γ干扰素对人NK细胞识别功能的负调节作用

目的 探讨γ干扰素(IFN-γ)对人NK细胞识别功能的负调节作用。方法 用MTT法测定人NK细胞系(NK92,NKL)的细胞毒活性及细胞增殖能力;用RT-PCR检测NK细胞受体(NKG2D、NKG2A／B、KIR2DLI、KIR2DSI)及NKG2D的识别配体主要组织相容性复合体Ⅰ类链相关分子A(MICA)的表达。结果 NK细胞系(NK92、NKL)对MICA表达阳性的肿瘤细胞杀伤活性明显高于对MICA表达阴性者;IFN-γ 1000U／ml以上可明显抑制NK细胞对MICA表达阳性肿瘤细胞的细胞毒活性,并轻度抑制NK细胞的增殖,而对MICA表达阴性肿瘤细胞的杀伤活性无明显抑制作用;IFN-γ可抑制NK细胞系活化受体NKG2D的表达,增强抑制性受体NKG2A／B和KIR2DLI的表达。结论 IFN-γ可能通过下调NK细胞活化受体的表达,上调抑制性受体的表达,使NK识别的信号平衡向抑制性方向倾斜,从而对NK细胞功能发挥负调节作用,这种作用可能是NK细胞自我调节功能的表现。     

Tumor suppressive microRNAs miR-34a/c control cancer cell expression of ULBP2, a stress-induced ligand of the natural killer cell receptor NKG2D

Malignant cells express ligands for the natural killer cell immunoreceptor NKG2D, which sensitizes to early recognition and elimination by cytotoxic lymphocytes and provides an innate barrier against tumor development. However, the mechanisms that control NKG2D ligand (NKG2DL) expression in tumor cells remain unknown. We recently identified the NKG2DL ULBP2 as strong prognostic marker in human malignant melanoma. Here, we provide evidence that the tumor-suppressive microRNAs (miRNA) miR-34a and miR-34c control ULBP2 expression. Reporter gene analyses revealed that both miRNAs directly targeted the 3'-untranslated region of ULBP2 mRNA and that levels of miR-34a inversely correlated with expression of ULBP2 surface molecules. Accordingly, treatment of cancer cells with miRNA inhibitors led to upregulation of ULBP2, whereas miR-34 mimics led to downregulation of ULBP2, diminishing tumor cell recognition by NK cells. Treatment with the small molecule inhibitor Nutlin-3a also decreased ULBP2 levels in a p53-dependent manner, which was due to a p53-mediated increase in cellular miR-34 levels. Taken together, our study shows that tumor-suppressive miR-34a and miR-34c act as ULBP2 repressors. These findings also implicate p53 in ULBP2 regulation, emphasizing the role of the specific NKG2DL in tumor immune surveillance.     

Interferon‐γ down‐regulates NKG2D ligand expression and impairs the NKG2D‐mediated cytolysis of MHC class I‐deficient melanoma by natural killer cells

NKG2D operates as an activating receptor on natural killer (NK) cells and costimulates the effector function of αβ CD8⁺ T cells. Ligands of NKG2D, the MHC class I chain‐related (MIC) and UL16 binding protein (ULBP) molecules, are expressed on a variety of human tumors, including melanoma. Recent studies in mice demonstrated that NKG2D mediates tumor immune surveillance, suggesting that antitumor immunity in humans could be enhanced by therapeutic manipulation of NKG2D ligand (NKG2DL) expression. However, signals and mechanisms regulating NKG2DL expression still need to be elucidated. Here, we asked whether the proinflammatory cytokine Interferon‐γ (IFN‐γ) affects NKG2DL expression in melanoma. Cell lines, established from MHC class I‐negative and ‐positive melanoma metastases, predominantly expressed MICA and ULBP2 molecules on their surface. Upon IFN‐γ treatment, expression of MICA, in some cases, also of ULBP2 decreased. Besides melanoma, this observation was made also for glioma cells. Down‐regulation of NKG2DL surface expression was dependent on the cytokine dose and the duration of treatment, but was neither due to an intracellular retention of the molecules nor to an increased shedding of ligands from the tumor cell surface. Instead, quantitative RT‐PCR revealed a decrease of MICA‐specific mRNA levels upon IFN‐γ treatment and siRNA experiments pointed to an involvement of STAT‐1 in this process. Importantly, IFN‐γ‐treated MHC class I‐negative melanoma cells were less susceptible to NKG2D‐mediated NK cell cytotoxicity. Our study suggests that IFN‐γ, by down‐regulating ligand expression, might facilitate escape of MHC class I‐negative melanoma cells from NKG2D‐mediated killing by NK cells. © 2008 Wiley‐Liss, Inc.     

