Biomedical subjects

Alexander Steinle

Publications and source records attributed to Alexander Steinle.

At least 19 recordsLinked to original sources

PubMed2006

Mutual activation of natural killer cells and monocytes mediated by NKp80-AICL interaction.

Receptors encoded by the natural killer (NK) cell gene complex (such as NKG2D) govern the reactivity of NK cells. However, the function and ligand(s) of the NK cell gene complex-encoded human NK cell receptor NKp80 remain elusive. Here we demonstrate that NKp80 binds to the genetically linked 'orphan' receptor AICL, which, like NKp80, is absent from rodents. We defined AICL as a myeloid-specific activating receptor that is upregulated by Toll-like receptor stimulation. AICL-NKp80 interactions promoted NK cell-mediated cytolysis of malignant myeloid cells. In addition, during crosstalk between NK cells and monocytes, NKp80 stimulated the release of proinflammatory cytokines from both cell types. Thus, by specifically bridging NK cells and myeloid cells, NKp80-AICL interactions may contribute to the initiation and maintenance of immune responses at sites of inflammation.

Animals↗

PubMed2006

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.

Animals↗

PubMed2006

TGF-beta and metalloproteinases differentially suppress NKG2D ligand surface expression on malignant glioma cells.

NKG2D ligands (NKG2DL) are expressed by infected and transformed cells. They transmit danger signals to NKG2D-expressing immune cells, leading to lysis of NKG2DL-expressing cells. We here report that the NKG2DL MHC class I-chain-related molecules A and B (MICA/B) and UL16-binding proteins (ULBP) 1-3 are expressed in human brain tumours in vivo, while expression levels are low or undetectable in normal brain. MICA and ULBP2 expression decrease with increasing WHO grade of malignancy, while MICB and ULBP1 are expressed independently of tumour grade. We further delineate two independent mechanisms that can explain these expression patterns: (i) transforming growth factor-beta (TGF-beta) is upregulated during malignant progression and selectively downregulates MICA, ULBP2 and ULBP4 expression, while MICB, ULBP1 and ULBP3 are unaffected. (ii) Cleavage of MICA and ULBP2 is reduced by inhibition of metalloproteinases (MP), whereas no changes in the expression levels of other NKG2DL were detected. Consequently, NKG2DL-dependent NK cell-mediated lysis is enhanced by depletion of TGF-beta or inhibition of MP. Thus, escape from NKG2D-mediated immune surveillance of malignant gliomas in vivo may be promoted by the inhibition of MICA and ULBP2 expression via an autocrine TGF-beta loop and by MP-dependent shedding from the cell surface. Loss of MICA and ULBP2, in contrast to other NKG2DL, may be particularly important in glioma immune escape, and differential regulation of human NKG2DL expression is part of the immunosuppressive properties of human malignant glioma cells.

Brain Neoplasms↗

PubMed2006

Soluble MICB in malignant diseases: analysis of diagnostic significance and correlation with soluble MICA.

Expression of ligands of the immunoreceptor NKG2D such as MICA and MICB has been proposed to play an important role in the immunosurveillance of tumors. Proteolytic shedding of NKG2D ligands from cancer cells therefore constitutes an immune escape mechanism impairing anti-tumor reactivity by NKG2D-bearing cytotoxic lymphocytes. Serum levels of sMICA have been shown to be of diagnostic significance in malignant diseases of various origins. Here, we investigated the potential of soluble MICB, the sister molecule of MICA, as a marker in cancer and its correlation with soluble MICA. Analysis of MICB in sera of 512 individuals revealed slightly higher MICB levels in patients with various malignancies (N = 296; 95th percentile 216 pg/ml; P = 0.069) than in healthy individuals (N = 62; 95th percentile 51 pg/ml). Patients with benign diseases (N = 154; 95th percentile 198 pg/ml) exhibited intermediate MICB levels. In cancer patients, elevated MICB levels correlated significantly with cancer stage and metastasis (P = 0.007 and 0.007, respectively). Between MICB and MICA levels, only a weak correlation was found (r = 0.24). Combination of both markers resulted only in a slightly higher diagnostic power in the high specificity range. The reduction of MICA and MICB surface expression on cells by shedding and the effects of sMICA and sMICB in serum on host lymphocyte NKG2D expression might play a role in late stages of tumor progression by overcoming the confining effect of NK cells and CD8 T cells. While MICB levels are not suited for the diagnosis of cancer in early stages, they may provide additional information for the staging of cancer disease.

