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1.  Chimeric NKG2D CAR-Expressing T Cell-Mediated Attack of Human Ovarian Cancer Is Enhanced by Histone Deacetylase Inhibition 
Human Gene Therapy  2013;24(3):295-305.
NKG2D ligands (NKG2DLs) are widely expressed on ovarian cancers to various degrees, making them attractive targets for immunotherapy. Here, we applied a chimeric antigen receptor (CAR) approach for the targeting of NKG2DLs expressed on human ovarian cancer cells and evaluated the impact of pharmacological upregulation of NKG2DLs on immune recognition. Various NKG2DLs, including MICA/B and ULBP-1, -2, -3, and -4, were expressed at various levels on the surface of all established ovarian cancer cell lines and primary ovarian cancer samples tested. To redirect human T cells against NKG2DLs, an NKG2DL-specific CAR was generated by fusing the extracellular domain of the NKG2D receptor to the 4-1BB costimulatory and CD3-ζ chain signaling domains. In vitro expansion of chimeric NKG2D CAR T cells was delayed compared with untransduced T cells and control CAR T cells; the likely result of fratricide among activated T cells expressing NKG2DLs. However, NKG2D CAR T cells did expand and were selectively enriched during prolonged culture. In coculture, CD4+ and CD8+ NKG2D CAR T cells specifically recognized and killed NKG2DL-expressing ovarian cancer cell lines but not NKG2DL-negative cells. Notably, pretreatment of ovarian cancer cells expressing moderate to low levels of NKG2DLs with the histone deacetylase inhibitor sodium valproate (VPA) upregulated NKG2DL cell surface expression and consequently enhanced their immune recognition by chimeric NKG2D CAR T cells. Our results demonstrate that VPA-induced upregulation of NKG2DL expression enhances the immune recognition of ovarian cancer cells by engineered NKG2D CAR T cells, and rationalizes the use of VPA in combination with NKG2DL-targeted immunotherapy in ovarian cancer.
Song and colleagues target NKG2D ligands (NGK2DLs) expressed on various human ovarian cancer cells, using a chimeric antigen receptor (CAR) approach. NGK2DL-specific CAR T cells selectively kill NKG2DL-expressing cells, and this immune recognition is enhanced by increasing surface NGK2DL expression, using a histone deacetylase inhibitor.
PMCID: PMC3609608  PMID: 23297870
2.  Chimeric NKG2D expressing T cells eliminate immunosuppression and activate immunity within the ovarian tumor microenvironment 
Adoptive transfer of T cells expressing chimeric NKG2D receptors (chNKG2D), a fusion of NKG2D and CD3ζ, can lead to long-term, tumor-free surv ival in a murine model of ovarian cancer. To determine the mechanisms of chNKG2D T cell anti-tumor efficacy, we analyzed how chNKG2D T cells altered the tumor microenvironment, including the tumor-infiltrating leukocyte populations. ChNKG2D T cell treatment of mice bearing ID8 tumor cells increased the number and activation of NK cells, and activation of host CD8+ T cells within the tumor. Foxp3+ regulatory T cells at the tumor site decreased more than 300-fold after chNKG2D T cell treatment. Tumor-associated regulatory T cells expressed cell-surface NKG2D ligands and were killed by chNKG2D T cells in a perforin-dependent manner. ChNKG2D T cells also altered the function of myeloid cells at the tumor site, changing these cells from being immunosuppressive to enhancing T cell responses. Cells isolated from the tumor produced elevated amounts of IFNγ, nitric oxide, and other proinflammatory cytokines after chNKG2D T cell treatment. ChNKG2D T cells required perforin, IFNγ, and GM-CSF to induce a full response at the tumor site. In addition, transfer of chNKG2D T cells into mice bearing tumors that were established for five weeks led to long-term survival of the mice. Thus, chNKG2D T cells altered the ovarian tumor microenvironment to eliminate immunosuppressive cells and induce infiltration and activation of anti-tumor immune cells and production of inflammatory cytokines. This induction of an immune response likely contributes chNKG2D T cells’ ability to eliminate established tumors.
PMCID: PMC2825039  PMID: 19915047
Tumor immunity; NK cells; Ovarian cancer; Gene therapy; Adoptive cell therapy
3.  Adenovirus serotype 5 E1A expressing tumor cells elicit a tumor-specific CD8+ T cell response independent of NKG2D 
Results in Immunology  2015;5:1-5.
The expression of the Adenovirus serotype 2 or serotype 5 (Ad2/5) E1A gene in tumor cells upregulates ligands that are recognized by the NKG2D activating receptor, which is expressed on NK cells and T cells, and reduces their tumorigenicity, a process dependent on NK cells and T cells. In some model systems, the forced overexpression of NKG2D ligands on tumor cells induced antigen-specific CD8+ T cells that mediated anti-tumor immunity. We wanted to determine if the interaction of NKG2D ligands on tumor cells that express E1A with NKG2D on immune cells contributed to the ability of E1A to induce a CD8+ T cell anti-tumor response or reduce tumorigenicity. To address these questions, we used the MCA-205 tumor cell line or MCA-205 cells that expressed Ad5 E1A (MCA-205-E1A cells), a fusion protein of E1A and ovalbumin (MCA-205-E1A-OVA) or OVA (MCA-205-OVA). We found that the expression of E1A or E1A–OVA, but not OVA, upregulated the expression of the NKG2D ligand RAE-1 on the surface of MCA-205 cells. Additionally, MCA-205-E1A cells and MCA-205-E1A-OVA cells were more sensitive to NK cell lysis than MCA-205 or MCA-205-OVA cells in WT B6 mice, but not NKG2D deficient B6 mice. Next, we adoptively transferred WT or NKG2D deficient OT-1 T cells (CD8 T cells that recognize OVA residues 257–264) into WT B6 mice or B6 mice that were deficient in NKG2D respectively and measured the expansion of OT-1 cells following immunization with MCA-205-E1A-OVA or MCA-205-OVA cells. We found that the expansion of OT-1 cells following immunization of either OVA-expressing MCA-205 cell lines was not affected by the presence or absence of NKG2D in B6 mice. Finally, we found that the capacity of E1A to reduce the tumorigenicity of MCA-205 cells was not impaired in B6-NKG2D deficient mice as compared to WT B6 mice. Our results suggest that the ability of E1A to reduce the tumorigenicity of MCA-205 cells, or induce an antigen-specific CD8+ T cell response, is independent of the interaction of NKG2D ligands with the NKG2D receptor.