MICA/NKG2D-mediated immunogene therapy of experimental gliomas

The failure of conventional cancer therapy renders glioblastoma an attractive target for immunotherapy. Tumor cells expressing ligands of the activating immunoreceptor NKG2D stimulate tumor immunity mediated by natural killer (NK), gammadelta T, and CD8(+) T cells. We report that human glioma cells express the NKG2D ligands MICA, MICB, and members of the UL16-binding protein family constitutively. However, glioma cells resist NK cell cytolysis because of high MHC class I antigen expression. Plasmid-mediated or adenovirus-mediated overexpression of MICA in glioma cells enhances their sensitivity to NK and T-cell responses in vitro and markedly delays the growth of s.c. and intracerebral LN-229 human glioma cell xenografts in nude mice and of SMA-560 gliomas in syngeneic VMDk mice. Glioma cells forming progressive tumors after implantation of stably MICA-transfected human LN-229 cells lost MICA expression, indicating a strong selection against MICA expression in vivo. Rejection of MICA-expressing SMA-560 cells in VMDk mice resulted in protective immunity to a subsequent challenge with wild-type tumor cells. Finally, the growth of syngeneic intracerebral SMA-560 tumors is inhibited by peripheral vaccination with adenovirus-mediated, MICA-infected irradiated tumor cells, and vaccination results in immune cell activation in the NK and T-cell compartments in vivo. These data commend MICA immunogene therapy as a novel experimental treatment for human malignant gliomas.     

Direct and natural killer cell-mediated antitumor effects of low-dose bortezomib in hepatocellular carcinoma.

PURPOSE: Hepatocellular carcinoma (HCC) displays particular resistance to conventional cytostatic agents. Alternative treatment strategies focus on novel substances exhibiting antineoplastic and/or immunomodulatory activity enhancing for example natural killer (NK) cell antitumor reactivity. However, tumor-associated ligands engaging activating NK cell receptors are largely unknown. Exceptions are NKG2D ligands (NKG2DL) of the MHC class I-related chain and UL16-binding protein families, which potently stimulate NK cell responses. We studied the consequences of proteasome inhibition with regard to direct and NK cell-mediated effects against HCC. EXPERIMENTAL DESIGN: Primary human hepatocytes (PHH) from different donors, hepatoma cell lines, and NK cells were exposed to Bortezomib. Growth and viability of the different cells, and immunomodulatory effects including alterations of NKG2DL expression on hepatoma cells, specific induction of NK cell cytotoxicity and IFN-gamma production were investigated. RESULTS: Bortezomib treatment inhibited hepatoma cell growth with IC(50) values between 2.4 and 7.7 nmol/L. These low doses increased MICA/B mRNA levels, resulting in an increase of total and cell surface protein expression in hepatoma cells, thus stimulating cytotoxicity and IFN-gamma production of cocultured NK cells. Importantly, although NK cell IFN-gamma production was concentration-dependently reduced, low-dose Bortezomib neither induced NKG2DL expression or cell death in PHH nor altered NK cell cytotoxicity. CONCLUSIONS: Low-dose Bortezomib mediates a specific dual antitumor effect in HCC by inhibiting tumor cell proliferation and priming hepatoma cells for NK cell antitumor reactivity. Our data suggest that patients with HCC may benefit from Bortezomib treatment combined with immunotherapeutic approaches such as adoptive NK cell transfer taking advantage of enhanced NKG2D-mediated antitumor immunity.     