Biomarkers, Tumor↗

PubMed2006

BCR/ABL oncogene directly controls MHC class I chain-related molecule A expression in chronic myelogenous leukemia.

MHC class I chain-related molecules (MIC) participate in immune surveillance of cancer through engagement of the NKG2D-activating receptor on NK and T cells. Decreased NKG2D expression and function upon chronic exposure to NKG2D ligands and/or soluble forms of MIC (sMIC) may participate in immune escape. In chronic myeloid leukemia, a malignancy caused by the BCR/ABL fusion oncoprotein, we showed cell surface expression of MICA on leukemic, but not healthy, donor hemopoietic CD34+ cells. At diagnosis, chronic myeloid leukemia patients had abnormally high serum levels of sMICA and weak NKG2D expression on NK and CD8+ T cells, which were restored by imatinib mesylate (IM) therapy. In the BCR/ABL+ cell line K562, IM decreased both surface MICA/B expression and NKG2D-mediated lysis by NK cells. Silencing BCR/ABL gene expression directly evidenced its role in the control of MICA expression. IM did not affect MICA mRNA levels, but decreased MICA protein production and release. Sucrose density gradient fractionation of K562 cytoplasmic extracts treated with IM showed a shift in the distribution of MICA mRNA from the polysomal toward the monosomal fractions, consistent with decreased translation. Among the major pathways activated by BCR/ABL that regulate translation, PI3K and mammalian target of rapamycin were shown to control MICA expression. These data provide evidence for direct control of MICA expression by an oncogene in human malignancy and indicate that posttranscriptional mechanisms may participate in the regulation of MICA expression.

Adult↗

PubMed2006

Release of MICB molecules by tumor cells: mechanism and soluble MICB in sera of cancer patients.

MICA, a ligand of the activating immunoreceptor NKG2D, is released by tumor cells in a soluble form and can be detected in sera of tumor patients at significant levels. Soluble MICA has been proposed to counteract NKG2D-mediated immunosurveillance of tumors. Here, we report that MICB, the second member of the human MIC protein family, is likewise shed by metalloproteases from tumor cells and is present in sera of patients with gastrointestinal tumors. While cell-bound MICB causes downregulation of surface NKG2D, soluble MICB did not alter NKG2D expression on NK cells in vitro. Thus, proteolytic shedding of MICB by tumor cells may impair immunogenicity of tumors primarily by reducing NKG2D-ligand densities on malignant cells.

Cell Line, Tumor↗

PubMed2006

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.

Carrier Proteins↗

PubMed2006

Soluble MICA in malignant diseases.

The immunoreceptor NKG2D activates natural killer cells and costimulates CD8 T cells. The MHC class I-related MICA molecules are ligands of NKG2D and are expressed on malignant, but not on normal, cells. As NKG2D plays an important role in the immunosurveillance of tumors, studies suggest that release of MICA from cancer cells constitutes an immune escape mechanism that systemically impairs antitumor immunity. Here, we investigated the potential of soluble MICA (sMICA) as a marker in cancer. Analysis of sMICA in sera of 512 individuals revealed significantly (p < 0.0001) higher levels in patients with various malignancies (n = 296, median 161 pg/ml) than in healthy individuals (n = 62, median <30 pg/ml). Patients with benign diseases (n = 154, median 84 pg/ml) exhibited intermediate sMICA levels. In cancer patients, elevated sMICA levels correlated significantly with cancer stage and metastasis (p = 0.015 and p = 0.007, respectively). While release of MICA is thought to impair tumor immunity, determination of sMICA levels may provide useful additional information in the diagnosis and staging of cancer.

