Natural Killer Cell Receptors and Cytotoxic Activity in Phosphomannomutase 2 Deficiency (PMM2-CDG)

BackgroundPMM2-CDG is the most common N-glycosylation defect and shows an increased risk of recurrent and/or severe, sometimes fatal, infections in early life. We hypothesized that natural killer (NK) cells, as important mediators of the immune response against microbial pathogens and regulators of adaptive immunity, might be affected in this genetic disorder.ObjectiveTo evaluate possible defects on PMM2-CDG NK peripheral blood cell number, killing activity and expression of membrane receptors.MethodsWe studied fresh and activated NK cells from twelve PMM2-CDG cells. The number and expression of lymphoid surface receptors were studied by flow cytometry. The NK responsiveness (frequency of degranulated NK cells) and killing activity against K562 target cells was determined in the NK cytotoxicity assay.ResultsWe found an increase of blood NK cells in three patients with a severe phenotype. Two of them, who had suffered from moderate/severe viral infections during their first year of life, also had reduced T lymphocyte numbers. Patient activated NK cells showed increased expression of CD54 adhesion molecule and NKG2D and NKp46 activating receptors. NKp46 and 2B4 expression was inversely correlated with the expression of NKG2D in activated PMM2-CDG cells. Maximal NK activity against K562 target cells was similar in control and PMM2-CDG cells. Interestingly, the NK cell responsiveness was higher in patient cells. NKG2D and specially CD54 increased surface expression significantly correlated with the increased NK cell cytolytic activity according to the modulation of the killer activity by expression of triggering receptors and adhesion molecules.ConclusionsOur results indicate that hypoglycosylation in PMM2-CDG altered NK cell reactivity against target cells and the expression of CD54 and NKG2D, NKp46 and 2B4 activating receptors during NK cell activation. This suggests a defective control of NK cell killing activity and the overall anti-viral immune response in PMM2-CDG patients. The present work improves our understanding of the immunological functions in PMM2-CDG and possibly in other CDG-I types.     

The activating NKG2D ligand MHC class I-related chain A transfers from target cells to NK cells in a manner that allows functional consequences

Recently, it has become apparent that surface proteins commonly transfer between immune cells in contact. Inhibitory receptors and ligands exchange between cells during NK cell surveillance and we report here that NK cells also acquire activating ligands from target cells. Specifically, the stress-inducible activating ligand for NKG2D, MHC class I-related chain A (MICA), transferred to NK cells upon conjugation with MICA-expressing target cells. Acquisition of MICA from target cells was dependent on cell contact and occurred after accumulation of MICA at the immunological synapse. Moreover, transfer of MICA was facilitated by specific molecular recognition via NKG2D and augmented by Src kinase signaling. Importantly, MICA associated with its new host NK cell membrane in an orientation that allowed engagement with NKG2D in trans and indeed could down-regulate NKG2D in subsequent homotypic interactions with other NK cells. MICA captured from target cells could subsequently transfer between NK cells and, more importantly, NK cell degranulation was triggered in such NK cell-NK cell interactions. Thus, NK cells can influence other NK cells with proteins acquired from target cells and our data specifically suggest that NK cells could lyse other NK cells upon recognition of activating ligands acquired from target cells. This mechanism could constitute an important function for immunoregulation of NK cell activity.     