PMCID: PMC4309920
Adenovirus E1A; NKG2D; NKG2D ligands; NK cells; CD8+ T cells; Tumor immunology; E1A, early region 1 A; Ad, adenovirus; NKG2D, natural killer group 2 D; MCA, methylcholanthrene; OVA, ovalbumin; NK, natural killer; WT, wildtype; RAE-1, retinoic acid early inducible; B6, C57BL/6; TPD50, tumor producing dose 50
4.  TLR and NKG2D Signaling Pathways Mediate CS-Induced Pulmonary Pathologies 
PLoS ONE  2013;8(10):e78735.
Long-term exposure to cigarette smoke (CS) can have deleterious effects on lung epithelial cells including cell death and the initiation of inflammatory responses. CS-induced cell injury can elaborate cell surface signals and cellular byproducts that stimulate immune system surveillance. Our previous work has shown that the expression of ligands for the cytotoxic lymphocyte activating receptor NKG2D is enhanced in patients with COPD and that the induction of these ligands in a mouse model can replicate COPD pathologies. Here, we extend these findings to demonstrate a role for the NKG2D receptor in CS-induced pathophysiology and provide evidence linking nucleic acid-sensing endosomal toll-like receptor (TLR) signaling to COPD pathology through NKG2D activation. Specifically, we show that mice deficient in NKG2D exhibit attenuated pulmonary inflammation and airspace enlargement in a model of CS-induced emphysema. Additionally, we show that CS exposure induces the release of free nucleic acids in the bronchoalveolar lavage and that direct exposure of mouse lung epithelial cells to cigarette smoke extract similarly induces functional nucleic acids as assessed by TLR3, 7, and 9 reporter cell lines. We demonstrate that exposure of mouse lung epithelial cells to TLR ligands stimulates the surface expression of RAET1, a ligand for NKG2D, and that mice deficient in TLR3/7/9 receptor signaling do not exhibit CS-induced NK cell hyperresponsiveness and airspace enlargement. The findings indicate that CS-induced airway injury stimulates TLR signaling by endogenous nucleic acids leading to elevated NKG2D ligand expression. Activation of these pathways plays a major role in the altered NK cell function, pulmonary inflammation and remodeling related to long-term CS exposure.
PMCID: PMC3793989  PMID: 24130907
5.  Cancer immunotherapy using a bi-specific NK receptor- fusion protein that engages both T cells and tumor cells1 
Cancer research  2011;71(6):2066-2076.
T cell immunotherapy is a promising strategy to treat cancer. The aim of this study was to investigate a new approach to deliver selective T cell activation against tumor cells. We have developed a bi-specific fusion protein that recognizes tumor cells through an NK activating receptor NKG2D and stimulates T cells through an anti-CD3 single chain fragment (scFv-NKG2D). In vitro, scFv-NKG2D engages both T cells and tumor cells, resulting in T cells producing IFN-γ and cytotoxicity against NKG2D ligand-positive tumor cells. In vivo, expression of scFv-NKG2D by tumor cells (NKG2D ligand-positive) significantly reduced tumor burden and, in some case, led to tumor-free survival. In vivo administration of scFv-NKG2D also significantly promoted survival in a murine lymphoma model. In addition, tumor-free mice were resistant to rechallenge with cognate tumor cells, suggesting the generation of a host specific immunological memory response. Host adaptive immunity (including γδ T cells) was required for scFv-NKG2D-mediated therapeutic efficacy. ScFv-NKG2D also inhibited the growth of NKG2D-ligand negative B16F10 tumors, reduced the percentage of myeloid-derived suppressor cells as well as regulatory T cells and increased T cell infiltration, suggesting that scFv-NKG2D target these immune suppressive cells. In summary, these results indicate that scFv-NKG2D represents a promising multi-tumor targeting reagent to induce anti-tumor immunity.
PMCID: PMC3095211  PMID: 21282338
Cancer immunotherapy; CD8 T cells; NKG2D; chimeric protein; myeloid suppressor; regulatory T cells
6.  Hepatitis C Virus (HCV) Evades NKG2D-Dependent NK Cell Responses through NS5A-Mediated Imbalance of Inflammatory Cytokines 
PLoS Pathogens  2010;6(11):e1001184.
Understanding how hepatitis C virus (HCV) induces and circumvents the host's natural killer (NK) cell-mediated immunity is of critical importance in efforts to design effective therapeutics. We report here the decreased expression of the NKG2D activating receptor as a novel strategy adopted by HCV to evade NK-cell mediated responses. We show that chronic HCV infection is associated with expression of ligands for NKG2D, the MHC class I-related Chain (MIC) molecules, on hepatocytes. However, NKG2D expression is downmodulated on circulating NK cells, and consequently NK cell-mediated cytotoxic capacity and interferon-γ production are impaired. Using an endotoxin-free recombinant NS5A protein, we show that NS5A stimulation of monocytes through Toll-like Receptor 4 (TLR4) promotes p38- and PI3 kinase-dependent IL-10 production, while inhibiting IL-12 production. In turn, IL-10 triggers secretion of TGFβ which downmodulates NKG2D expression on NK cells, leading to their impaired effector functions. Moreover, culture supernatants of HCV JFH1 replicating Huh-7.5.1 cells reproduce the effect of recombinant NS5A on NKG2D downmodulation. Exogenous IL-15 can antagonize the TGFβ effect and restore normal NKG2D expression on NK cells. We conclude that NKG2D-dependent NK cell functions are modulated during chronic HCV infection, and demonstrate that this alteration can be prevented by exogenous IL-15, which could represent a meaningful adjuvant for therapeutic intervention.
Author Summary
Natural killer (NK) cells are part of the innate immune response against virus infection. Their activation is the net result of signals emanating from a panel of inhibitory and activating receptors, among which the NKG2D activating receptor plays a major role. NKG2D ligands, the MHC class I related Chain (MIC) molecules, are induced on HCV-infected hepatocytes. In this paper, we show that NKG2D expression is decreased on NK cells from chronically infected HCV patients. As a consequence, NK cell cytolytic and IFNγ-producing functions are impaired. We show that this phenomenon is mediated by TGFβ produced by monocytes upon stimulation by the non-structural HCV-NS5A protein. NS5A could bind to TLR4 on monocytes, thus inducing the production of IL-10 and TGFβ, while inhibiting the production of IL-12. We further showed that TLR4-dependent IL-10 production by monocytes upon NS5A stimulation was mediated through the p38 and PI3 kinase pathways. In addition, we demonstrated that IL-15 could inhibit the TGFβ-mediated effects on NKG2D expression and NK cell functions. Collectively, these results identify a new dampening signal used by HCV to subvert innate immune response, and may provide new insights into the design of new strategies to restore NK cell functions in chronic hepatitis C.
