Human cytomegalovirus-encoded UL16 discriminates MIC molecules by their alpha2 domains

Human CMV infection results in MHC class I down-regulation and induction of NKG2D ligand expression favoring NK recognition of infected cells. However, human CMV-encoded UL16 counteracts surface expression of several NKG2D ligands by intracellular retention. Interestingly, UL16 interacts with MICB, but not with the closely related MICA, and with UL16-binding proteins (ULBP) ULBP1 and ULBP2, which are only distantly related to MICB, but not with ULPB3 or ULBP4, although all constitute ligands for NKG2D. Here, we dissected the molecular basis of MICA-MICB discrimination by UL16 to elucidate its puzzling binding behavior. We report that the UL16-MICB interaction is independent of glycosylation and demonstrate that selective MICB recognition by UL16 is governed by helical structures of the MICB alpha2 domain. Transplantation of the MICB alpha2 domain confers UL16 binding capacity to MICA, and thus, diversification of the MICA alpha2 domain may have been driven by the selective pressure exerted by UL16.     

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.     

HER2/HER3 signaling regulates NK cell-mediated cytotoxicity via MHC class I chain-related molecule A and B expression in human breast cancer cell lines

Overexpression of the receptor tyrosine kinases HER2 and HER3 is associated with a poor prognosis in several types of cancer. Presently, HER2- as well as HER3-targeted therapies are in clinical practice or evaluated within clinical trials, including treatment with mAbs mediating growth inhibition and/or activation of Ab-induced innate or adaptive cellular immunity. A better understanding of how HER2/HER3 signaling in tumors influences cellular immune mechanisms is therefore warranted. In this study, we demonstrate that HER2/HER3 signaling regulates the expression of MHC class I-related chain A and B (MICA and MICB) in breast cancer cell lines. The MICA and MICB (MICA/B) molecules act as key ligands for the activating receptor NK group 2, member D (NKG2D) and promote NK cell-mediated recognition and cytolysis. Genetic silencing of HER3 but not HER2 downregulated the expression of MICA/B, and HER3 overexpression significantly enhanced MICA expression. Among the major pathways activated by HER2/HER3 signaling, the PI3K/AKT pathway was shown to predominantly regulate MICA/B expression. Treatment with the HER3-specific ligand neuregulin 1β promoted the expression in a process that was antagonized by pharmacological and genetic interference with HER3 but not by the ataxia-telangiectasia-mutated (ATM) and ATM and Rad3-related protein kinases inhibitor caffeine. These observations further emphasize that HER2/HER3 signaling directly, and not via genotoxic stress, regulates MICA/B expression. As anticipated, stimulating HER2/HER3 enhanced the NKG2D-MICA/B-dependent NK cell-mediated cytotoxicity. Taken together, we conclude that signaling via the HER2/HER3 pathway in breast carcinoma cell lines may lead to enhanced NKG2D-MICA/B recognition by NK cells and T cells.     

Interactions of human NKG2D with its ligands MICA, MICB, and homologs of the mouse RAE-1 protein family

NKG2D is an activating receptor that is expressed on most natural killer (NK) cells, CD8 alphabeta T cells, and gammadelta T cells. Among its ligands is the distant major histocompatibility complex class I homolog MICA, which has no function in antigen presentation but is induced by cellular stress. To extend previous functional evidence, the NKG2D-MICA interaction was studied in isolation. NKG2D homodimers formed stable complexes with monomeric MICA in solution, demonstrating that no other components were required to facilitate this interaction. MICA glycosylation was not essential but enhanced complex formation. Soluble NKG2D also bound to cell surface MICB, which has structural and functional properties similar to those of MICA. Moreover, NKG2D stably interacted with surface molecules encoded by three newly identified cDNA sequences (N2DL-1, -2, and -3), which are identical to the human ULBP proteins and may represent homologs of the mouse retinoic acid-early inducible family of NKG2D ligands. Because of the substantial sequence divergence among these molecules, these results indicated promiscuous modes of receptor binding. Comparison of allelic variants of MICA revealed large differences in NKG2D binding that were associated with a single amino acid substitution at position 129 in the alpha2 domain. Varying affinities of MICA alleles for NKG2D may affect thresholds of NK-cell triggering and T-cell modulation.     