Human tumor-derived exosomes down-modulate NKG2D expression

NKG2D is an activating receptor for NK, NKT, CD8(+), and gammadelta(+) T cells, whose aberrant loss in cancer is a key mechanism of immune evasion. Soluble NKG2D ligands and growth factors, such as TGFbeta1 emanating from tumors, are mechanisms for down-regulating NKG2D expression. Cancers thereby impair the capacity of lymphocytes to recognize and destroy them. In this study, we show that exosomes derived from cancer cells express ligands for NKG2D and express TGFbeta1, and we investigate the impact of such exosomes on CD8(+) T and NK cell NKG2D expression and on NKG2D-dependent functions. Exosomes produced by various cancer cell lines in vitro, or isolated from pleural effusions of mesothelioma patients triggered down-regulation of surface NKG2D expression by NK cells and CD8(+) T cells. This decrease was rapid, sustained, and resulted from direct interactions between exosomes and NK cells or CD8(+) T cells. Other markers (CD4, CD8, CD56, CD16, CD94, or CD69) remained unchanged, indicating the selectivity and nonactivatory nature of the response. Exosomal NKG2D ligands were partially responsible for this effect, as down-modulation of NKG2D was slightly attenuated in the presence of MICA-specific Ab. In contrast, TGFbeta1-neutralizing Ab strongly abrogated NKG2D down-modulation, suggesting exosomally expressed TGFbeta as the principal mechanism. Lymphocyte effector function was impaired by pretreatment with tumor exosomes, as these cells exhibited poor NKG2D-dependent production of IFN-gamma and poor NKG2D-dependent killing function. This hyporesponsiveness was evident even in the presence of IL-15, a strong inducer of NKG2D. Our data show that NKG2D is a likely physiological target for exosome-mediated immune evasion in cancer.     

NK cells infiltrating a MHC class I-deficient lung adenocarcinoma display impaired cytotoxic activity toward autologous tumor cells associated with altered NK cell-triggering receptors

NK cells are able to discriminate between normal cells and cells that have lost MHC class I (MHC-I) molecule expression as a result of tumor transformation. This function is the outcome of the capacity of inhibitory NK receptors to block cytotoxicity upon interaction with their MHC-I ligands expressed on target cells. To investigate the role of human NK cells and their various receptors in the control of MHC-I-deficient tumors, we have isolated several NK cell clones from lymphocytes infiltrating an adenocarcinoma lacking beta2-microglobulin expression. Unexpectedly, although these clones expressed NKG2D and mediated a strong cytolytic activity toward K562, Daudi and allogeneic MHC-class I+ carcinoma cells, they were unable to lyse the autologous MHC-I- tumor cell line. This defect was associated with alterations in the expression of natural cytotoxicity receptor (NCR) by NK cells and the NKG2D ligands, MHC-I-related chain A, MHC-I-related chain B, and UL16 binding protein 1, and the ICAM-1 by tumor cells. In contrast, the carcinoma cell line was partially sensitive to allogeneic healthy donor NK cells expressing high levels of NCR. Indeed, this lysis was inhibited by anti-NCR and anti-NKG2D mAbs, suggesting that both receptors are required for the induced killing. The present study indicates that the MHC-I-deficient lung adenocarcinoma had developed mechanisms of escape from the innate immune response based on down-regulation of NCR and ligands required for target cell recognition.     

Comprehensive analysis of NKG2D ligand expression and release in leukemia: implications for NKG2D-mediated NK cell responses

Ligands of the prototypical activating NK receptor NKG2D render cancer cells susceptible to NK cell-mediated cytolysis if expressed at sufficiently high levels. However, malignant cells employ mechanisms to evade NKG2D-mediated immunosurveillance, such as NKG2D ligand (NKG2DL) shedding resulting in reduced surface expression levels. In addition, systemic downregulation of NKG2D on NK cells of cancer patients has been observed in many studies and was attributed to soluble NKG2DL (sNKG2DL), although there also are conflicting data. Likewise, relevant expression of NKG2DL in leukemia has been reported by some, but not all studies. Hence, we comprehensively studied expression, release, and function of the NKG2D ligands MHC class I chain-related molecules A and B and UL16-binding proteins 1-3 in 205 leukemia patients. Leukemia cells of most patients (75%) expressed at least one NKG2DL at the surface, and all investigated patient sera contained elevated sNKG2DL levels. Besides correlating NKG2DL levels with clinical data and outcome, we demonstrate that sNKG2DL in patient sera reduce NKG2D expression on NK cells, resulting in impaired antileukemia reactivity, which also critically depends on number and levels of surface-expressed NKG2DL. Together, we provide comprehensive data on the relevance of NKG2D/NKG2DL expression, release, and function for NK reactivity in leukemia, which exemplifies the mechanisms underlying NKG2D-mediated tumor immunosurveillance and escape.     