PMCID: PMC2978723  PMID: 21085608
7.  Human Herpesvirus 8 (HHV8) Sequentially Shapes the NK Cell Repertoire during the Course of Asymptomatic Infection and Kaposi Sarcoma 
PLoS Pathogens  2012;8(1):e1002486.
The contribution of innate immunity to immunosurveillance of the oncogenic Human Herpes Virus 8 (HHV8) has not been studied in depth. We investigated NK cell phenotype and function in 70 HHV8-infected subjects, either asymptomatic carriers or having developed Kaposi's sarcoma (KS). Our results revealed substantial alterations of the NK cell receptor repertoire in healthy HHV8 carriers, with reduced expression of NKp30, NKp46 and CD161 receptors. In addition, down-modulation of the activating NKG2D receptor, associated with impaired NK-cell lytic capacity, was observed in patients with active KS. Resolution of KS after treatment was accompanied with restoration of NKG2D levels and NK cell activity. HHV8-latently infected endothelial cells overexpressed ligands of several NK cell receptors, including NKG2D ligands. The strong expression of NKG2D ligands by tumor cells was confirmed in situ by immunohistochemical staining of KS biopsies. However, no tumor-infiltrating NK cells were detected, suggesting a defect in NK cell homing or survival in the KS microenvironment. Among the known KS-derived immunoregulatory factors, we identified prostaglandin E2 (PGE2) as a critical element responsible for the down-modulation of NKG2D expression on resting NK cells. Moreover, PGE2 prevented up-regulation of the NKG2D and NKp30 receptors on IL-15-activated NK cells, and inhibited the IL-15-induced proliferation and survival of NK cells. Altogether, our observations are consistent with distinct immunoevasion mechanisms that allow HHV8 to escape NK cell responses stepwise, first at early stages of infection to facilitate the maintenance of viral latency, and later to promote tumor cell growth through suppression of NKG2D-mediated functions. Importantly, our results provide additional support to the use of PGE2 inhibitors as an attractive approach to treat aggressive KS, as they could restore activation and survival of tumoricidal NK cells.
Author Summary
Natural Killer (NK) cells are part of the innate immune response against virus infections and tumors. Their activation is the net result of signals emanating from a panel of inhibitory and activating receptors recognizing specific ligands on target cells. Human Herpes Virus 8 (HHV8) is an oncogenic virus responsible of Kaposi Sarcoma (KS), a multifocal angiogenic tumor. How NK cells contribute to the control of infection by HHV8 infection and development of KS, is unclear. In this paper, we show different strategies used by HHV8 to escape NK cell response. Patients with asymptomatic infection or KS have down-modulated expression of NKp30, NKp46 and CD161 receptors. In addition, patients with active KS show additional down-modulation of the NKG2D activating receptor, associated with impaired NK-cell cytotoxicity against target cells. Resolution of KS correlates with regained NKG2D expression and cytotoxic function. We present evidence that down-modulation of NKG2D is mediated by inflammatory prostaglandin E2 (PGE2), known to be released by KS cells, and show that PGE2 acts by preventing IL-15-mediated activation of NK cells. These results strongly support the use of PGE2 inhibitors as an attractive approach to treat active KS.
PMCID: PMC3257307  PMID: 22253598
8.  Recognition of the Class Ib Molecule Qa-1b by Putative Activating Receptors Cd94/Nkg2c and Cd94/Nkg2e on Mouse Natural Killer Cells 
The Journal of Experimental Medicine  1999;190(12):1801-1812.
The heterodimeric CD94/NKG2A receptor, expressed by mouse natural killer (NK) cells, transduces inhibitory signals upon recognition of its ligand, Qa-1b, a nonclassical major histocompatibility complex class Ib molecule. Here we clone and express two additional receptors, CD94/NKG2C and CD94/NKG2E, which we show also bind to Qa-1b. Within their extracellular carbohydrate recognition domains, NKG2C and NKG2E share extensive homology with NKG2A (93–95% amino acid similarity); however, NKG2C/E receptors differ from NKG2A in their cytoplasmic domains (only 33% similarity) and contain features that suggest that CD94/NKG2C and CD94/NKG2E may be activating receptors. We employ a novel blocking anti-NKG2 monoclonal antibody to provide the first direct evidence that CD94/NKG2 molecules are the only Qa-1b receptors on NK cells. Molecular analysis reveals that NKG2C and NKG2E messages are extensively alternatively spliced and ∼20-fold less abundant than NKG2A message in NK cells. The organization of the mouse Cd94/Nkg2 gene cluster, presented here, shows striking similarity with that of the human, arguing that the entire CD94/NKG2 receptor system is relatively primitive in origin. Analysis of synonymous substitution frequencies suggests that within a species, NKG2 genes may maintain similarities with each other by concerted evolution, possibly involving gene conversion–like events. These findings have implications for understanding NK cells and also raise new possibilities for the role of Qa-1 in immune responses.
