BACKGROUND: Activating and inhibitory receptors of natural killer (NK) cells such as NKp, NKG2, or CLEC are highly relevant to cold tumors including glioblastoma (GBM). Here, we aimed to characterize the expression of these receptors in GBM to gain insight into their potential role as modulators of the intratumoral microenvironment.
METHODS: We performed a transcriptomic analysis of several NK receptors with a focus on the activating receptor encoded by KLRC2, NKG2C, among bulk and single-cell RNA sequencing GBM data sets. We also evaluated the effects of KLRC2-overexpressing GL261 cells in mice treated with or without programmed cell death protein-1 (PD-1) monoclonal antibody (mAb). Finally, we analyzed samples from two clinical trials evaluating PD-1 mAb effects in patients with GBM to determine the potential of NKG2C to serve as a biomarker of response.
RESULTS: We observed significant expression of several inhibitory NK receptors on GBM-infiltrating NK and T cells, which contrasts with the strong expression of KLRC2 on tumor cells, mainly at the infiltrative margin. Neoplastic KLRC2 expression was associated with a reduction in the number of myeloid-derived suppressor cells and with a higher level of tumor-resident lymphocytes. A stronger antitumor activity after PD-1 mAb treatment was observed in NKG2Chigh-expressing tumors both in mouse models and patients with GBM whereas the expression of inhibitory NK receptors showed an inverse association.
CONCLUSIONS: This study explored the role of neoplastic NKG2C/KLRC2 expression in shaping the immune profile of GBM and suggests that it is a predictive biomarker for positive responses to immune checkpoint inhibitor treatment in patients with GBM. Future studies could further validate this finding in prospective trials.

The immune system is integral to the surveillance and eradication of tumor cells. Interactions between the natural killer group 2 member D (NKG2D) receptor and its ligands (NKG2DLs) are vital for activating NKG2D receptor‑positive immune cells, such as natural killer cells. This activation enables these cells to identify and destroy tumor cells presenting with NKG2DLs, which is an essential aspect of tumor immunity. However, tumor immune escape is facilitated by soluble NKG2DL (sNKG2DL) shed from the surface of tumor cells. The production of sNKG2DL is predominantly regulated by metalloproteinases [a disintegrin and metalloproteinases (ADAM) and matrix metalloproteinase (MMP) families] and exosomes. sNKG2DL not only diminish immune recognition on the tumor cell surface but also suppress the function of immune cells, such as NK cells, and reduce the expression of the NKG2D receptor. This process promotes immune evasion, progression, and metastasis of tumors. In this review, an in‑depth summary of the mechanisms and factors that influence sNKG2DL production and their contribution to immune suppression within the tumor microenvironment are provided. Furthermore, due to the significant link between sNKG2DLs and tumor progression and metastasis, they have great potential as novel biomarkers. Detectable via liquid biopsies, sNKG2DLs could assess tumor malignancy and prognosis, and act as pivotal targets for immunotherapy. This could lead to the discovery of new drugs or the enhancement of existing treatments. Thus, the application of sNKG2DLs in clinical oncology was explored, offering substantial theoretical support for the development of innovative immunotherapeutic strategies for sNKG2DLs.

BACKGROUND: Cancer immunotherapy approaches that elicit immune cell responses, including T and NK cells, have revolutionized the field of oncology. However, immunosuppressive mechanisms restrain immune cell activation within solid tumors so additional strategies to augment activity are required.
METHODS: We identified the co-stimulatory receptor NKG2D as a target based on its expression on a large proportion of CD8+ tumor infiltrating lymphocytes (TILs) from breast cancer patient samples. Human and murine surrogate NKG2D co-stimulatory receptor-bispecifics (CRB) that bind NKG2D on NK and CD8+ T cells as well as HER2 on breast cancer cells (HER2-CRB) were developed as a proof of concept for targeting this signaling axis in vitro and in vivo.
RESULTS: HER2-CRB enhanced NK cell activation and cytokine production when co-cultured with HER2 expressing breast cancer cell lines. HER2-CRB when combined with a T cell-dependent-bispecific (TDB) antibody that synthetically activates T cells by crosslinking CD3 to HER2 (HER2-TDB), enhanced T cell cytotoxicity, cytokine production and in vivo antitumor activity. A mouse surrogate HER2-CRB (mHER2-CRB) improved in vivo efficacy of HER2-TDB and augmented NK as well as T cell activation, cytokine production and effector CD8+ T cell differentiation.
CONCLUSIONS: We demonstrate that targeting NKG2D with bispecific antibodies (BsAbs) is an effective approach to augment NK and CD8+ T cell antitumor immune responses. Given the large number of ongoing clinical trials leveraging NK and T cells for cancer immunotherapy, NKG2D-bispecifics have broad combinatorial potential.