An Fc‐optimized NKG2D‐immunoglobulin G fusion protein for induction of natural killer cell reactivity against leukemia

Recruitment of Fc‐receptor‐bearing effector cells, such as natural killer (NK) cells, is a feature critical for the therapeutic success of antitumor antibodies and can be improved by the modifications of an antibody's Fc part. The various ligands of the activating immunoreceptor NKG2D, NKG2DL) are selectively expressed on malignant cells including leukemia. We here took advantage of the tumor‐associated expression of NKG2DL for targeting leukemic cells by NKG2D–immunoglobulin G (IgG)1 fusion proteins containing modified Fc parts. Compared to NKG2D–Fc containing a wild‐type Fc part (NKG2D–Fc–WT), our mutants (S239D/I332E and E233P/L234V/L235A/ΔG236/A327G/A330S) displayed highly enhanced (NKG2D–Fc–ADCC) and abrogated (NKG2D–Fc–KO) affinity to the NK cell Fc receptor, respectively. Functional analyses with allogenic as well as autologous NK cells and primary malignant cells of leukemia patients revealed that NKG2D–Fc–KO significantly reduced NK reactivity by blocking immunostimulatory NKG2D–NKG2DL interaction. NKG2D–Fc–WT already enhanced antileukemia reactivity by inducing antibody‐dependent cellular cytotoxicity (ADCC) with NKG2D–Fc–ADCC mediating significantly stronger effects. Parallel application of NKG2D–Fc–ADCC with Rituximab caused additive effects in lymphoid leukemia. In line with the tumor‐associated expression of NKG2DL, no NK cell ADCC against resting healthy blood cells was induced. Thus, NKG2D–Fc–ADCC potently enhances NK antileukemia reactivity despite the inevitable reduction of activating signals upon binding to NKG2DL and may constitute an attractive means for immunotherapy of leukemia.     

Downregulation of miR-302c and miR-520c by 1,25(OH)2D3 treatment enhances the susceptibility of tumour cells to natural killer cell-mediated cytotoxicity

Background:

NKG2D recognises several ligands, including polymorphic major histocompatibility complex class I chain-related chain-related proteins A and B (MICA/B) and unique long 16-binding proteins (ULBPs). These ligands are present on cancer cells and are recognised by NKG2D in a cell-structure-sensing manner, triggering natural killer (NK) cell cytotoxicity. However, the mechanisms that control the expression of NKG2D ligands in malignant cells are poorly understood. 1-α,25-Dihydroxyvitamin D3 (1,25(OH)2D3) was recently shown to enhance the susceptibility of melanoma cells to the cytotoxicity of NK cells. However, the function of 1,25(OH)2D3 in other cancers and its potential mechanisms of action remain unknown.

Methods:

The expression levels of miR-302c and miR-520c in Kasumi-1, K562, MCF7 and MDA-MB-231 cells were evaluated using quantitative real-time PCR. The targets of miR-302c and miR-520c were confirmed by luciferase reporter assay. The killing effects of NK92 cells against Kasumi-1, K562, MCF7 and MDA-MB-231 cells were examined using the CytoTox 96 Non-Radioactive Cytotoxicity Assay. The levels of cytokines IFN-γ and granzyme B, which indicate the activation of NK cells, were also measured by enzyme-linked immunosorbent assay.

Results:

Treatment with 1,25(OH)2D3 enhanced the susceptibility of both the haematological tumour cell line Kasumi-1 and solid tumour cell line MDA-MB-231 to NK92 cells. miR-302c and miR-520c expression was induced, and their levels inversely correlated with the levels of NKG2D ligands MICA/B and ULBP2 upon 1,25(OH)2D3 treatment. A luciferase reporter assay revealed that miR-302c and miR-520c directly targeted the 3′-UTRs of MICA/B and ULBP2 and negatively regulated the expression of MIA/B and ULBP2. Moreover, upregulation of miR-302c or miR-520c by transfection of their mimics remarkably reduced the viability of Kasumi-1 cells upon NK cell co-incubation. By contrast, the suppression of the activity of miR-302c or miR-520c by their respective antisense oligonucleotides improved the resistance of Kasumi-1 cells to NK cells.

