NKG2D ligands expression and NKG2D-mediated cytotoxicity in human laryngeal squamous carcinoma cells

The NKG2D is an activating immunoreceptor expressed by NK cells and CD8⁺ T cells. Engagement of NKG2D by its ligands is critical for both innate and adoptive immunity. While the overexpression of NKG2D ligands on certain tumour cells has previously been demonstrated, little is known about NKG2D ligand expression on human laryngeal tumour cells. In this study, we first verified that the interaction between NKG2D and its ligands was critical for NK cell-based immune response to human laryngeal squamous carcinoma cells Hep-2. This NKG2D-mediated effect was observed by transfecting the recombinant eukaryotic expression vector pEGFP-N1/NKG2D as well as the NKG2D blockade. The mRNA and protein expression of NKG2D ligands, MHC class I-related chain molecules A (MICA) and UL16-binding proteins (ULBPs), in human laryngeal carcinoma cell line Hep-2 and fresh tumour tissues were evaluated. Compared with non-tumour tissues of vocal cords polyps, MICA and ULBP-3 were strongly overexpressed on both the human laryngeal carcinoma cell line Hep-2 and fresh human laryngeal carcinoma tissues. The mechanism and impact of NKG2D ligands overexpression on NK cell-mediated anti-laryngeal cancer immune response would require further investigation.     

NKG2D engagement of colorectal cancer-specific T cells strengthens TCR-mediated antigen stimulation and elicits TCR independent anti-tumor activity

The NKG2D receptor is expressed by human NK, gammadelta T and alpha/beta T lymphocytes and its engagement results in the stimulation of effector cells. We evaluated the role of NKG2D receptor in anti-colorectal cancer (CRC) immune response. The cell surface expression of stress-inducible NKG2D ligands MICA/B (MHC class I-related chain molecules A/B) and ULBP (UL16 binding protein) by a panel of CRC lines was evaluated by flow cytometry. MICA and ULBP2/3 were widely expressed by the analyzed lines, with a minority of them being also ULBP-1+, whereas MICB was undetectable. CD8+ and CD4+ HLA-restricted anti-tumor T cell clones of a CRC patient were used to evaluate whether NKG2D engagement could mediate tumor recognition. Three out of four CD8+ T cell clones recognized the autologous tumor with a marginal NKG2D engagement, a finding that was correlated with the weak expression of NKG2D ligands by the autologous tumor. On the contrary, NKG2D triggering of these CD8+ T cell clones induced recognition of allogeneic CRC lines showing high expression of MICA and ULBP. A costimulatory role of NKG2D was observed with one CD4+/NKG2D+ T cell clone when stimulated by tumors sharing the HLA class II alleles and expressing NKG2D ligands. Taken together these data indicate that the engagement of NKG2D, depending on the expression of its ligands by target cells, can influence the pattern of anti-tumor reactivity by T lymphocytes.     

Tumor-derived soluble MICs impair CD3(+)CD56(+) NKT-like cell cytotoxicity in cancer patients

Upon ligation with its ligands, the activating receptor NKG2D stimulates or costimulates CD8(+) T cells or NK cells. The inducible gene MHC class I chain-related molecules (MICs), which belong to the NKG2D ligand family and usually initiate the process of lymphocyte activation, have been found to be broadly expressed on epithelial tumor cells. Sustained localized expression or release of soluble forms of MICs (sMICs) by tumor cells play key roles in tumor evasion via the impairment of T cell and NK cell functions. NKG2D is also expressed on the surface of CD3(+)CD56(+) NKT-like cells, which participate in tumor rejection via direct cytolysis. We speculated whether sMICs have the same impact on NKT-like cells. In this study, we demonstrated that in vitro killing by freshly isolated NKT-like cells was principally mediated by NKG2D, and the cytotoxic function of NKT-like cells isolated from cancer patients was obviously compromised. We found a significant correlation between elevated tumor-derived sMICs and down-modulation of NKG2D expression on NKT-like cell surfaces in human ovarian cancer and prostate cancer patients. We determined that elevated serum sMIC impairs the lytic activity via downregulation of the NKG2D receptor because incubation of NKT-like cells with sera obtained from cancer patients down-modulated surface NKG2D expression, whereas the addition of neutralizing anti-MIC mAbs restored surface NKG2D expression. We suggest that tumors shedding MICs may promote immune evasion by impairing NKT-like cell cytotoxicity.     