Natural killer cells (NK) are the \"professional killer\" of tumors and play a crucial role in anti-tumor immunotherapy. NK cell desensitization is a key mechanism of tumor immune escape. Dysregulated NKG2D-NKG2DL signaling is a primary driver of this desensitization process. However, the factors that regulate NK cell desensitization remain largely uncharacterized. Here, we present the first report that circular RNA circARAP2 (hsa_circ_0069396) is involved in the soluble MICA (sMICA)-induced NKG2D endocytosis in the NK cell desensitization model. CircARAP2 was upregulated during NK cell desensitization and the loss of circARAP2 alleviated NKG2D endocytosis and NK cell desensitization. Using Chromatin isolation by RNA purification (ChIRP) and RNA pull-down approaches, we identified that RAB5A, a molecular marker of early endosomes, was its downstream target. Notably, transcription factor CTCF was an intermediate functional partner of circARAP2. Mechanistically, we discovered that circARAP2 interacted with CTCF and inhibited the recruitment of CTCF-Polycomb Repressive Complex 2 (PRC2) to the promoter region of RAB5A, thereby erasing histone H3K27 and H3K9 methylation suppression to enhance RAB5A transcription. These data demonstrate that inhibition of circARAP2 effectively alleviates sMICA-induced NKG2D endocytosis and NK cell desensitization, providing a novel target for therapeutic intervention in tumor immune evasion.

Endometriosis, affecting 10% of women, is defined as implantation, survival, and growth of endometrium-like/endometriotic tissue outside the uterine cavity, causing inflammation, infertility, pain, and susceptibility to ovarian cancer. Despite extensive studies, its etiology and pathogenesis are poorly understood and largely unknown. The prevailing view is that the immune system of endometriosis patients fails to clear ectopically disseminated endometrium from retrograde menstruation. Exosomes are small extracellular vesicles that exhibit immunomodulatory properties. We studied the role of endometriotic tissue-secreted exosomes in the pathophysiology of endometriosis. Two exosome-mediated mechanisms known to impair the immune response were investigated: 1) downregulation of NKG2D-mediated cytotoxicity and 2) FasL- and TRAIL-induced apoptosis of activated immune cells. We showed that secreted endometriotic exosomes isolated from supernatants of short-term explant cultures carry the NKG2D ligands MICA/B and ULBP1-3 and the proapoptotic molecules FasL and TRAIL on their surface, i.e., signature molecules of exosome-mediated immune suppression. Acting as decoys, these exosomes downregulate the NKG2D receptor, impair NKG2D-mediated cytotoxicity, and induce apoptosis of activated PBMCs and Jurkat cells through the FasL- and TRAIL pathway. The secreted endometriotic exosomes create an immunosuppressive gradient at the ectopic site, forming a \"protective shield\" around the endometriotic lesions. This gradient guards the endometriotic lesions against clearance by a cytotoxic attack and creates immunologic privilege by induction of apoptosis in activated immune cells. Taken together, our results provide a plausible, exosome-based mechanistic explanation for the immune dysfunction and the compromised immune surveillance in endometriosis and contribute novel insights into the pathogenesis of this enigmatic disease.

UNASSIGNED: Human Herpesvirus 6B (HHV-6B) impedes host immune responses by downregulating class I MHC molecules (MHC-I), hindering antigen presentation to CD8+ T cells. Downregulation of MHC-I disengages inhibitory receptors on natural killer (NK) cells, resulting in activation and killing of the target cell if NK cell activating receptors such as NKG2D have engaged stress ligands upregulated on the target cells. Previous work has shown that HHV-6B downregulates three MHC-like stress ligands MICB, ULBP1, and ULBP3, which are recognized by NKG2D. The U20 glycoprotein of the related virus HHV-6A has been implicated in the downregulation of ULBP1, but the precise mechanism remains undetermined.
UNASSIGNED: We set out to investigate the role of HHV-6B U20 in modulating NK cell activity. We used HHV-6B U20 expressed as a recombinant protein or transduced into target cells, as well as HHV-6B infection, to investigate binding interactions with NK cell ligands and receptors and to assess effects on NK cell activation. Small-angle X-ray scattering was used to align molecular models derived from machine-learning approaches.
UNASSIGNED: We demonstrate that U20 binds directly to ULBP1 with sub-micromolar affinity. Transduction of U20 decreases NKG2D binding to ULBP1 at the cell surface but does not decrease ULBP1 protein levels, either at the cell surface or in toto. HHV-6B infection and soluble U20 have the same effect. Transduction of U20 blocks NK cell activation in response to cell-surface ULBP1. Structural modeling of the U20 - ULBP1 complex indicates some similarities to the m152-RAE1γ complex.