Conclusion:

1,25(OH)2D3 facilitates the immuno-attack of NK cells against malignant cells partly through downregulation of miR-302c and miR-520c and hence upregulation of the NKG2D ligands MICA/B and ULBP2.     

Resistance to Cytarabine Induces the Up-regulation of NKG2D Ligands and Enhances Natural Killer Cell Lysis of Leukemic Cells

Prolonged treatment of leukemic cells with chemotherapeutic agents frequently results in development of drug resistance. Moreover, selection of drug-resistant cell populations may be associated with changes in malignant properties such as proliferation rate, invasiveness, and immunogenicity. In the present study, the sensitivity of cytarabine (1-β-d-arabinofuranosylcytosine, araC)-resistant and parental human leukemic cell lines (T-lymphoid H9 and acute T-lymphoblastic leukemia Molt-4) to natural killer (NK) cell-mediated killing was investigated. The results obtained demonstrate that araC-resistant H9 and Molt-4 (H9^rARAC¹⁰⁰ and Molt-4^rARAC¹⁰⁰) cell lines are more sensitive to NK cell-mediated lysis than their respective parental cell lines. This increased sensitivity was associated with a higher surface expression of ligands for the NK cell-activating receptor NKG2D, notably UL16 binding protein-2 (ULBP-2) and ULBP-3 in H9^rARAC¹⁰⁰ and Molt-4^rARAC¹⁰⁰ cell lines. Blocking ULBP-2 and ULBP-3 or NKG2D with monoclonal antibody completely abrogated NK cell lysis. Constitutive phosphorylated extracellular signal-regulated kinase (ERK) but not pAKT was higher in araC-resistant cells than in parental cell lines. Inhibition of ERK using ERK inhibitor PD98059 decreased both ULBP-2/ULBP-3 expression and NK cell cytotoxicity. Furthermore, overexpression of constitutively active ERK in H9 parental cells resulted in increased ULBP-2/ULBP-3 expression and enhanced NK cell lysis. These results demonstrate that increased sensitivity of araC-resistant leukemic cells to NK cell lysis is caused by higher NKG2D ligand expression, resulting from more active ERK signaling pathway.     

HO-1 downregulation favors BRAFV600 melanoma cell death induced by Vemurafenib/PLX4032 and increases NK recognition

Heme oxygenase 1 (HO-1) plays a pivotal role in preventing cell damage. Indeed, through the antioxidant, antiapoptotic and anti-inflammatory properties of its metabolic products, it favors cell adaptation against different stressors. However, HO-1 induction has also been related to the gain of resistance to therapy in different types of cancers and its involvement in cancer immune-escape has been hypothesized. We have investigated the role of HO-1 expression in Vemurafenib-treated BRAF^V600 melanoma cells in modulating their susceptibility to NK cell-mediated recognition. Different cell lines, isolated in house from melanoma patients, have been exposed to 1–10 μM PLX4032, which efficiently reduced ERK phosphorylation. In three lines, Vemurafenib was able to induce only a limited decrease in cell viability, while HO-1 expression was upregulated. HO-1 silencing/inhibition was able to induce a further significant reduction of Vemurafenib-treated melanoma viability. Moreover, while NK cell degranulation and killing activity were decreased upon interaction with melanoma exposed to Vemurafenib, HO-1 silencing was able to completely restore NK cell ability to degranulate and kill. Furthermore, melanoma cell treatment with Vemurafenib downregulated the expression of ligands of NKp30 and NKG2D activating receptors, and HO-1 silencing/inhibition was able to restore their expression. Our results indicate that HO-1 downregulation can both improve the efficacy of Vemurafenib on melanoma cells and favor melanoma susceptibility to NK cell-mediated recognition and killing.     