Down-regulation of the NKG2D ligand MICA by the human cytomegalovirus glycoprotein UL142

Human cytomegalovirus (HCMV) employs a variety of strategies to modify or evade the host immune response, and natural killer (NK) cells play a crucial role in controlling cytomegalovirus infections in mice and humans. Activation of NK cells through the receptor NKG2D/DAP10 leads to killing of NKG2D ligand-expressing cells. We have previously shown that HCMV is able to down-regulate the surface expression of some NKG2D ligands, ULBP1, ULBP2, and MICB via the viral glycoprotein UL16. Here, we show that the viral gene product UL142 is able to down-regulate another NKG2D ligand, MICA, leading to protection from NK cytotoxicity. UL142 is not able to affect surface expression of all MICA alleles, however, which may reflect selective pressure on the host to thwart viral immune evasion, further supporting an important role for the MICA-NKG2D interaction in immune surveillance.     

Immunohistochemical Validation and Expression Profiling of NKG2D Ligands in a Wide Spectrum of Human Epithelial Neoplasms

The MHC class I-chain-related proteins (MICs) and the UL16-binding proteins (ULBPs) are inducible stress response molecules that work as activators of a specific receptor, NKG2D, which is expressed on effector cells, such as NK cells and subsets of T cells. In this study, we sought to explore the biological significance of NKG2D ligands in human neoplasms by comprehensively examining the immunohistochemical expression profile of NKG2D ligands in a variety of human epithelial neoplasms. Following careful validation of the immunohistochemical specificity and availability of anti-human ULBP antibodies for formalin-fixed paraffin-embedded (FFPE) materials, the expression of NKG2D ligands was analyzed in FFPE tissue microarrays comprising 22 types of epithelial neoplastic tissue with their non-neoplastic counterpart from various organs. Hierarchical cluster analysis demonstrated a positive relationship among ULBP2/6, ULBP3, ULBP1, and ULBP5, whose expression patterns were similar across all of the neoplastic tissues examined. In contrast, MICA/B, as well as ULBP4, did not appear to be related to any other ligand. These expression profiles of NKG2D ligands in human neoplasms based on well-validated specific antibodies, followed by hierarchical cluster analysis, should help to clarify some functional aspects of these molecules in cancer biology, and also provide a path to the development of novel tumor-type-specific treatment strategies.     

Systemic NKG2D down-regulation impairs NK and CD8 T cell responses in vivo

The immunoreceptor NKG2D stimulates activation of cytotoxic lymphocytes upon engagement with MHC class I-related NKG2D ligands of which at least some are expressed inducibly upon exposure to carcinogens, cell stress, or viruses. In this study, we investigated consequences of a persistent NKG2D ligand expression in vivo by using transgenic mice expressing MHC class I chain-related protein A (MICA) under control of the H2-K(b) promoter. Although MICA functions as a potent activating ligand of mouse NKG2D, H2-K(b)-MICA mice appear healthy without aberrations in lymphocyte subsets. However, NKG2D-mediated cytotoxicity of H2-K(b)-MICA NK cells is severely impaired in vitro and in vivo. This deficiency concurs with a pronounced down-regulation of surface NKG2D that is also seen on activated CD8 T cells. As a consequence, H2-K(b)-MICA mice fail to reject MICA-expressing tumors and to mount normal CD8 T cell responses upon Listeria infection emphasizing the importance of NKG2D in immunity against tumors and intracellular infectious agents.     

Structure of the HCMV UL16-MICB Complex Elucidates Select Binding of a Viral Immunoevasin to Diverse NKG2D Ligands

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.     

The heat shock protein HSP70 promotes mouse NK cell activity against tumors that express inducible NKG2D ligands

The stress-inducible heat shock protein (HSP) 70 is known to function as an endogenous danger signal that can increase the immunogenicity of tumors and induce CTL responses. We show in this study that HSP70 also activates mouse NK cells that recognize stress-inducible NKG2D ligands on tumor cells. Tumor size and the rate of metastases derived from HSP70-overexpressing human melanoma cells were found to be reduced in T and B cell-deficient SCID mice, but not in SCID/beige mice that lack additionally functional NK cells. In the SCID mice with HSP70-overexpressing tumors, NK cells were activated so that they killed ex vivo tumor cells that expressed NKG2D ligands. In the tumors, the MHC class I chain-related (MIC) A and B molecules were found to be expressed. Interestingly, a counter selection was observed against the expression of MICA/B in HSP70-overexpressing tumors compared with control tumors in SCID, but not in SCID/beige mice, suggesting a functional relevance of MICA/B expression. The melanoma cells were found to release exosomes. HSP70-positive exosomes from the HSP70-overexpressing cells, in contrast to HSP70-negative exosomes from the control cells, were able to activate mouse NK cells in vitro to kill YAC-1 cells, which express NKG2D ligands constitutively, or the human melanoma cells, in which MICA/B expression was induced. Thus, HSP70 and inducible NKG2D ligands synergistically promote the activation of mouse NK cells resulting in a reduced tumor growth and suppression of metastatic disease.     