Biomarkers, Tumor↗

PubMed2006

Natural killer cell-mediated rejection of experimental human lung cancer by genetic overexpression of major histocompatibility complex class I chain-related gene A.

Studies have highlighted molecular and cell biological mechanisms of the NKG2D-NKG2D ligand system in the activation of natural killer (NK) cell and T cell functions. In this study we explore the potential of genetic overexpression of human major histocompatibility complex class I chain-related gene A (MICA), a powerful NKG2D ligand, for the induction of NK cell-mediated antitumor immunity in a humanized murine model of non-small cell lung cancer. Using tissue microarray technology we detected expression of MICA in only 30% of the samples from patients with lung cancer. Staining was always weak and focal, indicating that expression of MICA is detectable but limited in human lung cancer. Genetic overexpression of MICA by means of adenoviral transduction or transfection of expression plasmids was feasible in cell lines in vitro, primary human cancer tissue ex vivo, and in experimental human cancers in vivo. The presence of MICA on the surface of largely NK cell-resistant human lung cancer cells reestablished NK cell susceptibility and provoked NK cell-mediated antitumor immunity by murine and human NK cells in two different experimental therapy models. In this study we analyze the interaction of human NK cells with MICA-positive human cancer cells in an in vivo setting. Our data demonstrate that MICA overexpression can function as NK cell-mediated immunotherapy in experimental lung cancer.

Animals↗

PubMed2005

Expression of toll-like receptors by human muscle cells in vitro and in vivo: TLR3 is highly expressed in inflammatory and HIV myopathies, mediates IL-8 release and up-regulation of NKG2D-ligands.

The particular microenvironment of the skeletal muscle can be the site of complex immune reactions. Toll-like receptors (TLRs) mediate inflammatory stimuli from pathogens and endogenous danger signals and link the innate and adaptive immune system. We investigated innate immune responses in human muscle. Analyzing TLR1-9 mRNA in cultured myoblasts and rhabdomyosarcoma cells, we found constitutive expression of TLR3. The TLR3 ligand Poly (I:C), a synthetic analog of dsRNA, and IFN-gamma increased TLR3 levels. TLR3 was mainly localized intracellularly and regulated at the protein level. Poly (I:C) challenge 1) activated nuclear factor-kappaB (NF-kappaB), 2) increased IL-8 release, and 3) up-regulated NKG2D ligands and NK-cell-mediated lysis of muscle cells. We examined muscle biopsy specimens of 6 HIV patients with inclusion body myositis/polymyositis (IBM/PM), 7 cases of sporadic IBM and 9 nonmyopathic controls for TLR3 expression. TLR3 mRNA levels were elevated in biopsy specimens from patients with IBM and HIV-myopathies. Muscle fibers in inflammatory myopathies expressed TLR3 in close proximity of infiltrating mononuclear cells. Taken together, our study suggests an important role of TLR3 in the immunobiology of muscle, and has substantial implications for the understanding of the pathogenesis of inflammatory myopathies or therapeutic interventions like vaccinations or gene transfer.

Cell Line↗

PubMed2005

Activation of V gamma 9V delta 2 T cells by NKG2D.

Human Vgamma9 Vdelta2 T cells recognize phosphorylated nonpeptide Ags (so called phosphoantigens), certain tumor cells, and cells treated with aminobisphosphonates. NKG2D, an activating receptor for NK cells, has been described as a potent costimulatory receptor in the Ag-specific activation of gammadelta and CD8 T cells. This study provides evidence that Vgamma9 Vdelta2 T cells may also be directly activated by NKG2D. Culture of PBMC with immobilized NKG2D-specific mAb or NKG2D ligand MHC class I related protein A (MICA) induces the up-regulation of CD69 and CD25 in NK and Vgamma9 Vdelta2 but not in CD8 T cells. Furthermore, NKG2D triggers the production of TNF-alpha but not of IFN-gamma, as well as the release of cytolytic granules by Vgamma9 Vdelta2 T cells. Purified Vgamma9 Vdelta2 T cells kill MICA-transfected RMA mouse cells but not control cells. Finally, DAP10, which mediates NKG2D signaling in human NK cells, was detected in resting and activated Vgamma9 Vdelta2 T cells. These remarkable similarities in NKG2D function in NK and Vgamma9 Vdelta2 T cells may open new perspectives for Vgamma9 Vdelta2 T cell-based immunotherapy, e.g., by Ag-independent killing of NKG2D ligand-expressing tumors.

