NKG2D In NK Cells Research Articles

Natural killer cells activated through NKG2D mediate lung ischemia-reperfusion injury.

Pulmonary ischemia-reperfusion injury (IRI) is a clinical syndrome of acute lung injury that occurs after lung transplantation or remote organ ischemia. IRI causes early mortality and has no effective therapies. While NK cells are innate lymphocytes capable of recognizing injured cells, their roles in acute lung injury are incompletely understood. Here, we demonstrated that NK cells were increased in frequency and cytotoxicity in 2 different IRI mouse models. We showed that NK cells trafficked to the lung tissue from peripheral reservoirs and were more mature within lung tissue. Acute lung ischemia-reperfusion injury was blunted in a NK cell-deficient mouse strain but restored with adoptive transfer of NK cells. Mechanistically, NK cell NKG2D receptor ligands were induced on lung endothelial and epithelial cells following IRI, and antibody-mediated NK cell depletion or NKG2D stress receptor blockade abrogated acute lung injury. In human lung tissue, NK cells were increased at sites of ischemia-reperfusion injury and activated NK cells were increased in prospectively collected human bronchoalveolar lavage in subjects with severe IRI. These data support a causal role for recipient peripheral NK cells in pulmonary IRI via NK cell NKG2D receptor ligation. Therapies targeting NK cells may hold promise in acute lung injury.

Expressions of natural cytotoxicity receptor, NKG2D and NKG2D ligands in endometriosis

Pathogenesis of endometriosis is still unknown, and the relationship between NK cell activating receptors and endometriosis remains to be explored. We investigated the expression of NCRs and NKG2D in NK cells in peripheral blood (PB) and peritoneal fluid (PF) as well as expression of NKG2D ligands in endometrial cells, and illuminated their relationship with ovarian endometriosis. 20 patients with ovarian endometriosis and 13 subjects for control group were recruited. Flow cytometry was used for examining expressions of NCRs and NKG2D on NK cells. In PF with endometriosis, the expressions of NKp30 (P = 0. 006) and NKG2D (P = 0. 010) on CD56+NK cells were decreased, whereas the expression of NKp46 (P = 0. 040) on CD16+NK cells was higher than that of control. Real time PCR and Western blotting were used for detecting expression of NKG2D ligands. mRNA level of NKG2D ligands on endometrial cells showed no noticeable difference. As for protein expression, the ULBP-2 expression on eutopic endometrial cells with pelvic endometriosis was lower than that on ectopic endometrial cells and eutopic endometrial cells without endometriosis (P < 0.05), and the ULBP-3 expression on ectopic endometrial cells was lower than that on eutopic endometrial cells with or without endometriosis (P < 0.05). These findings indicate that change of NKp30, NKp46 and NKG2D on NK cells in PF and ULBP-2, 3 on endometrial cells may relate to the pathogenesis of pelvic endometriosis. Especially, change of NK cell activating receptors in PF implies that pelvic endometriosis is probably due to local immune changes.

PF268 DECIPHERING THE ABERRANT IMMUNE MICROENVIRONMENT IN ACUTE MYELOID LEUKEMIA DEVELOPMENT AND ITS ROLE IN CHEMOTHERAPY RESPONSE

Background:Comprehensive illustration of phenotypic and functional signatures of abnormal immune microenvironment in acute myeloid leukemia (AML) is lacking.Aims:In this study, we dissected the aberrant immune microenvironment in AML development and its association with chemotherapy response, which may contribute to personalized therapy and provide new insight into novel immunotherapies to augment anti‐leukemia immunity.Methods:Phenotypic and functional analysis of various lymphocytes (eg. CD4+ T cells, CD8+ T cells, NK cells, TNK cells, γδ T cells and B cells) in peripheral blood (PB) and their prognostic implications were performed by multiparameter flow cytometry in 49 patients with AML (before or/and after induction chemotherapy) and 22 healthy controls (HCs).Results:The proportions of CD3+ T cells, NK cells, Th cells, Tc cells and γδ T cells in PB were higher in 25 newly‐diagnosed AML (ND‐AML) patients versus 22 HCs (each P &lt; 0.05). A significant increase in the proportion of CD4+ T (relative to CD3+ T cells), Tfh and Treg cells was also noted, but B cells and TNK cells were similar for ND‐AML and HCs (Fig.1A). As for memory subsets, we found a significantly reduced percentage of naïve subset (CD45RA+CCR7+, relative to CD4+ or CD8+ T cells) in AML at diagnosis. An aberrant unbalance among Th1, Th2, Th17 and Tfh1 cells was also demonstrated in ND‐AML patients. Additionally, the expression of chemokine receptor CCR4, CCR6 and CXCR3 was shown to be down‐regulated. To functionally characterize T cells and NK cells from newly diagnosed AML patients, we assessed several function‐related markers. Further analysis showed a significantly lower expression of CD28 and CD127 in CD4+ or CD8+ T cells, but a higher expression of senescence marker CD57 for ND‐AML versus HCs (Fig.1B). Meanwhile, the homing receptor CD38 and memory marker HLADR demonstrated higher expression in CD8+ T cells in ND‐AML versus HCs. NKG2D expression was found to be significantly lower in NK cells, Vδ1 cells and Vδ2 cells, as well as NKP30 expression in NK cells. The percentage of CD4+ T, CD8+ T, Vδ1 T and Vδ2 T cells expressing inhibitory receptor PD‐1 was significantly higher in AML compared with HCs. Following induction chemotherapy, the immune landscape of AML patients was altered as immune reconstitution occurred. The proportion of CD3+ T cells, CD8+ Tnaïve cells and Th2 cells increased after induction chemotherapy, with the elevated expression of NKG2D in NK and Vδ2 cells, CD28 and CD25 in CD4+ T cells and CD127 in CD8+ T cells. Several phenotypic and functional markers diverged between good responders (GR, n = 19) and refractory or relapsed (RR, n = 14) patients. Good response to induction chemotherapy significantly correlated with downregulation of PD‐1 and CD57 in CD8+ T cells, upregulation of NKG2D in NK cells and Vδ2 cells and increased proportion of CD8+ Tnaïve and Th17 cells (Fig.1C).Summary/Conclusion:Our findings suggest that exhaustion and senescence are the dominant aspects of aberrant immune states in AML at diagnosis. Chemotherapy response and relapse significantly correlate with suppressed immune microenvironment, indicating that immunological markers may be novel prognostic risk factor in acute myeloid leukemia.image