Lenalidomide enhances antibody-dependent cellular cytotoxicity of solid tumor cells in vitro: influence of host immune and tumor markers

We evaluated the effect of combining lenalidomide with therapeutic antibodies on antibody-dependant cell-mediated cytotoxicity (ADCC) of solid tumor cells, and the requirement for expression of natural killer (NK) cell-activating receptors and their solid tumor surface ligands. Twenty-three human tumor cell lines (colon, breast, lung, head and neck, ovary, and bone sarcoma) were analyzed. NK effector cells were isolated from healthy donors, pre-treated with and without lenalidomide, and incubated with antibody-coated tumor cells to determine ADCC. In blocking experiments, NK cells were pre-incubated with anti-DNAM-1 or anti-NKG2D antibodies, and target colorectal cells were pre-incubated with anti-CD155 (PVR), anti-MIC-A/B, or anti-ULBP 3 antibodies. Differences between groups were assessed using unpaired and paired Student’s t test and one-way ANOVA. Lenalidomide enhanced NK cell-mediated ADCC of trastuzumab- and cetuximab-coated tumor cells. Activity against colorectal cancer cells was dependent on target antigen expression, but independent of KRAS status and FcγRIIIa genotype. The extent of ADCC and its enhancement by lenalidomide correlated with NK cell expression of NKG2D and DNAM-1, and tumor cell expression of PVR and MIC-A. Blocking of NKG2D and, to a lesser extent, DNAM-1 inhibited ADCC. Anti-MIC-A/B monoclonal antibody blocked natural cytotoxicity, but not ADCC. Lenalidomide enhances the ability of IgG1-isotype antibodies to mediate ADCC of solid tumor cells, the extent of which is largely dependent on NKG2D–NKG2D ligand interactions, but appears to be independent of MIC-A/B. This provides a rationale for exploratory clinical studies and an assessment of potential biomarkers predictive of clinical benefit.     

NK cells use NKG2D to recognize a mouse renal cancer (Renca), yet require intercellular adhesion molecule-1 expression on the tumor cells for optimal perforin-dependent effector function

The NKG2D receptor on NK cells can recognize a variety of ligands on the tumor cell surface. Using a mouse renal cancer (Renca), we show that NKG2D recognition by NK cells was crucial for their ability to limit tumor metastases in vivo in both liver and lungs using perforin-dependent effector mechanisms. However, for the R331 cell line established from Renca, NKG2D recognition and perforin-dependent lysis played no role in controlling liver metastases. R331 cells were also more resistant to perforin-dependent lysis by NK cells in vitro. We therefore used these phenotypic differences between Renca and R331 to further investigate the crucial receptor:ligand interactions required for triggering lytic effector functions of NK cells. Reconstitution of R331 cells with ICAM-1, but not Rae-1gamma, restored NKG2D-mediated, perforin-dependent lysis. Interestingly, R331 cells were efficiently lysed by NK cells using death ligand-mediated apoptosis. This death ligand-mediated killing did not depend on NKG2D recognition of its ligands on tumor cells. This result suggests that the intracellular signaling in NK cells required for perforin and death ligand-mediated lysis of tumor target cell are quite distinct, and activation of both of these antitumor lytic effector functions of NK cells could improve therapeutic benefits for certain tumors.     

双歧杆菌脂磷壁酸对B16荷瘤小鼠NK细胞受体NKG2D及其配体的影响

目的探讨双歧杆菌脂磷壁酸（LTA）对黑色素瘤B16荷瘤小鼠NK细胞受体NKG2D及其配体的影响。方法将黑色素瘤B16细胞接种于C57BL／6小鼠皮下,待触及肿块后于荷瘤小鼠皮下注射双歧杆菌LTA。采用MTT、流式细胞术（FCM）、RT-PCR方法分别检测经双歧杆菌LTA处理后B16荷瘤小鼠NK细胞杀伤活性、NK细胞NKG2D受体蛋白表达以及肿瘤组织内Rae-1、H60 mRNA表达的变化。结果与对照组相比,经双歧杆菌LTA处理后,B16荷瘤小鼠的NK细胞杀伤活性增强（P〈0．05）,NK细胞受体NKG2D表达明显增加（P〈0．05）,肿瘤组织Rae-1、H60 mRNA表达上升（P〈0．05）,并具有浓度依赖性。结论双歧杆菌LTA能够增强B16荷瘤小鼠NK细胞的杀伤活性,其机制可能与上调NK细胞受体NKG2D的蛋白表达和肿瘤组织Rae-1、H60 mRNA的表达有关。     