Animals↗

PubMed2005

Natural killer cell-mediated lysis of hepatoma cells via specific induction of NKG2D ligands by the histone deacetylase inhibitor sodium valproate.

Natural killer (NK) cells as components of the innate immunity substantially contribute to antitumor immune responses. However, the tumor-associated ligands engaging activating NK cell receptors are largely unknown. An exception are the MHC class I chain-related molecules MICA and MICB and the UL16-binding proteins (ULBP) which bind to the activating immunoreceptor NKG2D expressed on cytotoxic lymphocytes. A therapeutic induction of NKG2D ligands that primes cancer cells for NK cell lysis has not yet been achieved. By microarray studies, we found evidence that treatment of human hepatocellular carcinoma cells with the histone deacetylase inhibitor (HDAC-I) sodium valproate (VPA) mediates recognition of cancer cells by cytotoxic lymphocytes via NKG2D. VPA induced transcription of MICA and MICB in hepatocellular carcinoma cells, leading to increased cell surface, soluble and total MIC protein expression. No significant changes in the expression of the NKG2D ligands ULBP1-3 were observed. The induction of MIC molecules increased lysis of hepatocellular carcinoma cells by NK cells which was abolished by addition of a blocking NKG2D antibody. Importantly, in primary human hepatocytes, VPA treatment did not induce MIC protein expression. Taken together, our data show that the HDAC-I VPA mediates specific priming of malignant cells for innate immune effector mechanisms. These results suggest the clinical evaluation of HDAC-I in solid tumors such as hepatocellular carcinoma, especially in combination with immunotherapy approaches employing adoptive NK cell transfer.

Carcinoma, Hepatocellular↗

PubMed2005

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.

Animals↗

PubMed2004

Vdelta1 T lymphocytes from B-CLL patients recognize ULBP3 expressed on leukemic B cells and up-regulated by trans-retinoic acid.

We analyzed 38 untreated patients with chronic lymphocytic leukemia of B-cell type (B-CLL): 24 low-, 8 intermediate-, and 6 high-risk stage. In 15 patients (13 low risk and 2 intermediate risk), circulating Vdelta1 T lymphocytes were significantly increased (100 to 300 cells/muL) compared with most intermediate, all high-risk stage, and 15 healthy donors (50 to 100 cells/muL). We studied these Vdelta1 T lymphocytes and observed that they proliferated in vitro and produced tumor necrosis factor alpha or IFN-gamma in response to autologous leukemic B cells but not to normal lymphocytes. However, they were unable to kill resting autologous B cells, which lack the MHC-related MIC-A antigen and express low levels of the UL16-binding protein (ULBP) 3 and undetectable levels of ULBP1, ULBP2, and ULBP4. All these molecules are reported ligands for the NKG2D receptor, which is expressed by gammadelta T cells and activates their cytolytic function. The Vdelta1 T lymphocytes studied were able to lyse the ULBP3(+) C1R B-cell line upon transfection with MIC-A. More importantly, they also lysed autologous B-CLL cells when transcription and expression of MIC-A or up-regulation of ULBP3 were achieved either by activation or by exposure to trans-retinoic acid. The NKG2D receptor expressed on Vdelta1 T cells was involved in the recognition of B-CLL. Finally, in six patients with low numbers of circulating Vdelta1 T cells and undetectable ULBP3, the disease progressed over 1 year, whereas no progression occurred in patients with high Vdelta1 T lymphocytes and detectable/inducible ULBP3. These data suggest that Vdelta1 T lymphocytes may play a role in limiting the progression of B-CLL.

Aged↗

PubMed2004

RNA interference targeting transforming growth factor-beta enhances NKG2D-mediated antiglioma immune response, inhibits glioma cell migration and invasiveness, and abrogates tumorigenicity in vivo.