Defects in NKG2D ligand expression result in failed tolerance induction at the maternal–fetal interface: A possible cause for recurrent miscarriage

Certain maternal immune responses against the fetus at the maternal–fetal interface may contribute to recurrent miscarriage (RM). Trophoblast cells involve in forming of maternal–fetal interface and interact with many immune cells, including NK cells. NK cells accumulate at the maternal–fetal interface and play a critical role during pregnancy. NKG2D ligands can activate NK cells through engaging with corresponding receptors. The 5′-end flanking regions of DNA sequence of some NKG2D ligands contain heat shock elements. It is very possible that oxidative stress, produced in pathological process of RM, induces abnormal NKG2D ligand expression in the trophoblast cells, which stimulate cytotoxicity of NK cells. Moreover, in normal pregnancy, soluble NKG2D ligands are secreted into sera by syncytiotrophoblast cells, which disturb NKG2D-mediated maternal anti-fetus immunity. Reduction of soluble NKG2D ligand levels in association with upregulation of NKG2D on immune cells may also contribute to pathogenesis of RM.     

UL16 binding proteins

According to present concepts, innate immunity plays an important role in tumor surveillance and immune modulation. The state of NK cells depends on the balance between inhibitory and activating signals from corresponding receptors. As one of the activating receptors, NKG2D recognises some self ligands such as MICA/B in human and Rae1 in mice, which is dissimilar to those toll-like receptors that recognise some pathogen-derived ligands. NKG2D is expressed not only on NK cells, but on gammadelta T cells, CD8+ alphabeta T cells in normal individuals and CD4+ alphabeta T cells in rheumatoid arthritis patients and plays a different role on respective cells. Whereas NKG2D can only function as a costimulatory receptor on CD8+ alphabeta T cells under the domination of alphabeta TCR in spite of a deficiency of costimulatory molecule CD28, NKG2D can directly activate NK cells even in the presence of inhibitory signals from MHC-I and corresponding receptor complexes. Experiments in mice have identified that alternative splicing produces two distinct NKG2D polypeptides that associate differentially with the DAP10 and DAP12 signaling subunits and that differential expression of these isoforms and of signaling proteins determines whether NKG2D only functions as a costimulatory receptor in the adaptive immune system (CD8+ T cells) or as both a primary recognition unit and a costimulatory receptor in the innate immune system (natural killer cells and macrophages). This review summarizes the research achievements in a new ligand family (UL16 binding proteins) of NKG2D in human and shows the possible prospects of ULBP function and application.     

免疫受体NKG2D介导的细胞免疫及其与疾病的关系

NKG2D是一种重要的淋巴细胞活化受体.它以同源二聚体形式表达于NK细胞、某些T细胞或巨噬细胞表面.NKG2D启动的信号对淋巴细胞起着刺激或共刺激的作用.NKG2D受体拥有多种配体,这些配体在正常细胞表面不表达或低水平表达,当细胞处于应激状态或恶变时,这些配体在细胞表面诱导性表达.NKG2D介导的细胞免疫应答对于肿瘤或感染起着重要的免疫监视作用,同时与某些自身免疫性疾病也密切相关.     

The NKG2D receptor: immunobiology and clinical implications

NK cells are critical components of our immune system functioning, in part, to recognize and then eradicate virally infected or tumorigenic cells without previous sensitization. One of the best-characterized activating receptors expressed on NK cells is the NKG2D receptor, which is capable of transmitting co-stimulatory signals by subsets of T cells. Viruses and tumors have evolved strategies to evade NKG2D-mediated immune recognition thus highlighting the importance of this receptor in immunity. This review will focus on the structure of NKG2D and its interaction with its diverse array of ligands, as well as highlighting current knowledge regarding NKG2D signal transduction and biological mechanisms that govern its cell surface expression. The impact that NKG2D has in disease pathologies is also assessed.     