PMCID: PMC2195720  PMID: 10601355
CD94; NKG2; Qa-1; natural killer cell; MHC class I
9.  A gluten-free diet lowers NKG2D and ligand expression in BALB/c and non-obese diabetic (NOD) mice 
The interplay between diet and immune parameters which could affect type 1 diabetes (T1D) pathogenesis is not sufficiently clarified. Intestinal up-regulation of the activating receptor natural killer group 2D (NKG2D) (CD314) and its ligands is a hallmark of coeliac disease. However, the direct effect of gluten on NKG2D expression is not known. We studied, by fluorescence activated cell sorter (lymphoid tissues) and reverse transcription–quantitative polymerase chain reaction (intestine and pancreatic islets), if a gluten-free diet (GF diet) from 4 weeks of age or a gluten-free diet introduced in breeding pairs (SGF diet), induced changes in NKG2D expression on DX5+(CD49b) natural killer (NK) cells, CD8+ T cells and in intestinal and islet levels of NKG2D and ligands in BALB/c and non-obese diabetic (NOD) mice. Gluten-free NOD mice had lower insulitis (P < 0·0001); reduced expression of NKG2D on DX5+ NK cells in spleen and auricular lymph nodes (P < 0·05); and on CD8+ T cells in pancreas-associated lymph nodes (P = 0·04). Moreover, the level of CD71 on DX5+ NK cells and CD8+ T cells (P < 0·005) was markedly reduced. GF and SGF mice had reduced expression of NKG2D and DX5 mRNA in intestine (P < 0·05). Differences in intestinal mRNA expression were found in mice at 8, 13 and 20 weeks. Intestinal expression of NKG2D ligands was reduced in SGF mice with lower expression of all ligands. In isolated islets, a SGF diet induced a higher expression of specific NKG2D ligands. Our data show that a gluten-free diet reduces the level of NKG2D and the expression of NKG2D ligands. These immunological changes may contribute to the lower T1D incidence associated with a gluten-free diet.
PMCID: PMC4226590  PMID: 24673402
coeliac disease; gluten; gluten-free diet; NKG2D; type 1 diabetes
10.  A gluten-free diet lowers NKG2D and ligand expression in BALB/c and non-obese diabetic (NOD) mice 
The interplay between diet and immune parameters which could affect type 1 diabetes (T1D) pathogenesis is not sufficiently clarified. Intestinal up-regulation of the activating receptor natural killer group 2D (NKG2D) (CD314) and its ligands is a hallmark of coeliac disease. However, the direct effect of gluten on NKG2D expression is not known. We studied, by fluorescence activated cell sorter (lymphoid tissues) and reverse transcription–quantitative polymerase chain reaction (intestine and pancreatic islets), if a gluten-free diet (GF diet) from 4 weeks of age or a gluten-free diet introduced in breeding pairs (SGF diet), induced changes in NKG2D expression on DX5+(CD49b) natural killer (NK) cells, CD8+ T cells and in intestinal and islet levels of NKG2D and ligands in BALB/c and non-obese diabetic (NOD) mice. Gluten-free NOD mice had lower insulitis (P < 0·0001); reduced expression of NKG2D on DX5+ NK cells in spleen and auricular lymph nodes (P < 0·05); and on CD8+ T cells in pancreas-associated lymph nodes (P = 0·04). Moreover, the level of CD71 on DX5+ NK cells and CD8+ T cells (P < 0·005) was markedly reduced. GF and SGF mice had reduced expression of NKG2D and DX5 mRNA in intestine (P < 0·05). Differences in intestinal mRNA expression were found in mice at 8, 13 and 20 weeks. Intestinal expression of NKG2D ligands was reduced in SGF mice with lower expression of all ligands. In isolated islets, a SGF diet induced a higher expression of specific NKG2D ligands. Our data show that a gluten-free diet reduces the level of NKG2D and the expression of NKG2D ligands. These immunological changes may contribute to the lower T1D incidence associated with a gluten-free diet.
PMCID: PMC4226590  PMID: 24673402
coeliac disease; gluten; gluten-free diet; NKG2D; type 1 diabetes
11.  Expression of the RAE-1 Family of Stimulatory NK-Cell Ligands Requires Activation of the PI3K Pathway during Viral Infection and Transformation 
PLoS Pathogens  2011;7(9):e1002265.
Natural killer (NK) cells are lymphocytes that play a major role in the elimination of virally-infected cells and tumor cells. NK cells recognize and target abnormal cells through activation of stimulatory receptors such as NKG2D. NKG2D ligands are self-proteins, which are absent or expressed at low levels on healthy cells but are induced upon cellular stress, transformation, or viral infection. The exact molecular mechanisms driving expression of these ligands remain poorly understood. Here we show that murine cytomegalovirus (MCMV) infection activates the phosphatidylinositol-3-kinase (PI3K) pathway and that this activation is required for the induction of the RAE-1 family of mouse NKG2D ligands. Among the multiple PI3K catalytic subunits, inhibition of the p110α catalytic subunit blocks this induction. Similarly, inhibition of p110α PI3K reduces cell surface expression of RAE-1 on transformed cells. Many viruses manipulate the PI3K pathway, and tumors frequently mutate the p110α oncogene. Thus, our findings suggest that dysregulation of the PI3K pathway is an important signal to induce expression of RAE-1, and this may represent a commonality among various types of cellular stresses that result in the induction of NKG2D ligands.
Author Summary
Human and mouse cytomegaloviruses (HCMV and MCMV) are members of the Herpesvirus family. Both viruses cause disease in individuals with a compromised immune system, such as transplant patients and AIDS patients. Natural killer (NK) cells are essential players in the immune response against these viruses. NK cells recognize self-proteins, such as NKG2D ligands, that are poorly expressed on healthy cells but are upregulated on cells that are undergoing stress, such as infection and tumor development. The biological processes associated with NKG2D ligand expression in infected cells are unknown. The PI3K pathway, which controls many cellular processes, is activated by a variety of viruses to prime cells for efficient viral replication. We observed that MCMV activates the PI3K pathway and that this activation is required for NKG2D ligand expression. We also found that the expression of NKG2D ligands on cancer cell lines is dependent on this pathway. Our data suggest that NKG2D ligand expression, and thus recognition of infected and cancer cells by NK cells, is associated with a dysregulation in the PI3K pathway.
PMCID: PMC3178570  PMID: 21966273
12.  HIV-1 Vpr Triggers Natural Killer Cell–Mediated Lysis of Infected Cells through Activation of the ATR-Mediated DNA Damage Response 
PLoS Pathogens  2009;5(10):e1000613.