Transforming growth factor (TGF)-beta is the key molecule implicated in impaired immune function in human patients with malignant gliomas. Here we report that patients with glioblastoma, the most common and lethal type of human glioma, show decreased expression of the activating immunoreceptor NKG2D in CD8(+) T and natural killer (NK) cells. TGF-beta is responsible for the down-regulation of NKG2D expression in CD8(+) T and NK cells mediated by serum and cerebrospinal fluid of glioma patients in vitro. Moreover, TGF-beta inhibits the transcription of the NKG2D ligand MICA. Interference with the synthesis of TGF-beta1 and TGF-beta2 by small interfering RNA technology prevents the down-regulation of NKG2D on immune cells mediated by LNT-229 glioma cell supernatant and strongly enhances MICA expression in the glioma cells and promotes their recognition and lysis by CD8(+) T and NK cells. Furthermore, TGF-beta silencing results in a less migratory and invasive glioma cell phenotype in vitro. LNT-229 glioma cells deficient in TGF-beta exhibit a loss of subcutaneous and orthotopic tumorigenicity in nude mice, and NK cells isolated from these mice show an activated phenotype. RNA interference targeting TGF-beta1,2 results in a glioma cell phenotype that is more sensitive to immune cell lysis and less motile in vitro and nontumorigenic in nude mice, strongly confirming TGF-beta antagonism as a major therapeutic strategy for the future treatment of malignant gliomas.

Aged↗

PubMed2004

Prevalent expression of the immunostimulatory MHC class I chain-related molecule is counteracted by shedding in prostate cancer.

The MHC class I chain-related molecules (MICs) have previously been shown to be induced on most epithelial tumor cells. Engagement of MIC by the activating immune receptor NKG2D triggers NK cells and augments antigen-specific CTL anti-tumor immunity. The MIC-NKG2D system was proposed to participate in epithelial tumor immune surveillance. Paradoxically, studies suggest that tumors may evade MIC-NKG2D-mediated immunity by MIC shedding-induced impairment of effector cell function. Here we demonstrate the first evidence to our knowledge of a significant correlation of MIC shedding and deficiency in NK cell function with the grade of disease in prostate cancer. MIC is widely expressed in prostate carcinoma. The presence of surface target MIC, however, is counteracted by shedding. A significant increase in serum levels of soluble MIC (sMIC) and deficiency in NK cell function was shown in patients with advanced cancer. Finally, the deficiency in NK cell function can be overcome by treatment with IL-2 or IL-15 in vitro. Our results suggest that (a) deficiency in MIC-NKG2D immune surveillance may contribute to prostate cancer progression, (b) sMIC may be a novel biomarker for prostate cancer, and (c) using cytokines to restore MIC-NKG2D-mediated immunity may have clinical significance for prostate cancer in cell-based adaptive immunotherapy.

Aged↗

PubMed2004

Novel APC-like properties of human NK cells directly regulate T cell activation.

Initiation of the adaptive immune response is dependent on the priming of naive T cells by APCs. Proteomic analysis of unactivated and activated human NK cell membrane-enriched fractions demonstrated that activated NK cells can efficiently stimulate T cells, since they upregulate MHC class II molecules and multiple ligands for TCR costimulatory molecules. Furthermore, by manipulating antigen administration, we show that NK cells possess multiple independent unique pathways for antigen uptake. These results highlight NK cell-mediated cytotoxicity and specific ligand recognition by cell surface-activating receptors on NK cells as unique mechanisms for antigen capturing and presentation. In addition, we analyzed the T cell-activating potential of human NK cells derived from different clinical conditions, such as inflamed tonsils and noninfected and CMV-infected uterine decidual samples, and from transporter-associated processing antigen 2-deficient patients. This in vivo analysis revealed that proinflammatory, but not immune-suppressive, microenvironmental requirements can selectively dictate upregulation of T cell-activating molecules on NK cells. Taken together, these observations offer new and unexpected insights into the direct interactions between NK and T cells and suggest novel APC-like activating functions for human NK cells.

Antigen-Presenting Cells↗

PubMed2003

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.

Animals↗