5-氟尿嘧啶增强HeLa细胞对自然杀伤细胞敏感性研究

目的用5-氟尿嘧啶（5-fluorouracil,5-FU）处理HeLa细胞,检测其NKG2D配体MICA的表达及其对NK92细胞杀伤敏感性的变化。方法不同浓度的5-Fu处理HeLa细胞,在不同时间点用半定量PCR及流式细胞术检测HeLa细胞表面的NKG2D配体MICA在RNA及蛋白水平的表达变化情况,用MTT法检测NKG2D抗体封闭NK92细胞的NKG2D受体前后,NK92细胞对HeLa细胞的杀伤作用。结果不同浓度的5．Fu作用于HeLa细胞后,半定量RT—PCR结果显示MICA表达随5-Fu作用浓度增加逐渐增高。而且40μg／ml5．Fu作用于HeLa细胞后随着作用时间的延长（0、8、16、24h）MICA表达增加,流式细胞术检测结果表明,MICA表达的增加主要依赖于未凋亡细胞的MICA表达。在40μg／ml5-FU作用24h,效靶比为2．5：1,5：1,10：1,20：1时都检测到NK92细胞对HeLa细胞的杀伤增强,杀伤作用可部分被NKG2D抗体抑制。结论5-FU能够上调HeLa细胞表面NKG2D配体MICA的表达,增强HeLa细胞对NK92细胞的敏感性,提示化疗联合NK细胞免疫治疗宫颈癌可产生协同作用,提高治疗效果。     

NKG2D discriminates diverse ligands through selectively mechano‐regulated ligand conformational changes

Stimulatory immune receptor NKG2D binds diverse ligands to elicit differential anti‐tumor and anti‐virus immune responses. Two conflicting degeneracy recognition models based on static crystal structures and in‐solution binding affinities have been considered for almost two decades. Whether and how NKG2D recognizes and discriminates diverse ligands still remain unclear. Using live‐cell‐based single‐molecule biomechanical assay, we characterized the in situ binding kinetics of NKG2D interacting with different ligands in the absence or presence of mechanical force. We found that mechanical force application selectively prolonged NKG2D interaction lifetimes with the ligands MICA and MICB, but not with ULBPs, and that force‐strengthened binding is much more pronounced for MICA than for other ligands. We also integrated steered molecular dynamics simulations and mutagenesis to reveal force‐induced rotational conformational changes of MICA, involving formation of additional hydrogen bonds on its binding interface with NKG2D, impeding MICA dissociation under force. We further provided a kinetic triggering model to reveal that force‐dependent affinity determines NKG2D ligand discrimination and its downstream NK cell activation. Together, our results demonstrate that NKG2D has a discrimination power to recognize different ligands, which depends on selective mechanical force‐induced ligand conformational changes.     

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.     

The endogenous danger signals HSP70 and MICA cooperate in the activation of cytotoxic effector functions of NK cells

Although natural killer (NK) cells are often described as first line defence against infected or malignant cells which act without the need of prior activation, it is known now that the NK cell activity is tightly regulated by other cells and soluble factors. We show here that the stress‐inducible heat shock protein (HSP) 70 activates human NK cells to kill target cells expressing major histocompatibility complex class I chain‐related molecule A (MICA) in a natural killer group 2 member D (NKG2D‐) dependent manner. The HSP70‐derived peptide TKD (TKDNNLLGRFELSG) was able to replace the full‐length HSP70 and to exert the same function. Interestingly, the expression of the cytotoxic effector protease granzyme B in NK cells was increased after TKD stimulation. When MICA and MICB expression was induced in human tumour cells by a histone deacetylase inhibitor and NK cells were activated by HSP70 or TKD, both treatments jointly improved the killing of the tumour cells. Thus, the synergistic activity of two stress‐inducible immunological danger signals, HSP70 and MICA/B, leads to activation and enhanced cytotoxicity of human NK cells against tumour cells.     

