions of unrelated NK receptors and thereby compromise NK responsiveness to third-party antigens.

Natural Killer Group 2D Ligand Depletion Reconstitutes Natural Killer Cell Immunosurveillance of Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a highly heterogeneous and aggressive tumor originating from the epithelial lining of the upper aero-digestive tract accounting for 300,000 annual deaths worldwide due to failure of current therapies. The natural killer group 2D (NKG2D) receptors on natural killer (NK) cells and several T cell subsets play an important role for immunosurveillance of HNSCC and are thus targeted by tumor immune evasion strategies in particular by shedding of various NKG2D ligands (NKG2DLs). Based on plasma and tumor samples of 44 HNSCC patients, we found that despite compositional heterogeneity the total plasma level of NKG2DLs correlates with NK cell inhibition and disease progression. Strikingly, based on tumor spheroids and primary tumors of HNSCC patients, we found that NK cells failed to infiltrate HNSCC tumors in the presence of high levels of NKG2DLs, demonstrating a novel mechanism of NKG2DL-dependent tumor immune escape. Therefore, the diagnostic acquisition of the plasma level of all NKG2DLs might be instrumental for prognosis and to decipher a patient cohort, which could benefit from restoration of NKG2D-dependent tumor immunosurveillance. Along these lines, we could show that removal of shed NKG2DLs (sNKG2DLs) from HNSCC patients' plasma restored NK cell function in vitro and in individual patients following surgical removal of the primary tumor. In order to translate these findings into a therapeutic setting, we performed a proof-of-concept study to test the efficacy of adsorption apheresis of sNKG2DLs from plasma after infusion of human MICA in rhesus monkeys. Complete removal of MICA was achieved after three plasma volume exchanges. Therefore, we propose adsorption apheresis of sNKG2DLs as a future preconditioning strategy to improve the efficacy of autologous and adoptively transferred immune cells in cellular cancer immunotherapy.