Natural killer (NK) cells are stimulated by ligands on virus-infected cells. We have recently demonstrated that NK cells respond to human immunodeficiency virus type-1 (HIV-1)-infected autologous T-cells, in part, through the recognition of ligands for the NK cell activating receptor NKG2D on the surface of the infected cells. Uninfected primary CD4pos T-cell blasts express little, if any, NKG2D ligands. In the present study we determined the mechanism through which ligands for NKG2D are induced on HIV-1-infected cells. Our studies reveal that expression of vpr is necessary and sufficient to elicit the expression of NKG2D ligands in the context of HIV-1 infection. Vpr specifically induces surface expression of the unique-long 16 binding proteins (ULBP)-1 and ULBP-2, but not ULBP-3, MHC class I-related chain molecules (MIC)-A or MIC-B. In these studies we also demonstrated that Vpr increases the level of ULBP-1 and ULBP-2 mRNA in primary CD4pos T-cell blasts. The presence of ULBP-1 and ULBP-2 on HIV-1 infected cells is dependent on the ability of Vpr to associate with a protein complex know as Cullin 4a (Cul4a)/damaged DNA binding protein 1 (DDB1) and Cul4a-associated factor-1(DCAF-1) E3 ubiquitin ligase (Cul4aDCAF-1). ULBP-1 and -2 expression by Vpr is also dependent on activation of the DNA damage sensor, ataxia telangiectasia and rad-3-related kinase (ATR). When T-cell blasts are infected with a vpr-deficient HIV-1, NK cells are impaired in killing the infected cells. Thus, HIV-1 Vpr actively triggers the expression of the ligands to the NK cell activation receptor.
Author Summary
Natural killer (NK) cells are part of the innate immune response against virus infection and cancer. Recently we demonstrated that ligands for the NK cell activation receptor, NKG2D, trigger NK cell-mediated response to infected cells. These ligands are expressed on HIV-1-infected cells and not on uninfected cells. Despite the observation that NKG2D ligands are expressed on infected cells, it is unclear how HIV-1 induces their expression. In the present study, we demonstrate that HIV induces the ligands of the NKG2D receptor through the viral gene product Vpr. Vpr triggers a DNA damage response in infected cells, which in turn, increases virus production. We also demonstrate that by blocking the activity of ATR, a major component in the DNA damage response, we were able to prevent NKG2D ligand expression. When Vpr was removed from the virus genome, NK cells lost their ability to lyse the HIV-infected cells. Thus, HIV-1 actively triggers NK cells through the activity of its viral gene product, Vpr.
PMCID: PMC2747015  PMID: 19798433
13.  Sustained CTL activation by murine pulmonary epithelial cells promotes the development of COPD-like disease 
Chronic obstructive pulmonary disease (COPD) is a lethal progressive lung disease culminating in permanent airway obstruction and alveolar enlargement. Previous studies suggest CTL involvement in COPD progression; however, their precise role remains unknown. Here, we investigated whether the CTL activation receptor NK cell group 2D (NKG2D) contributes to the development of COPD. Using primary murine lung epithelium isolated from mice chronically exposed to cigarette smoke and cultured epithelial cells exposed to cigarette smoke extract in vitro, we demonstrated induced expression of the NKG2D ligand retinoic acid early transcript 1 (RAET1) as well as NKG2D-mediated cytotoxicity. Furthermore, a genetic model of inducible RAET1 expression on mouse pulmonary epithelial cells yielded a severe emphysematous phenotype characterized by epithelial apoptosis and increased CTL activation, which was reversed by blocking NKG2D activation. We also assessed whether NKG2D ligand expression corresponded with pulmonary disease in human patients by staining airway and peripheral lung tissues from never smokers, smokers with normal lung function, and current and former smokers with COPD. NKG2D ligand expression was independent of NKG2D receptor expression in COPD patients, demonstrating that ligand expression is the limiting factor in CTL activation. These results demonstrate that aberrant, persistent NKG2D ligand expression in the pulmonary epithelium contributes to the development of COPD pathologies.
PMCID: PMC2648699  PMID: 19197141
14.  NKG2D Receptor Signaling Enhances Cytolytic Activity by Virus-Specific CD8+ T Cells: Evidence for a Protective Role in Virus-Induced Encephalitis▿  
Journal of Virology  2007;82(6):3031-3044.
Inoculation with the neurotropic JHM strain of mouse hepatitis virus (JHMV) into the central nervous system (CNS) of mice results in an acute encephalitis associated with an immune-mediated demyelinating disease. During acute disease, infiltrating CD8+ T cells secrete gamma interferon (IFN-γ) that controls replication in oligodendrocytes, while infected astrocytes and microglia are susceptible to perforin-mediated lysis. The present study was undertaken to reveal the functional contributions of the activating NKG2D receptor in host defense and disease following JHMV infection. NKG2D ligands RAE-1, MULT1, and H60 were expressed within the CNS following JHMV infection. The immunophenotyping of infiltrating cells revealed that NKG2D was expressed on ∼90% of infiltrating CD8+ T cells during acute and chronic disease. Blocking NKG2D following JHMV infection resulted in increased mortality that correlated with increased viral titers within the CNS. Anti-NKG2D treatment did not alter T-cell infiltration into the CNS or the generation of virus-specific CD8+ T cells, and the expression of IFN-γ was not affected. However, cytotoxic T-lymphocyte (CTL) activity was dependent on NKG2D expression, because anti-NKG2D treatment resulted in a dramatic reduction in lytic activity by virus-specific CD8+ T cells. Blocking NKG2D during chronic disease did not affect either T-cell or macrophage infiltration or the severity of demyelination, indicating that NKG2D does not contribute to virus-induced demyelination. These findings demonstrate a functional role for NKG2D in host defense during acute viral encephalitis by selectively enhancing CTL activity by infiltrating virus-specific CD8+ T cells.
PMCID: PMC2259000  PMID: 18160433
15.  Prostate Tumor-Derived Exosomes Down-Regulate NKG2D Expression on Natural Killer Cells and CD8+ T Cells: Mechanism of Immune Evasion 
PLoS ONE  2014;9(9):e108925.
Tumor-derived exosomes, which are nanometer-sized extracellular vesicles of endosomal origin, have emerged as promoters of tumor immune evasion but their role in prostate cancer (PC) progression is poorly understood. In this study, we investigated the ability of prostate tumor-derived exosomes to downregulate NKG2D expression on natural killer (NK) and CD8+ T cells. NKG2D is an activating cytotoxicity receptor whose aberrant loss in cancer plays an important role in immune suppression. Using flow cytometry, we found that exosomes produced by human PC cells express ligands for NKG2D on their surface. The NKG2D ligand-expressing prostate tumor-derived exosomes selectively induced downregulation of NKG2D on NK and CD8+ T cells in a dose-dependent manner, leading to impaired cytotoxic function in vitro. Consistent with these findings, patients with castration-resistant PC (CRPC) showed a significant decrease in surface NKG2D expression on circulating NK and CD8+ T cells compared to healthy individuals. Tumor-derived exosomes are likely involved in this NKG2D downregulation, since incubation of healthy lymphocytes with exosomes isolated from serum or plasma of CRPC patients triggered downregulation of NKG2D expression in effector lymphocytes. These data suggest prostate tumor-derived exosomes as down-regulators of the NKG2D-mediated cytotoxic response in PC patients, thus promoting immune suppression and tumor escape.