NKG2D and cytotoxic effector function in tumor immune surveillance

NKG2D is a type II transmembrane-anchored glycoprotein expressed as a disulfide-linked homodimer on the surface of all mouse and human natural killer cells (NK cells). Stimulation of NK cells through NKG2D triggers cell-mediated cytotoxicity and in some cases induces the production of cytokines. NKG2D binds to family of ligands with structural homology to MHC class I, however, unlike conventional MHC class I molecules, NKG2D ligands often display up-regulated surface expression on stressed cells and are frequently over expressed by tumors. Recent evidence clearly implicates that NKG2D recognition plays an important role in tumor immune surveillance and that NKG2D primarily acts to trigger perforin-mediated apoptosis. The data begin to place the NKG2D pathway into the context of other recognition-effector systems used by NK cells.     

Expression of the NKG2D ligand UL16 binding protein-1 (ULBP-1) on dendritic cells

Innate and adaptive immunity have not evolved separately. In this regard, the NKG2D molecule first identified on NK cells and classified as an activating NK cell receptor is also an important receptor for CD8(+) T cells. Functional analyses of human NKG2D and its ligands, i.e. UL16 binding proteins (ULBP) and MHC class I chain-related (MIC), have so far focused on immune cell-target cell situations because of the expression of NKG2D ligands on infected, stressed or transformed cells. Here, however, we address a possible function of NKG2D/ULBP-1 during the initiation of T cell responses. ULBP-1 can be detected on mature dendritic cells both in situ in the T cell areas of lymph nodes as well as in vitro after artificial maturation. FCM analysis further demonstrated that although NKG2D is expressed to some degree on all analyzed T cell subsets from peripheral blood, in vitro stimulation of T cells results in up-regulation of NKG2D on proliferating T cells. Using the sentinel lymph nodes of primary melanoma as a model for induction of defined T cell responses in vivo, we were able to demonstrate the expression of NKG2D on melanoma-associated antigen-specific T cells. Thus, our results suggest a role for NGK2D-ULBP-1 in the induction or reactivation of T cell responses.     

Generation of Soluble NKG2D Ligands: Proteolytic Cleavage,Exosome Secretion and Functional Implications

The activating natural killer group 2 member D (NKG2D) receptor is expressed on NK cells, cytotoxic T cells and additional T cell subsets. Ligands for human NKG2D comprise two groups of MHC class I‐related molecules, the MHC class I chain‐related proteins A and B (MICA/B) and 6 UL16‐binding proteins (ULBP1‐6). While NKG2D ligands are absent from most normal cells, expression is induced upon stress and malignant transformation. In fact, most solid tumours and leukaemia/lymphomas constitutively express at least one NKG2D ligand and thereby are susceptible to NKG2D‐dependent immunosurveillance. However, soluble NKG2D ligands are released from tumour cells and can down‐modulate NKG2D activation as a means of tumour immune escape. In some tumour entities, levels of soluble NKG2D ligands in the serum correlate with tumour progression. NKG2D ligands can be proteolytically shed from the cell surface or liberated from the membrane by phospholipase C in the case of glycosylphosphatidylinositol (GPI)‐anchored molecules. Moreover, NKG2D ligands can be secreted in exosomal microvesicles together with other tumour‐derived molecules. Depending on the specific tumour/immune cell setting, these various forms of soluble and/or exosome‐bound NKG2D ligands can exert multiple effects on NKG2D/NKG2D ligand interactions. In this review, we focus on the role of various proteases in the shedding of human NKG2D ligands from tumour cells and discuss the not completely unanimous reported functional implications of soluble and exosome‐secreted NKG2D ligands for immunosurveillance.     

NKG2D与NKG2DL在肺癌免疫调节中的作用及临床意义

NKG2D与NKG2DL是目前研究的热点,NKG2D可表达于几乎所有的NK细胞、CD8+αβT细胞、γδ T细胞及少量CD4+αβT细胞的细胞膜表面,与其配体NKG2DL结合后,可激活NK细胞及T细胞,诱导机体的抗肿瘤免疫应答,杀伤表达NKG2DL的肿瘤细胞,因此,NKG2D及其配体NKG2DL在肿瘤的免疫调节过程中起着极其重要的作用.目前肺癌的预后仍不容乐观,迫切需要发展一种新型的治疗方法来达到特异杀灭肿瘤细胞而不损伤或仅轻微损伤正常细胞的目的,这使得免疫疗法成为一种极具吸引力及应用前景的治疗肺癌的方法.     