生物肽SP对NK细胞NKG2D/NKG2A受体表达的影响

选择NK92-MI细胞为研究体系,研究SP对NK细胞的杀伤活性及功能性受体NKG2D/NKG2A表达的影响,以探讨SP对NK细胞功能的调节作用机制。采用MTT法测定NK92-MI细胞对K562细胞的杀伤活性;采用Real-Time PCR和流式细胞术检测NK92-MI细胞活化性受体NKG2D和抑制性受体NKG2A的基因表达和膜表达。10-14～10-8 mol/L的SP在体外可明显增强NK92-MI细胞的杀伤活性。该浓度范围的SP均可上调NKG2D/NKG2A的mRNA水平;10-14～10-8 mol/L的SP均上调NKG2D/NKG2A的膜表达,较低浓度（10-14 mol/L）的SP仅使NKG2D表达上调,而NKG2A表达无明显变化;SP刺激NKG2D膜表达增加的程度高于NKG2A。生物肽SP调节NK细胞功能性受体NKG2D/NKG2A的表达,可能是SP增强NK细胞杀伤活性的一种原因。     

The Traffic of the NKG2D/Dap10 Receptor Complex during Natural Killer (NK) Cell Activation

NKG2D is an important activating receptor for triggering the NK cell cytotoxic activity, although chronic engagement of specific ligands by NKG2D is also known to provoke decreased cell surface expression of the receptor and compromised NK cell function. We have studied the dynamics of surface NKG2D expression and how exposure to the specific ligand major histocompatibility complex class I chain-related molecule B (MICB) affects receptor traffic and fate. While in the NKL cell line and “resting” NK cells NKG2D was found principally at the cell surface, in activated primary NK cells an intracellular pool of receptor could also be found recycling to the plasma membrane. Exposure of NK cells to targets expressing MICB resulted in degradation of ∼50% of total NKG2D protein and lysosomal degradation of the DAP10 adaptor molecule. Consistent with these observations, confocal microscopy experiments demonstrated that DAP10 trafficked to secretory lysosomes in both transfected NKL cells and in activated primary NK cells upon interaction with MICB-expressing target cells. Interestingly, polarization to the synapse of secretory lysosomes containing DAP10 was also observed. The implications of the intracellular traffic of the NKG2D/DAP10 receptor complex for NK cell activation are discussed. We propose that the rapid degradation of NKG2D/DAP10 observed coincident with recruitment of the receptor to the cytotoxic immune synapse may explain the loss of NKG2D receptor expression after chronic exposure to NKG2D ligands.The killer cell lectin-like receptor NKG2D is one of the best characterized NK³ cell-activating receptors. Signaling via NKG2D depends on its association with DAP10, a transmembrane adaptor molecule containing the sequence YINM, which signals via recruitment of phosphatidylinositol 3-kinase and Grb2 (growth factor receptor-bound protein 2) (1, 2). Effector cell activation mediated by NKG2D has been described as immune recognition of the “induced self,” because the cellular ligands for NKG2D (NKG2D-L): the polymorphic MHC class I chain-related molecules (MIC) A and MICB and the UL16-binding proteins are not normally expressed but instead are up-regulated on target cells after pathogen infection or tumor transformation to render these cells susceptible to NK cell lysis (3). Strikingly, however, although induced expression of NKG2D-L acts as a danger signal to provoke an immune response, a number of studies performed in mouse models have shown that chronic exposure to NKG2D-L can also lead to down-modulation of the surface expression of NKG2D and impaired NK cell cytotoxic function (4–6).In humans, a common feature of patients with multiple different tumors is the presence in the serum of high levels of soluble MICA and -B or UL16-binding proteins, released by tumor cells, that are associated with an impairment of CTL and NK cell cytotoxic function (7–10). These observations have been interpreted as suggesting that the release of soluble NKG2D-L is a strategy of tumor immune evasion (11). However, recent data show that receptor interaction with cell membrane-anchored MICB can also lead to impaired NKG2D function. We have shown that brief cytotoxic interactions between NK cells and MICB-expressing target cells trigger a synaptic interchange of NKG2D and MICB as well as a rapid down-modulation of surface NKG2D and compromised NK cell cytotoxicity suggesting that NKG2D traffic is rapidly altered upon recognition of MICB expressed on target cell (12).The surface level of a receptor is dictated by the relative rates of synthesis and transport to the plasma membrane and endocytosis, recycling, and degradation. The loss of cell surface NKG2D observed after NKG2D-L binding (7–10, 12) raises the question of what is the intracellular fate of the receptor on interaction with NKG2D-L. However, the traffic of this receptor has not been previously studied. Here we describe the dynamics of surface NKG2D expression and examine how cytotoxic interactions between NK cells and the MHC class I- 721.221 (here called 221) cells that express MICB (here called 221B) affect the traffic and fate of the NKG2D/DAP10 receptor complex. In NKL cells and resting primary NK cells NKG2D is mainly expressed at the cell surface; however, in activated primary NK cells an intracellular pool of receptor recycling to the cell surface is detected. During cytotoxic interactions the recognition of MICB expressed on target cells results in a rapid degradation of NKG2D/DAP10 that is associated with the traffic of DAP10 to secretory lysosomes (SLs) (13, 14). Our data provide new insights into the dynamics of NKG2D receptor expression in NK cells and suggest a plausible model to explain how chronic exposure to NKG2D-L could lead to NKG2D down-modulation and compromised NK cell function.     