Constitutive expression of ULBP-4 on monocytes regulates NK cell NKG2D expression

Key Points The NKG2D ligand ULBP-4 is expressed on healthy monocytes. Monocyte ULBP-4 expression regulates NKG2D expression by NK cells.

MICA-Expressing Monocytes Enhance Natural Killer Cell Fc Receptor-Mediated Antitumor Functions

Natural killer (NK) cells are large granular lymphocytes that promote the antitumor response via communication with other cell types in the tumor microenvironment. Previously, we have shown that NK cells secrete a profile of immune stimulatory factors (e.g., IFNγ, MIP-1α, and TNFα) in response to dual stimulation with the combination of antibody (Ab)-coated tumor cells and cytokines, such as IL12. We now demonstrate that this response is enhanced in the presence of autologous monocytes. Monocyte enhancement of NK cell activity was dependent on cell-to-cell contact as determined by a Transwell assay. It was hypothesized that NK cell effector functions against Ab-coated tumor cells were enhanced via binding of MICA on monocytes to NK cell NKG2D receptors. Strategies to block MICA-NKG2D interactions resulted in reductions in IFNγ production. Depletion of monocytes in vivo resulted in decreased IFNγ production by murine NK cells upon exposure to Ab-coated tumor cells. In mice receiving trastuzumab and IL12 therapy, monocyte depletion resulted in significantly greater tumor growth in comparison to mock-depleted controls (P < 0.05). These data suggest that NK cell-monocyte interactions enhance NK cell antitumor activity in the setting of monoclonal Ab therapy for cancer. Cancer Immunol Res; 5(9); 778-89. ©2017 AACR.

Magnesium alterations of graft immune cells (MAGIC).

e18541 Background: Magnesium plays important roles in cellular processes. Allogeneic hematopoietic stem cell transplantation provides potentially curative therapy to patients with a variety of hematologic malignancies. A common abnormality post transplant is hypomagnesaemia. Recent studies identified magnesium transporter 1 deficiency in association with XMEN syndrome. These patients exhibit low intracellular [Mg++] and low T cell and NK cell NKG2D expression. Therefore, we investigated this potential functional link between magnesium deficiency and immune function in patients post transplant. Methods: A pilot study investigated whether detectable changes in T and NK cells were associated with IV magnesium repletion post hematopoietic transplant. Blood was obtained before and after magnesium transfusions. Immunophenotypic analysis of T and NK cell NKG2D levels using commercially available monoclonal antibodies (CD3, CD16, CD56, CD4, CD8, CD314 [NKG2D]) was performed. Samples from patients demonstrating an increase in NKG2D expression were subsequently analyzed for ex vivo NK activity. Here, NK activity and cytotoxicity of Jurkat cells were assayed using standard laboratory techniques and changes &gt; 2 fold were considered significant. A total of 7 patients were enrolled. Results: NKG2D expression on T and NK cell subsets were determined by flow cytometry using commercially available monoclonal antibodies. NK reactivity and killing of K562 were undertaken using standard cytotoxicity assays. Our findings indicate a significant response in five of the seven enrolled patients. NKG2D expression on CD8+ T cells increased by up to 31%. Conclusions: Our results suggest that magnesium infusions post allogeneic transplant are associated with augmented NKG2D expression on immune cells. Additional studies are needed to clarify the mechanisms by which therapeutic magnesium repletion restores the cytotoxicity of NK cells and T lymphocytes. Although the clinical utility of magnesium supplementation remains to be validated, our data indicate that NKG2D is regulated by magnesium post transplant and magnesium may play a key role in immune regulation.