The role of regulatory T cells in the control of natural killer cells: relevance during tumor progression

Summary:  Tumor immunosurveillance relies on cognate immune effectors [lymphocytes and interferon-γ (IFN-γ)] and innate immunity [natural killer (NK) cells, natural killer group 2, member D (NKG2D) ligands, perforin/granzyme, and tumor necrosis factor-related apoptosis-inducing ligand]. In parallel, tumor cells promote the expansion of CD4⁺CD25⁺ regulatory T cells (Tregs) that counteract T-cell-based anti-tumor immunity. Moreover, accumulating evidence points to a critical role for Tregs in dampening NK cell immune responses. This review summarizes the findings showing that Tregs suppress NK cell effector functions in vitro and in vivo , i.e. homeostatic proliferation, cytotoxicity, and interleukin-12-mediated IFN-γ production. The molecular mechanism involve selective expression of membrane-bound transforming growth factor-β on Tregs, which downregulate NKG2D expression on NK cells in vitro and in vivo . The regulatory events dictating NK cell suppression by Tregs have been studied and are discussed. The pathological relevance of the Treg–NK cell interaction has been brought up in tumor models and in patients with cancer. Consequently, inhibition of Tregs through pharmacological interventions should be considered during NK-cell-based immunotherapy of cancer.     

Inosine pranobex enhances human NK cell cytotoxicity by inducing metabolic activation and NKG2D ligand expression

Inosine pranobex (IP) is a synthetic immunomodulating compound, indicated for use in the treatment of human papillomavirus-associated warts and subacute sclerosing panencephalitis. Previous studies demonstrate that the immunomodulatory activity of IP is characterized by enhanced lymphocyte proliferation, cytokine production, and NK cell cytotoxicity. The activation of NKG2D signaling on NK cells, CD8⁺ T cells, and γδ T cells also produces these outcomes. We hypothesized that IP alters cellular immunity through the induction of NKG2D ligand expression on target cells, thereby enhancing immune cell activation through the NKG2D receptor. We tested this hypothesis and show that exposure of target cells to IP leads to increased expression of multiple NKG2D ligands. Using both targeted metabolic interventions and unbiased metabolomic studies, we found that IP causes an increase in intracellular concentration of purine nucleotides and tricarboxylic acid (TCA) cycle intermediates and NKG2D ligand induction. The degree of NKG2D ligand induction was functionally significant, leading to increased NKG2D-dependent target cell immunogenicity. These findings demonstrate that the immunomodulatory properties of IP are due to metabolic activation with NKG2D ligand induction.     

Engineering of chimeric natural killer cell receptors to develop precision adoptive immunotherapies for cancer

Natural killer (NK) cells are innate immune effectors which play a crucial role in recognizing and eliminating virally infected and cancerous cells. They effectively distinguish between healthy and distressed self through the integration of signals delivered by germline-encoded activating and inhibitory cell surface receptors. The frequent up-regulation of stress markers on genetically unstable cancer cells has prompted the development of novel immunotherapies that exploit such innate receptors. One prominent example entails the development of chimeric antigen receptors (CAR) that detect cell surface ligands bound by NK receptors, coupling this engagement to the delivery of tailored immune activating signals. Here, we review strategies to engineer CARs in which specificity is conferred by natural killer group 2D (NKG2D) or other NK receptor types. Multiple preclinical studies have demonstrated the remarkable ability of chimeric NK receptor-targeted T cells and NK cells to effectively and specifically eliminate cancer cells and to reject established tumour burdens. Importantly, such systems act not only acutely but, in some cases, they also incite immunological memory. Moreover, CARs targeted with the NKG2D ligand binding domain have also been shown to disrupt the tumour microenvironment, through the targeting of suppressive T regulatory cells, myeloid-derived suppressor cells and tumour vasculature. Collectively, these findings have led to the initiation of early-phase clinical trials evaluating both autologous and allogeneic NKG2D-targeted CAR T cells in the haematological and solid tumour settings.