Persistent human papillomavirus infection is associated with the development of premalignant lesions that can eventually lead to cervical cancer. In this study, we evaluated the expression of activating (NKG2D, DNAM-1) and inhibitory immune checkpoints receptors (PD-1, TIGIT, and Tim-3) in peripheral blood NKT-like (CD3+CD56+) lymphocytes from patients with cervical carcinoma (CC, n = 19), high-grade lesions (HG, n = 8), low-grade lesions (LG, n = 19) and healthy donors (HD, n = 17) using multiparametric flow cytometry. Dimensional data analysis showed four clusters within the CD3+CD56+ cells with different patterns of receptor expression. We observed upregulation of CD16 in CC and HG patients in one of the clusters. In another, TIGIT was upregulated, while DNAM-1 was downregulated. Throughout manual gating, we observed that NKT-like cells expressing activating receptors also co-express inhibitory receptors (PD-1 and TIGIT), which can affect the activation of these cells. A deeper characterization of the functional state of the cells may help to clarify their role in cervical cancer, as will the characterization of the NKT-like cells as cytotoxic CD8+ T cells or members of type I or type II NKT cells.

The short-lived nature and heterogeneity of Natural Killer (NK) cells limit the development of NK cell-based therapies, despite their proven safety and efficacy against cancer. Here, we describe the biological basis, detailed phenotype and function of long-lived anti-tumour human NK cells (CD56highCD16+), obtained without cell sorting or feeder cells, after priming of peripheral blood cells with Bacillus Calmette-Guérin (BCG). Further, we demonstrate that survival doses of a cytokine combination, excluding IL18, administered just weekly to BCG-primed NK cells avoids innate lymphocyte exhaustion and leads to specific long-term proliferation of innate cells that exert potent cytotoxic function against a broad range of solid tumours, mainly through NKG2D. Strikingly, a NKG2C+CD57-FcεRIγ+ NK cell population expands after BCG and cytokine stimulation, independently of HCMV serology. This strategy was exploited to rescue anti-tumour NK cells even from the suppressor environment of cancer patients\' bone marrow, demonstrating that BCG confers durable anti-tumour features to NK cells.

OBJECTIVE: Natural killer (NK) cells are an integral part of the staunch defense line against malignant tumors within the tumor microenvironment. Existing research indicates that miRNAs can influence the development of NK cells by negatively modulating gene expression. In this study, we aim to explore how the miR-17-5p in Hepatocellular Carcinoma (HCC) exosomes regulates the killing function of NK cells towards HCC cells through the transcription factor RNX1.
METHODS: The exosomes were isolated from HCC tissues and cell lines, followed by a second generation sequencing to compare differential miRNAs. Verification was performed using qRT-PCR and Western blot methods. The mutual interactions between miR-17-5p and RUNX1, as well as between RUNX1 and NKG2D, were authenticated using techniques like luciferase reporter gene assays, Western blotting, and Chromatin Immunoprecipitation (ChIP). The cytotoxic activity of NK cells towards HCC cells in vitro was measured using methods such as RTCA and ELISPOT. The zebrafish xenotransplantation was utilized to assess the in vivo killing capacity of NK cells against HCC cells.
RESULTS: The level of miR-17-5p in exosomes from HCC tissue increased compared to adjacent tissues. We verified that RUNX1 was a target of miR-17-5p and that RUNX1 enhances the transcription of NKG2D. MiR-17-5p was found to downregulate the expression of RUNX1 and NKG2D, subsequently reducing the in vitro and in vivo cytotoxic capabilities of NK cells against HCC cells.
CONCLUSIONS: The miR-17-5p found within HCC exosomes can target RUNX1, subsequently attenuating the cytotoxic activity of NK cells.

In our previous studies, a novel cryothermal therapy (CTT) was developed to induce systemic long-term anti-tumor immunity. Natural killer (NK) cells were found to play an important role in CTT-induced long-term immune-mediated tumor control at the late stage after CTT, but the underlying mechanism is unclear. Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells that have potent immunosuppressive effects on T cells and weaken the long-term benefits of immunotherapy. Consequently, overcoming MDSC immunosuppression is essential for maintaining the long-term efficacy of immunotherapy. In this study, we revealed that NK cells considerably diminish MDSC accumulation at the late stage after CTT, boost T cell production, increase T cell activation, and promote MDSC maturation, culminating in Th1-dominant CD4+ T cell differentiation and enhancing NK and CD8+ T cell cytotoxicity. Additionally, NK cells activate ERK signaling in MDSCs through NKG2D-ligand interaction to increase the activity of tumor necrosis factor (TNF)-α converting enzyme (TACE)-cleaved membrane TNF-α. Furthermore, Increased TACE activity releases more soluble TNF-α from MDSCs to promote MDSC maturation. In our studies, we propose a novel mechanism by which NK cells can overcome MDSC-induced immunosuppression and maintain CTT-induced persistent anti-tumor immunity, providing a prospective therapeutic option to improve the performance of cancer immunotherapy.