Valproic Acid Upregulates NKG2D Ligand Expression through an ERK-dependent Mechanism and Potentially Enhances NK Cell-mediated Lysis of Myeloma

Modulation of the antitumor immune response through the engagement of NKG2D receptors with their ligands (L) on targets represents a promising therapeutic approach against cancer. In this study, we tested the effect of valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, on the expression of NKG2D ligands in myeloma cells. We demonstrated that VPA was able to upregulate both protein and mRNA expression of major histocompatibility complex class I-related chain (MIC) A/B and UL16-binding protein (ULBP) 2 without any significant effect on the expression of ULBP1, ULBP3, and ULBP4 or induction of other natural killer (NK) cell ligands, such as NKp30-L, NKp44-L, and NKp46-L in myeloma cells. A ⁵¹Cr release assay and degranulation assay indicated that the induction of MICA/B and ULBP2 augmented NK cell-mediated lysis of myeloma cells, which was abolished by the addition of a blocking NKG2D antibody. Activation of constitutively phosphorylated extracellular signal-regulated kinase (ERK) by VPA is essential for the up-regulation of MICA/B and ULBP2 expressions. Inhibition of ERK using ERK inhibitor PD98059 decreased both MICA/B and ULBP2 expressions and NK cell cytotoxicity. Furthermore, overexpression of constitutively active ERK in ARK resulted in increased MICA/B and ULBP2 expressions and enhanced NK cell lysis. These data indicate that increased sensitivity of VPA-treated myeloma cells to NK cell lysis is caused by higher NKG2D ligand expression, resulting from more active ERK signaling pathway. Our results provide evidence that targeting ERK signaling pathway may be an additional mechanism supporting the antimyeloma activity of HDAC inhibitors and suggest its possible immunotherapeutic value for myeloma treatment.     

顺铂对鼻咽癌细胞NKG2D配体表达和NK细胞杀伤活性的增强作用

 目的 研究顺铂(Cisplatin, DDP)作用前后人鼻咽癌细胞CNE2 NKG2D配体和HLA-Ⅰ类分子表达的改变及NK细胞杀伤活性的变化。 方法 MTT法测定DDP对CNE2细胞的50%抑制量（IC₅₀）;以此浓度DDP作用CNE2细胞24h,乳酸脱氢酶释放法检测效靶比20∶1时,NK细胞对DDP作用前后的CNE2细胞的杀伤活性;流式细胞仪检测DDP作用前后的CNE2细胞表面NKG2D配体(MICA/B、ULBP1、ULBP2、 ULBP3)和HLA-Ⅰ类分子表达的变化。 结果 DDP对CNE2细胞的IC₅₀为5mg/L。效靶比20∶1时,NK细胞对5mg/L DDP作用前后的CNE2细胞的杀伤活性分别为 （38.11±1.41）%,（47.71±1.53）%,差异有统计学意义（P＜0.05）,DDP作用后CNE2细胞表面MICA/B、ULBP1、 ULBP3表达显著升高,与作用前相比差异有统计学意义（P＜0.05）。ULBP2、HLA-Ⅰ类分子无明显变化(P＞0.05)。 结论 DDP能提高CNE2细胞NKG2D配体(MICA/B、ULBP1、ULBP3)的表达,从而增强CNE2细胞对NK细胞杀伤的敏感度。     

Human glioma growth is controlled by microRNA-10b

MicroRNA (miRNA) expression profiling studies revealed a number of miRNAs dysregulated in the malignant brain tumor glioblastoma. Molecular functions of these miRNAs in gliomagenesis are mainly unknown. We show that inhibition of miR-10b, a miRNA not expressed in human brain and strongly upregulated in both low-grade and high-grade gliomas, reduces glioma cell growth by cell-cycle arrest and apoptosis. These cellular responses are mediated by augmented expression of the direct targets of miR-10b, including BCL2L11/Bim, TFAP2C/AP-2γ, CDKN1A/p21, and CDKN2A/p16, which normally protect cells from uncontrolled growth. Analysis of The Cancer Genome Atlas expression data set reveals a strong positive correlation between numerous genes sustaining cellular growth and miR-10b levels in human glioblastomas, while proapoptotic genes anticorrelate with the expression of miR-10b. Furthermore, survival of glioblastoma patients expressing high levels of miR-10 family members is significantly reduced in comparison to patients with low miR-10 levels, indicating that miR-10 may contribute to glioma growth in vivo. Finally, inhibition of miR-10b in a mouse model of human glioma results in significant reduction of tumor growth. Altogether, our experiments validate an important role of miR-10b in gliomagenesis, reveal a novel mechanism of miR-10b-mediated regulation, and suggest the possibility of its future use as a therapeutic target in gliomas.     