PMCID: PMC4182531  PMID: 25268476
16.  Structure of the HCMV UL16-MICB Complex Elucidates Select Binding of a Viral Immunoevasin to Diverse NKG2D Ligands 
PLoS Pathogens  2010;6(1):e1000723.
The activating immunoreceptor NKG2D promotes elimination of infected or malignant cells by cytotoxic lymphocytes through engagement of stress-induced MHC class I-related ligands. The human cytomegalovirus (HCMV)-encoded immunoevasin UL16 subverts NKG2D-mediated immune responses by retaining a select group of diverse NKG2D ligands inside the cell. We report here the crystal structure of UL16 in complex with the NKG2D ligand MICB at 1.8 Å resolution, revealing the molecular basis for the promiscuous, but highly selective, binding of UL16 to unrelated NKG2D ligands. The immunoglobulin-like UL16 protein utilizes a three-stranded β-sheet to engage the α-helical surface of the MHC class I-like MICB platform domain. Intriguingly, residues at the center of this β-sheet mimic a central binding motif employed by the structurally unrelated C-type lectin-like NKG2D to facilitate engagement of diverse NKG2D ligands. Using surface plasmon resonance, we find that UL16 binds MICB, ULBP1, and ULBP2 with similar affinities that lie in the nanomolar range (12–66 nM). The ability of UL16 to bind its ligands depends critically on the presence of a glutamine (MICB) or closely related glutamate (ULBP1 and ULBP2) at position 169. An arginine residue at this position however, as found for example in MICA or ULBP3, would cause steric clashes with UL16 residues. The inability of UL16 to bind MICA and ULBP3 can therefore be attributed to single substitutions at key NKG2D ligand locations. This indicates that selective pressure exerted by viral immunoevasins such as UL16 contributed to the diversification of NKG2D ligands.
Author Summary
Cytotoxic lymphocytes such as natural killer (NK) cells or CD8 T cells have the ability to detect and destroy cells infected by viruses. They therefore are tools on which the human immune system critically depends in order to control viral infections. To avoid discovery by cytotoxic lymphocytes and to allow for longtime persistence in the human host, the human cytomegalovirus (HCMV) has developed a multitude of immune evasive strategies that are mediated by so-called immunoevasins. We present here a structure-function analysis of one of the best-known HCMV immunevasins, UL16, and its interaction with a cellular ligand for NK cells, MICB. The normal function of MICB is to activate NK cells by engaging the most well-known NK receptor, NKG2D. Our results provide molecular evidence for the strategy used by UL16 to disable NK cell activation. In a rare example of structural mimicry that has likely arisen through convergent evolution, UL16 mimics a central binding motif of the structurally unrelated NKG2D protein. This allows UL16 to engage and disable several diverse NKG2D ligands, while others have apparently evolved to escape recognition by UL16 through alteration of key residues at strategic locations.
PMCID: PMC2797645  PMID: 20090832
17.  NKG2D CAR T cell therapy inhibits the growth of NKG2D ligand heterogeneous tumors 
Immunology and cell biology  2013;91(6):435-440.
Tumor heterogeneity presents a substantial barrier to increasing clinical responses mediated by targeted therapies. Broadening the immune response elicited by treatments that target a single antigen is necessary for the elimination of tumor variants that fail to express the targeted antigen. In this study, it is shown that adoptive transfer of T cells bearing a chimeric antigen receptor (CAR) inhibited the growth of target-expressing and –deficient tumor cells within ovarian and lymphoma tumors. Mice bearing the ID8 ovarian or RMA lymphoma tumors were treated with T cells transduced with a NKG2D-based CAR (chNKG2D). NKG2D CAR T cell therapy protected mice from heterogeneous RMA tumors. Moreover, adoptive transfer of chNKG2D T cells mediated tumor protection against highly heterogeneous ovarian tumors in which 50%, 20%, or only 7% of tumor cells expressed significant amounts of NKG2D ligands. CAR T cells did not mediate an in vivo response against tumor cells that did not express sufficient amounts of NKG2D ligands, and the number of ligand-expressing tumor cells correlated with therapeutic efficacy. In addition, tumor-free surviving mice were protected against a tumor re-challenge with NKG2D ligand-negative ovarian tumor cells. These data indicate that NKG2D CAR T cell treatment can be an effective therapy against heterogeneous tumors and induce tumor-specific immunity against ligand-deficient tumor cells.
PMCID: PMC3700668  PMID: 23628805
chNKG2D; adoptive T cell therapy; immunotherapy; chimeric antigen receptors; CD8 T cells; epitope spreading
18.  Generation, affinity maturation, and characterization of a human anti-human NKG2D monoclonal antibody with dual antagonistic and agonistic activity 
Journal of molecular biology  2008;384(5):1143-1156.
In humans, NKG2D is an activating receptor on NK cells and a costimulatory receptor on certain T cells and plays a central role in mediating immune responses in autoimmune diseases, infectious diseases, and cancer. Monoclonal antibodies that antagonize or agonize immune responses mediated by human NKG2D are considered to be of broad and potent therapeutic utility. Nonetheless, monoclonal antibodies to NKG2D that are suitable for clinical investigations have not been published yet. Here we describe the generation, affinity maturation, and characterization of a fully human monoclonal antibody to human NKG2D. Using phage display technology based on a newly generated naïve human Fab library in phage display vector pC3C followed by a tandem chain shuffling process designed for minimal deviation from natural human antibody sequences, we selected a human Fab, designated KYK-2.0, with high specificity and affinity to human NKG2D. KYK-2.0 Fab blocked the binding of the natural human NKG2D ligands MICA, MICB, and ULBP2 as potently as a commercially available mouse anti-human NKG2D monoclonal antibody in IgG format. Conversion of KYK-2.0 Fab to IgG1 resulted in subnanomolar avidity for human NKG2D. KYK-2.0 IgG1 was found to selectively recognize defined subpopulations of human lymphocytes known to express NKG2D, i.e. the majority of human CD8+, CD16+, and CD56+ cells as well as a small fraction of human CD4+ cells. In solution, KYK-2.0 IgG1 interfered with the cytolytic activity of ex vivo expanded human NK cells. By contrast, immobilized KYK-2.0 IgG1 was found to strongly induce human NK cell activation. The dual antagonistic and agonistic activity promises a wide range of therapeutic applications for KYK-2.0 IgG1 and its derivatives.