Contrasting Effects of the Cytotoxic Anticancer Drug Gemcitabine and the EGFR Tyrosine Kinase Inhibitor Gefitinib on NK Cell-Mediated Cytotoxicity via Regulation of NKG2D Ligand in Non-Small-Cell Lung Cancer Cells

IntroductionSeveral cytotoxic anticancer drugs inhibit DNA replication and/or mitosis, while EGFR tyrosine kinase inhibitors inactivate EGFR signalling in cancer cell. Both types of anticancer drugs improve the overall survival of the patients with non-small-cell lung cancer (NSCLC), although tumors often become refractory to this treatment. Despite several mechanisms by which the tumors become resistant having been described the effect of these compounds on anti-tumor immunity remains largely unknown.MethodsThis study examines the effect of the cytotoxic drug Gemcitabine and the EGFR tyrosine kinase inhibitor Gefitinib on the expression of NK group 2 member D (NKG2D) ligands as well as the sensitivity of NSCLC cells to the NK-mediated lysis.ResultsWe demonstrate that Gemcitabine treatment leads to an enhanced expression, while Gefitinib downregulated the expression of molecules that act as key ligands for the activating receptor NKG2D and promote NK cell-mediated recognition and cytolysis. Gemcitabine activated ATM and ATM- and Rad-3-related protein kinase (ATR) pathways. The Gemcitabine-induced phosphorylation of ATM as well as the upregulation of the NKG2D ligand expression could be blocked by an ATM-ATR inhibitor. In contrast, Gefitinib attenuated NKG2D ligand expression. Silencing EGFR using siRNA or addition of the PI3K inhibitor resulted in downregulation of NKG2D ligands. The observations suggest that the EGFR/PI3K pathway also regulates the expression of NKG2D ligands. Additionally, we showed that both ATM-ATR and EGFR regulate MICA/B via miR20a.ConclusionIn keeping with the effect on NKG2D expression, Gemcitabine enhanced NK cell-mediated cytotoxicity while Gefitinib attenuated NK cell killing in NSCLC cells.     

Interaction of monocytes with NK cells upon Toll-like receptor-induced expression of the NKG2D ligand MICA

Reciprocal interactions between NK cells and dendritic cells have been shown to influence activation of NK cells, maturation, or lysis of dendritic cells and subsequent adaptive immune responses. However, little is known about the crosstalk between monocytes and NK cells and the receptors involved in this interaction. We report in this study that human monocytes, upon TLR triggering, up-regulate MHC class I-Related Chain (MIC) A, but not other ligands for the activating immunoreceptor NKG2D like MICB or UL-16 binding proteins 1-3. MICA expression was associated with CD80, MHC class I and MHC class II up-regulation, secretion of proinflammatory cytokines, and apoptosis inhibition, but was not accompanied by release of MIC molecules in soluble form. TLR-induced MICA on the monocyte cell surface was detected by autologous NK cells as revealed by NKG2D down-regulation. Although MICA expression did not render monocytes susceptible for NK cell cytotoxicity, LPS-treated monocytes stimulated IFN-gamma production of activated NK cells which was substantially dependent on MICA-NKG2D interaction. No enhanced NK cell proliferation or cytotoxicity against third-party target cells was observed after stimulation of NK cells with LPS-activated monocytes. Our data indicate that MICA-NKG2D interaction constitutes a mechanism by which monocytes and NK cells as an early source of IFN-gamma may communicate directly during an innate immune response to infections in humans.     