Low miR-16 expression induces regulatory CD4+NKG2D+ T cells involved in colorectal cancer progression

MiR-15a/16 is a member of the miRNA cluster that exhibits tumor suppression and immune modulation via targeting multiple genes. Decreased miR-15a/16 expression is involved in many cancer cells. Here, miR-16 had decreased expression in NK1.1^-CD4⁺NKG2D⁺ T cells and bound with the 3’-UTR of NKG2D gene. MiR-15a/16-deficient mice had many CD4⁺NKG2D⁺ T cells, which produced TGF-β1 and IL-10 and inhibited the IFN-γ production of CD8⁺ T cells. Adoptive transfer of NK1.1^-CD4⁺NKG2D⁺ T cells from miR-15a/16-deficient mice promoted tumor growth in vivo. However, no changes for NK1.1^-CD4⁺NKG2D⁺ T cells were found in the miR-15a/16-transgenic mice. Although the miR-15a/16 transgenic mice transplanted with B16BL6 or MC38 cells exhibited rapid growth, these tumor-bearing mice did not show changes in NK1.1^-CD4⁺NKG2D⁺ T cell distributions in either spleens or tumors. When NK1.1^-CD4⁺ T cells were stimulated by α-CD3/sRAE-1 ex vivo, the NKG2D expression was difficult to induce in the T cells of miR-15a/16-transgenic mice. Finally, increased frequencies of regulatory CD4⁺NKG2D⁺ T cells with low miR-16 levels were observed in patients with late-stage colorectal cancer (Duke’s C, D). Thus, miR-16 modulates NK1.1^-CD4⁺NKG2D⁺ T cell functions via targeting NKG2D. Low miR-16 expression in CD4⁺ T cells induces the regulatory CD4⁺NKG2D⁺ T subpopulation, which promotes tumor evasion via the secretion of immune-suppressive molecules.     

Reduced Expression of Natural Killer Cell-Related Activating Receptors by Peripheral Blood Mononuclear Cells from Patients with Breast Cancer and Their Improvement by Zoledronic Acid

Background/aim: Natural killer (NK) cell receptors affect the NK cell-mediated elimination of malignant cells. In this experimental study the effect of Zoledronic acid (ZOL) was investigated on the expression of NK activating- (NKP46 and NKG2D) and inhibitory (KIR2DL1) receptors by Phytohaemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMCs) from breast cancer (BC) patients. Materials and Methods: Peripheral blood mononuclear cell-extracted RNA from thirty breast cancer women and twenty-five healthy subjects was analyzed for gene expression of NKP46, NKG2D and KIR2DL1 using real time-PCR. Then, the PBMCs from BC patients were cultured in the presence of PHA with 5 μg/ml, 10 or 20 μg/ml of ZOL for 32 hours and expression of the aforementioned receptors was determined. Results: Expression of NKP46, NKG2D and NKP46/KIR2DL1 ratio in BC women were lower than healthy group (P<0.01, P<0.04 and P<0.05, respectively). NKP46 expression was up-regulated by PHA-stimulated PBMCs treated with 10 μg/ml and 20 μg/ml of ZOL compared with PHA-stimulated cultures (P<0.01 and P<0.05, respectively). NKG2D expression remarkably increased by PHA-stimulated cultures treated with 5 μg/ml, 10 μg/ml and 20 μg/ml of ZOL compared with PHA-stimulated cultures (P<0.05 and P<0.02 and P<0.04, respectively). Conclusion: Expression of NK cell-related activating receptors decreased in BC patients. ZOL can improve the expression of NK activating receptors.     