PMCID: PMC2659651  PMID: 18809410
NKG2D; NK cells; human monoclonal antibodies; phage display; antibody engineering
19.  NKG2D Is Critical for NK Cell Activation in Host Defense Against Pseudomonas aeruginosa Respiratory Infection 
Pseudomonas aeruginosa is a major cause of nosocomial respiratory infections. The eradication of P. aeruginosa from the lung involves the orchestrated actions of the pulmonary epithelium and both resident and recruited immune cells. The NKG2D receptor is constitutively expressed on the surface of circulating and tissue-resident NK cells (and other cytotoxic lymphocytes), and is capable of controlling NK cell activation and production of cytokines such as IFN-γ via interactions with ligands expressed on the surface of stressed cells. Previously, we demonstrated that NKG2D mediates pulmonary clearance of P. aeruginosa. In the present study, we investigated the cellular and molecular mechanisms of NKG2D-mediated clearance of P. aeruginosa using a novel transgenic mouse model of doxycycline (DOX)-inducible conditional expression of NKG2D ligands (Raet1a) in pulmonary epithelial cells. NKG2D ligand expression in this model increased pulmonary clearance, cellular phagocytosis, and survival following P. aeruginosa respiratory infection. Additionally, NK cell sensitivity to ex vivo LPS stimulation was greater in lung cells isolated from naive transgenic mice administered DOX. We also showed that NK cells are the primary source of lymphocyte-derived IFN-γ in response to P. aeruginosa respiratory infection. Significantly, we demonstrated that NKG2D is critical to the non-redundant IFN-γ production by pulmonary NK cells following acute P. aeruginosa infection. These results represent the principal report of NKG2D-mediated activation of lung NK cells following respiratory infection with an opportunistic pathogen and further establish the importance of NKG2D in the host response against P. aeruginosa respiratory infection.
PMCID: PMC2567053  PMID: 18832705
Natural killer cells; bacterial; lung; transgenic/knockout mice
20.  Immune Activation Resulting from NKG2D/Ligand Interaction Promotes Atherosclerosis 
Circulation  2011;124(25):2933-2943.
The interplay between the immune system and abnormal metabolic conditions sustains and propagates a vicious feedback cycle of chronic inflammation and metabolic dysfunction that is critical for atherosclerotic progression. It is well established that abnormal metabolic conditions, such as dyslipidemia and hyperglycemia, cause various cellular stress responses that induce tissue inflammation and immune cell activation, which in turn exacerbate the metabolic dysfunction. However, molecular events linking these processes are not well understood.
Methods and Results
Tissues and organs of humans and mice with hyperglycemia and hyperlipidemia were examined for expression of ligands for NKG2D, a potent immune activating receptor expressed by several types of immune cells, and the role of NKG2D in atherosclerosis and metabolic diseases was probed using mice lacking NKG2D or by blocking NKG2D with monoclonal antibodies. NKG2D ligands were upregulated in multiple organs, particularly atherosclerotic aortae and inflamed livers. Ligand upregulation was induced in vitro by abnormal metabolites associated with metabolic dysfunctions. Using ApoE-/- mouse models we demonstrated that preventing NKG2D functions resulted in a dramatic reduction in plaque formation, suppressed systemic and organ inflammation mediated by multiple immune cell types, and alleviated abnormal metabolic conditions.
The NKG2D/ligand interaction is a critical molecular link in the vicious cycle of chronic inflammation and metabolic dysfunction that promotes atherosclerosis and might be a useful target for therapeutic intervention in the disease.
PMCID: PMC3289255  PMID: 22104546
NKG2D; atherosclerosis; immune activation; inflammation; metabolic dysfunction
21.  NKG2C Deletion Is a Risk Factor of HIV Infection 
NK cell function is important in the immune response to HIV infection. NKG2C and NKG2A are activating and inhibitory NK cell receptors, respectively, and their only known ligand, HLA-E, demonstrates increased expression in HIV infection and presents at least one HIV-derived peptide. A variation in chromosome 12 exists in which the 16-kb section of DNA encompassing the nkg2c gene is completely absent. DNA samples of 433 HIV-1-infected patients and 280 controls were genotyped by PCR, and revealed an association of the absence variation with a higher risk of HIV infection, as well as faster progression and higher pretreatment viral loads (p<0.05, respectively). Surface NKG2C expression, analyzed by FACS, on the freshly isolated lymphocytes of 20 control and 19 HIV-infected donors revealed that NKG2C expression is genotype dependent in both populations: no NKG2C expression in the −/− groups, intermediate expression in the +/− groups, and highest expression in the +/+ groups. The comparison of NKG2C and NKG2A expression in HIV and control groups (+/− and +/+ included) indicates an increased NKG2C expression on HIV patient NK cells (p<0.05) and decreased inhibitory NKG2A expression on CD8 T cells (p<0.001), and both these effects are more striking in the +/+ genotype (p<0.005). Furthermore, a positive correlation was found between HIV viral load and the proportion of NKG2C+ NK cells. The increased expression of NKG2C in HIV patients, in combination with the genetic association of the absence variation with an increased susceptibility to HIV infection, higher HIV viral set point, and a faster progression, indicate that NKG2C is important in the defense against HIV infection and progression.