Elevated TGF-beta1 secretion and down-modulation of NKG2D underlies impaired NK cytotoxicity in cancer patients

NK cell function in cancer patients is severely impaired, but the mechanism underlying this impairment is not clearly understood. In this study we show evidence that TGF-beta1 secreted by tumors is responsible for the poor NK lytic activity via down-regulating an NK-activating receptor, NKG2D. The plasma level of TGF-beta1 in human lung cancer or colorectal cancer patients was elevated compared with that in normal volunteers, and this elevation was inversely correlated with surface expression of NKG2D on NK cells in these patients. Incubation of NK cells with plasma obtained from cancer patients specifically down-modulated surface NKG2D expression, whereas addition of neutralizing anti-TGF-beta1 mAbs completely restored surface NKG2D expression. Likewise, incubation of NK cells and lymphokine-activated killer cells with TGF-beta1 resulted in dramatic reduction of surface NKG2D expression associated with impaired NK cytotoxicity. Modulation of NKG2D by TGF-beta1 was specific, as expression of other NK receptors, CD94/NKG2A, CD44, CD16, 2B4, or CD56, was not affected by TGF-beta1. Impaired NK cytotoxicity by TGF-beta1 was not due to alteration of lytic moieties, such as perforin or Fas, or apoptotic pathway, but, rather, appeared to be due to lack of NKG2D expression. Taken together, our data suggest that impaired NK function in cancer patients can be attributed to down-modulation of activating receptors, such as NKG2D, via secretion of TGF-beta1.     

Expansion of NK cells by engineered K562 cells co-expressing 4-1BBL and mMICA,combined with soluble IL-21

NK cells hold promise for protecting hosts from cancer and pathogen infection through direct killing and expressing immune-regulatory cytokines. In our study, a genetically modified K562 cell line with surface expression of 4-1BBL and MICA was constructed to expand functional NK cells in vitro for further adoptive immunotherapy against cancer. After a long-term up to 21 day co-culture with newly isolated peripheral blood mononuclear cells (PBMCs) in the presence of soluble IL-21 (sIL-21), notable increase in proportion of expanded NK cells was observed, especially the CD56^brightCD16⁺ subset. Apparent up-regulation of activating receptors CD38, CD69 and NKG2D was detected on expanded NK cells, so did inhibitory receptor CD94; the cytotoxicity of expanded NK cells against target tumor cells exceeded that of NK cells within fresh PBMCs. The intracellular staining showed expanded NK cells produced immune-regulatory IFN-γ. Taken together, we expanded NK cells with significant up-regulation of activating NKG2D and moderate enhancement of cytotoxicity, with IFN-γ producing ability and a more heterogeneous population of NK cells. These findings provide a novel perspective on expanding NK cells in vitro for further biology study and adoptive immunotherapy of NK cells against cancer.     

自然杀伤细胞受体及其配体表达的转录和表观遗传调控

自然杀伤(natural killer cell,NK)细胞受体及其配体在NK细胞发挥抗病毒、抗肿瘤和免疫调节作用中起重要作用.NK细胞功能的发挥取决于NK细胞受体及其配体的表达水平和其所传递信号的综合.病毒、肿瘤和热休克等刺激可以通过激活相应的转录调节因子,提高启动子活性而上调NKG2家族受体及其配体的表达,而启动子区DNA的甲基化状态、组蛋白的乙酰化和甲基化等表观遗传调控,在NK细胞受体及其配体的表达方面亦起重要作用,并决定NK细胞受体的克隆性分布.深入探讨NK细胞受体及其配体的表达调控机制,将为提高NK.细胞抗肿瘤和抗感染疗效提供新的策略.