Proteolytic release of soluble UL16-binding protein 2 from tumor cells

The MHC class I-related ligands of the immunoreceptor NKG2D are frequently expressed by tumor cells and stimulate tumor immunity mediated by CD8 T cells and natural killer (NK) cells. In humans, NKG2D ligands (NKG2DL) are encoded by the MHC-encoded MIC and non-MHC-encoded UL16-binding protein (ULBP) families of proteins. Recently, we and others showed that tumor cells release soluble MICA (sMICA), thereby counteracting NKG2D-mediated tumor immunosurveillance. Here, we now report that ULBP2 molecules are likewise released from tumor cells in a processed soluble form, and that soluble ULBP2 (sULBP2) can be detected in sera of some patients with hematopoietic malignancies. Tumor cell-derived sULBP2 as opposed to cell-bound ULBP2 does not down-regulate NKG2D on NK cells. Unexpectedly, the glycosylphosphatidylinositol-anchored ULBP2 molecules are not released by phospholipases but by the action of metalloproteases. Proteolytic shedding of both NKG2D ligands MICA and ULBP2 by tumor cells was strongly enhanced after phorbol 12-myristate 13-acetate treatment and paralleled by a markedly reduced susceptibility to NKG2D-mediated cytotoxicity. Shedding of MICA and ULBP2 can be blocked by the same inhibitors, suggesting the involvement of related metalloproteases. Thus, our data suggest that reducing NKG2DL surface densities is due to a common cleavage process executed by metalloproteases that promotes escape of tumors from NKG2D-mediated immunosurveillance.     

Ataxia–telangiectasia mutated kinase‐mediated upregulation of NKG2D ligands on leukemia cells by resveratrol results in enhanced natural killer cell susceptibility

The powerful activating receptor NKG2D is expressed by natural killer (NK) cells and promotes cytotoxic lysis of cancer cells expressing NKG2D ligands (NKG2D‐Ls). We report the effective induction of NKG2D‐Ls, achieved with the naturally occurring polyphenol resveratrol, in a broad range of leukemia cells. In this study, resveratrol upregulated the NKG2D‐Ls MHC class I chain‐related proteins MICA and MICB, and UL16‐binding proteins ULBP1, ULBP2, and ULBP3 in most of the leukemia cells analyzed. Ligand upregulation induced by resveratrol was impaired by pharmacological and genetic disruption of ataxia–telangiectasia mutated kinase, the main regulator of NKG2D‐L expression. Leukemia cells treated with resveratrol were more susceptible to killing by NK cells than untreated cells, and the enhanced cytotoxicity of NK cells was blocked by treatment of NK cells with anti‐NKG2D mAbs. Interestingly, resveratrol consistently upregulated the NKG2D receptor expression and enhanced NKG2D‐mediated functions in resting NK cells obtained from healthy individuals. Therefore, resveratrol has attractive immunotherapeutic potential.     

The requirement for DNAM-1, NKG2D, and NKp46 in the natural killer cell-mediated killing of myeloma cells

Recent evidence suggests a role for natural killer (NK) cells in the control of multiple myeloma. We show that expression of the NK cell receptor DNAM-1 (CD226) is reduced on CD56(dim) NK cells from myeloma patients with active disease compared with patients in remission and healthy controls. This suggested that this receptor might play a role in NK-myeloma interactions. The DNAM-1 ligands Nectin-2 (CD112) and the poliovirus receptor (PVR; CD155) were expressed by most patient myeloma samples analyzed. NK killing of patient-derived myelomas expressing PVR and/or Nectin-2 was DNAM-1 dependent, revealing a functional role for DNAM-1 in myeloma cell killing. In myeloma cell lines, cell surface expression of PVR was associated with low levels of NKG2D ligands, whereas cells expressing high levels of NKG2D ligands did not express PVR protein or mRNA. Furthermore, NK cell-mediated killing of myeloma cell lines was dependent on either DNAM-1 or NKG2D but not both molecules. In contrast, the natural cytotoxicity receptor NKp46 was required for the killing of all myeloma cell lines analyzed. Thus, DNAM-1 is important in the NK cell-mediated killing of myeloma cells expressing the cognate ligands. The importance of NKp46, NKG2D, and DNAM-1 in myeloma killing mirrors the differential expression of NK cell ligands by myeloma cells, reflecting immune selection during myeloma disease progression.