PMCID: PMC3399562  PMID: 22074011
22.  NKG2D Blockade Inhibits Poly(I:C)-Triggered Fetal Loss in Wild Type but Not IL-10−/− Mice 
Infection and inflammation can disturb immune tolerance at the maternal-fetal interface, resulting in adverse pregnancy outcomes. However, the underlying mechanisms for detrimental immune responses remain ill-defined. Here, we provide evidence for immune programming of fetal loss in response to poly(I:C), a viral mimic and an inducer of inflammatory milieu. IL-10 and uterine NK (uNK) cells expressing the activating receptor NKG2D play a critical role in poly(I:C)-induced fetal demise. In wild type (WT) mice, poly(I:C) treatment induced expansion of NKG2D+ uNK cells and expression of Rae-1 (an NKG2D ligand) on uterine macrophages and led to fetal resorption. In IL-10−/− mice, NKG2D− T cells instead became the source of fetal resorption during the same gestation period. Interestingly, both uterine NK and T cells produced TNF-α as the key cytotoxic factor contributing to fetal loss. Treatment of WT mice with poly(I:C) resulted in excessive trophoblast migration into the decidua and increased TUNEL positive signal. IL-10−/− mice supplemented with recombinant IL-10 induced fetal loss through NKG2D+ uNK cells, similar to the response in WT mice. Blockade of NKG2D in poly(I:C)-treated WT mice led to normal pregnancy outcome. Thus, we demonstrate for the first time that pregnancy disrupting inflammatory events mimicked by poly(I:C) are regulated by IL- 10 and depend on the effector function of uterine NKG2D+ NK cells in WT mice and NKG2D− T cells in IL-10 null mice.
PMCID: PMC3608719  PMID: 23455498
IL-10; TLR-3; uterine T cells; uterine NK cells; NKG2D
23.  NKG2D and its ligands: active factors in the outcome of solid organ transplantation? 
The role of natural killer (NK) cells in solid organ transplantation is not well established, although several recent reports highlight the importance of the activating receptor NKG2D and its ligands in the development of rejection during transplantation. The human NKG2D ligands (MICA and MICB) are induced in allografts during acute and chronic rejection, and the presence of anti-MICA antibodies is correlated with a higher incidence of rejection. The binding of these ligands to its receptor NKG2D activates NK cells, enhances the functions of effectors, and allows NK cells to function as a bridge between innate and adaptive immunity associated with the transplantation. In fact, blockage of NKG2D with the anti-NKG2D monoclonal antibodies prolongs graft survival and prevents CD28-independent rejection in heart and skin allograft mouse models. Furthermore, the current immunosuppressive therapies can modulate the expression of NK cell receptors and consequently the effector functions of NK cells. That is particularly important during the first few months after transplantation, when the susceptibility to opportunistic viral infections is higher and NKG2D has an essential role. In this review, we analyze in detail the potential role of the NKG2D-activating receptor and its ligands in the immune responses during the outcome of solid organ transplantation. These findings open a new pathway for therapeutic intervention that can contribute to tolerance in solid organ transplantation.
PMCID: PMC4089716  PMID: 25018903
MICA; NKG2D; natural killer cells; rejection; solid organ transplantation
24.  Redirecting T Cells to Ewing's Sarcoma Family of Tumors by a Chimeric NKG2D Receptor Expressed by Lentiviral Transduction or mRNA Transfection 
PLoS ONE  2012;7(2):e31210.
We explored the possibility to target Ewing's sarcoma family of tumors (ESFT) by redirecting T cells. To this aim, we considered NKG2D-ligands (NKG2D-Ls) as possible target antigens. Detailed analysis of the expression of MICA, MICB, ULBP-1, -2, and -3 in fourteen ESFT cell lines revealed consistent expression of at least one NKG2D-L. Thus, for redirecting T cells, we fused a CD3ζ/CD28-derived signaling domain to the ectodomain of NKG2D, however, opposite transmembrane orientation of this signaling domain and NKG2D required inverse orientation fusion of either of them. We hypothesized that the particularly located C-terminus of the NKG2D ectodomain should allow reengineering of the membrane anchoring from a native N-terminal to an artificial C-terminal linkage. Indeed, the resulting chimeric NKG2D receptor (chNKG2D) was functional and efficiently mediated ESFT cell death triggered by activated T cells. Notably, ESFT cells with even low NKG2D-L expression were killed by CD8pos and also CD4pos cells. Both, mRNA transfection and lentiviral transduction resulted in high level surface expression of chNKG2D. However, upon target-cell recognition receptor surface levels were maintained by tranfected RNA only during the first couple of hours after transfection. Later, target-cell contact resulted in strong and irreversible receptor down-modulation, whereas lentivirally mediated expression of chNKG2D remained constant under these conditions. Together, our study defines NKG2D-Ls as targets for a CAR-mediated T cell based immunotherapy of ESFT. A comparison of two different methods of gene transfer reveals strong differences in the susceptibility to ligand-induced receptor down-modulation with possible implications for the applicability of RNA transfection.
PMCID: PMC3280271  PMID: 22355347
25.  Cervical cancer cell lines expressing NKG2D-ligands are able to down-modulate the NKG2D receptor on NKL cells with functional implications 
BMC Immunology  2012;13:7.
Cervical cancer represents the third most commonly diagnosed cancer and the fourth leading cause of cancer-related deaths in women worldwide. Natural killer (NK) cells play an important role in the defense against viruses, intracellular bacteria and tumors. NKG2D, an activating receptor on NK cells, recognizes MHC class I chain-related molecules, such as MICA/B and members of the ULBP/RAET1 family. Tumor-derived soluble NKG2D-ligands have been shown to down-modulate the expression of NKG2D on NK cells. In addition to the down-modulation induced by soluble NKG2D-ligands, it has recently been described that persistent cell-cell contact can also down-modulate NKG2D expression. The goal of this study was to determine whether the NKG2D receptor is down-modulated by cell-cell contact with cervical cancer cells and whether this down-modulation might be associated with changes in NK cell activity.
We demonstrate that NKG2D expressed on NKL cells is down-modulated by direct cell contact with cervical cancer cell lines HeLa, SiHa, and C33A, but not with non-tumorigenic keratinocytes (HaCaT). Moreover, this down-modulation had functional implications. We found expression of NKG2D-ligands in all cervical cancer cell lines, but the patterns of ligand distribution were different in each cell line. Cervical cancer cell lines co-cultured with NKL cells or fresh NK cells induced a marked diminution of NKG2D expression on NKL cells. Additionally, the cytotoxic activity of NKL cells against K562 targets was compromised after co-culture with HeLa and SiHa cells, while co-culture with C33A increased the cytotoxic activity of the NKL cells.
Our results suggest that differential expression of NKG2D-ligands in cervical cancer cell lines might be associated with the down-modulation of NKG2D, as well as with changes in the cytotoxic activity of NKL cells after cell-cell contact with the tumor cells.
PMCID: PMC3364150  PMID: 22316211
NK cells; NKG2D; MICA; MICB; ULBP; Cervical cancer

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