Natural killer cell NKG2D and granzyme B are critical for allergic pulmonary inflammation⋆

BackgroundThe diverse roles of innate immune cells in the pathogenesis of asthma remain to be fully defined. Natural killer (NK) cells are innate lymphocytes that can regulate adaptive immune responses. NK cells are activated in asthma; however, their role in allergic airway inflammation is not fully understood.ObjectiveWe investigated the importance of NK cells in house dust mite (HDM)-triggered allergic pulmonary inflammation. Specifically, we aimed to determine the role of the major NK-cell activating receptor NKG2D and NK-cell effector functions mediated by granzyme B.MethodsAllergic airway inflammation was induced in the airways of mice by repeated intranasal HDM extract administration and responses in wild-type and NKG2D-deficient mice were compared. Adoptive transfer studies were used to identify the cells and mechanisms involved.ResultsMice that lacked NKG2D were resistant to the induction of allergic inflammation and showed little pulmonary eosinophilia, few airway TH2 cells, and no rise in serum IgE after multiple HDM-allergen exposures. However, NKG2D was not required for pulmonary inflammation after a single inoculation of allergen. NKG2D-deficient mice showed no alteration in responses to respiratory virus infection. Transfer of wild-type NK cells (but not CD3+ cells) into NKG2D-deficient mice restored allergic inflammatory responses only if the NK cells expressed granzyme B.ConclusionsThese studies established a pivotal role for NK-cell NKG2D and granzyme B in the pathogenesis of HDM-induced allergic lung disease, and identified novel therapeutic targets for the prevention and treatment of asthma.

IDO metabolite produced by EBV-transformed B cells inhibits surface expression of NKG2D in NK cells via the c-Jun N-terminal kinase (JNK) pathway

Natural Killer cells are known to play a major role in the innate immune response against viral infections and tumor cells. Several viruses, such as CMV, EBV and HIV-1, have acquired strategies to escape elimination by NK cells. In this study, we observed that EBV infection increased expression of IDO on B cells. To evaluate the function of IDO associated with EBV infection, we investigated whether EBV-induced IDO could modulate expression of NK cell-activation receptor, NKG2D. When NK cells were co-incubated with EBV transformed B cells, surface expression of NKG2D was significantly reduced in NK cells. Incubation with L-kynurenine, an IDO metabolite, down-modulated NKG2D expression in NK cells in a dose- and time-dependent manner. Incubation with the JNK inhibitor SP600125 also inhibited NKG2D expression in NK cells. In addition, we observed that the effect of L-kynurenine was blocked by JNK agonist, anisomycin, suggesting the involvement of the JNK pathway in the signal transduction of L-kynurenine-reduced NKG2D expression. Furthermore, IL-18 significantly reduced L-kynurenine-induced down-regulation of NKG2D expression in NK cells. Taken together, these data indicate that down-regulation of NKG2D by EBV-induced IDO metabolite provides a potential mechanism by which EBV escapes NKG2D-mediated attack by immune cells.

Oxidative Stress Mediates a Reduced Expression of the Activating Receptor NKG2D in NK Cells from End-Stage Renal Disease Patients

To characterize the immune defect of patients with end-stage renal disease (ESRD), we performed NK cell subset analysis in 66 patients with ESRD treated by hemodialysis (n = 59) or peritoneal dialysis (n = 7). Compared with healthy blood donors, patients undergoing chronic dialysis showed a profound decrease in NKG2D(+) cells within both the CD8(+) T cell (58% vs 67%, p = 0.03) and NK cell (39% vs 56%, p = 0.002) populations. CD56(dim) cells, which comprise the majority of NK cells in the periphery, were more affected in this regard than were CD56(bright) cells. Uremic serum could decrease NKG2D expression on NK cells from healthy donors. Among factors that could contribute to the decrease in NKG2D expression in ESRD patients, reactive oxygen species (ROS) play a major role. We found that catalase could reverse the effects of uremic serum on NKG2D expression (p < 0.001) and that ROS down-regulated NKG2D at the mRNA level and at the NK cell surface. Additionally, ESRD patients had both increased membrane-bound MHC class I-related chain A (MICA) on monocytes (p = 0.04) and increased soluble MICA (203 pg/ml vs 110 pg/ml; p < 0.001). Both ROS and uremic serum could significantly increase in vitro the expression of the NKG2D ligand MICA on the renal epithelial cell line HK-2. Taken together, these studies suggest for the first time that both low NKG2D expression and up-regulation of its ligand MICA are related to ROS production and may be involved in the immune deficiency of ESRD patients.

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.