N-Glycosylation of Asparagine 8 Regulates Surface Expression of Major Histocompatibility Complex Class I Chain-related Protein A (MICA) Alleles Dependent on Threonine 24

NKG2D is an activating receptor expressed on several types of human lymphocytes. NKG2D ligands can be induced upon cell stress and are frequently targeted post-translationally in infected or transformed cells to avoid immune recognition. Virus infection and inflammation alter protein N-glycosylation, and we have previously shown that changes in cellular N-glycosylation are involved in regulation of NKG2D ligand surface expression. The specific mode of regulation through N-glycosylation is, however, unknown. Here we investigated whether direct N-glycosylation of the NKG2D ligand MICA itself is critical for cell surface expression and sought to identify the essential residues. We found that a single N-glycosylation site (Asn⁸) was important for MICA018 surface expression. The frequently expressed MICA allele 008, with an altered transmembrane and intracellular domain, was not affected by mutation of this N-glycosylation site. Mutational analysis revealed that a single amino acid (Thr²⁴) in the extracellular domain of MICA018 was essential for the N-glycosylation dependence, whereas the intracellular domain was not involved. The HHV7 immunoevasin, U21, was found to inhibit MICA018 surface expression by affecting N-glycosylation, and the retention was rescued by T24A substitution. Our study reveals N-glycosylation as an allele-specific regulatory mechanism important for regulation of surface expression of MICA018, and we pinpoint the residues essential for this N-glycosylation dependence. In addition, we show that this regulatory mechanism of MICA surface expression is likely targeted during different pathological conditions.     

MMP9 mediates MICA shedding in human osteosarcomas

The MICA (MHC class I chain-related molecule A) is a ligand for the activating immunoreceptor NKG2D (natural killer group 2, member D). NKG2D recognizes MICA expressing at the cell surface for cell elimination. Although MICA is overexpressed in many kinds of tumours, tumour cells can cleverly escape immunosurveillance. One underlying mechanism for immunoescape is tumour-derived MICA shedding. In this study, we report that osteosarcoma-derived MICA results from proteolytic cleavage of MICA α3 ectodomain. sMICA (soluble MICA) might be released in the early stage of disease. A MMP9 (matrix metalloproteinase 9, gelatinase B)-specific inhibitor suppressed sMICA release, indicating that MMP9 is critically involved in the osteosarcoma-associated proteolytic release of sMICA, which facilitates tumour immune escape. Using a specific MMP inhibitor might represent a double-edged sword, where it can inhibit tumour invasion and restore antitumour immune response.     

Intracellular retention of the NKG2D ligand MHC class I chain-related gene A in human melanomas confers immune privilege and prevents NK cell-mediated cytotoxicity

Most tumors grow in immunocompetent hosts despite expressing NKG2D ligands (NKG2DLs) such as the MHC class I chain-related genes A and B (MICA/B). However, their participation in tumor cell evasion is still not completely understood. Here we demonstrate that several human melanomas (cell lines and freshly isolated metastases) do not express MICA on the cell surface but have intracellular deposits of this NKG2DL. Susceptibility to NK cell-mediated cytotoxicity correlated with the ratio of NKG2DLs to HLA class I molecules but not with the amounts of MICA on the cell surface of tumor cells. Transfection-mediated overexpression of MICA restored cell surface expression and resulted in an increased in vitro cytotoxicity and IFN-gamma secretion by human NK cells. In xenografted nude mice, these melanomas exhibited a delayed growth and extensive in vivo apoptosis. Retardation of tumor growth was due to NK cell-mediated antitumor activity against MICA-transfected tumors, given that this effect was not observed in NK cell-depleted mice. Also, mouse NK cells killed MICA-overexpressing melanomas in vitro. A mechanistic analysis revealed the retention of MICA in the endoplasmic reticulum, an effect that was associated with accumulation of endoH-sensitive (immature) forms of MICA, retrograde transport to the cytoplasm, and degradation by the proteasome. Our study identifies a novel strategy developed by melanoma cells to evade NK cell-mediated immune surveillance based on the intracellular sequestration of immature forms of MICA in the endoplasmic reticulum. Furthermore, this tumor immune escape strategy can be overcome by gene therapy approaches aimed at overexpressing MICA on tumor cells.