Biomedical subjects

Barbara Seliger

Publications and source records attributed to Barbara Seliger.

At least 37 records · Page 2Linked to original sources

PubMed2004

CD4+ T cell-mediated HER-2/neu-specific tumor rejection in the absence of B cells.

HER-2/neu (HER-2) is a cell surface proto-oncogene that is often overexpressed in carcinomas. Passive administration of anti-HER-2 antibodies in breast cancer patients has achieved promising results, but less is known about the role of antibodies in active immunization. We asked whether B cells/antibodies are needed for tumor immunity induced by plasmid (HER-2 and GM-CSF) immunization. HER-2 specific tumor immunity relied completely on both CD4+ and CD8+ T cells. IFN-gamma, and to a lesser extent IL-4, seemed to be crucial cytokines during tumor rejection. Protection was associated with production of anti-HER-2 IgG antibodies in B cell competent mice. After immunization, however, B cell-deficient mice rejected HER-2-expressing tumors as efficiently as control littermates. We conclude that T cells are the main effector cells in DNA vaccine induced immunity against HER-2 and that anti HER-2 antibodies are not necessary to elicit a protective anti tumor immune response in this model.

Animals↗

PubMed2004

MHC class I antigen processing pathway defects, ras mutations and disease stage in colorectal carcinoma.

Colorectal tumorigenesis has been associated with the progressive acquisition of a variety of genetic alterations. These include mutations of the Ki-ras proto-oncogene in codons 12 and 13, which account for 85% of genetic changes in colorectal cancer. In murine in vitro models of oncogenic transformation, an association between ras-mediated transformation and downregulation of different components of the MHC class I antigen processing machinery (APM) has been described. In order to investigate whether this association also exists in human tumors, 10 cases of high-grade intraepithelial neoplasia (HIN), as well as primary tumors and autologous lymph node metastases from 42 patients with colorectal carcinoma, were monitored by allele-specific restriction analysis for Ki-ras mutations. In parallel, APM component expression and tumor cell proliferation were analyzed by immunohistochemistry. In comparison to autologous colorectal mucosa, TAP1, LMP2 and tapasin loss was found in 68%, 67% and 80% of HIN, respectively. In contrast, impaired TAP1, LMP2 and tapasin expression was found in 42%, 42% and 63% of primary adenocarcinomas of stage III disease and in 63%, 47% and 79% of the matched lymph node metastases, respectively. More than 60% of colorectal tumor lesions with TAP1, LMP2 and/or tapasin defects displayed Ki-ras mutations. The frequency of TAP1, LMP2 and tapasin loss varied between 33% of primary adenocarcinomas, 40% of HIN to approximately 67% of metastases. These data suggest that i) APM component deficiencies occur more frequently in Ki-ras-mutated colorectal carcinoma lesions and ii) APM abnormalities in conjunction with Ki-ras mutations appear to be associated with disease stage. These findings support the hypothesis that Ki-ras mutations may contribute to immune escape mechanisms of tumors by downregulating the MHC class I APM component expression.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2004

Small interfering RNA (siRNA) inhibits the expression of the Her2/neu gene, upregulates HLA class I and induces apoptosis of Her2/neu positive tumor cell lines.

Silencing of a specific mRNA using double stranded RNA oligonucleotides represents one of the newest technologies for suppressing a specific gene product. Small interfering RNA (siRNA) are 21 nucleotides long, double stranded RNA fragments that are identical in sequence to the target mRNA. We designed 3 such siRNA against the Her2/neu (HER2) gene. The HER2 gene is known to play an important role in the oncogenesis of several types of cancers, such as breast, ovarian, colon and gastric cancers. Introduction of the siRNA into HER2 positive tumor lines in vitro greatly reduced the cell surface expression of the HER2 protein. Concurrently, a range of effects on cell physiology, such as growth inhibition or apoptosis, was observed. The expression of HLA class I was observed to be upregulated when HER2 was silenced with siRNA. Treatment of SKBr3 and MCF7/HER2 tumor cell lines with the HER2 siRNA resulted in growth arrest of cells in the late G(1)/S-phase. Our results suggest that siRNA may be an effective method of abrogating the effect of HER2 in tumorigenesis.

Antibodies, Monoclonal↗

PubMed2004

HER-2/neu-mediated regulation of components of the MHC class I antigen-processing pathway.

Because of its amplification and/or overexpression in many human tumors, the HER-2/neu proto-oncogene represents an attractive target for T-cell-mediated vaccination strategies. However, overexpression of oncogenes is often associated with defective expression of components of the MHC class I antigen-processing machinery (APM), thereby resulting in an immune escape phenotype of oncogene-transformed cells. To determine whether HER-2/neu influences the MHC class I antigen-processing pathway, the expression pattern of different APM components was examined in murine in vitro models of constitutive and tetracycline-controlled HER-2/neu expression. In comparison with HER-2/neu(-) control cells, HER-2/neu(+) fibroblasts exhibit reduced levels of MHC class I surface antigens that were associated with impaired expression and/or function of the peptide transporter associated with antigen processing, the proteasome subunits low molecular weight protein 2 and low molecular weight protein 10, the proteasome activators PA28alpha and PA28beta, and tapasin. These APM abnormalities resulted in reduced sensitivity to lysis by CTLs. The HER-2/neu-mediated immune escape phenotype could be corrected by IFN-gamma treatment. The clinical relevance of this finding was supported by an inverse correlation between HER-2/neu and the peptide transporter associated with antigen-processing protein expression as determined by immunhistochemical analysis of a series of HER-2/neu(-) and HER-2/neu(+) breast cancer specimens. Thus, a functional link between deficient APM component expression and HER-2/neu overexpression is proposed that might influence the design of HER-2/neu-targeted T-cell-based immunotherapeutic strategies.

3T3 Cells↗

PubMed2004

Down-regulation of HLA class I antigen processing molecules: an immune escape mechanism of renal cell carcinoma?

PURPOSE: Proper HLA class I antigen processing and presentation is a prerequisite for the recognition of tumor cells by cytotoxic T lymphocytes. To date there exist only limited information on the expression of components of the HLA class I associated antigen processing machinery (APM) in surgically removed benign renal cell adenoma, and primary and metastatic renal cell carcinoma (RCC). MATERIALS AND METHODS: A total of 133 primary RCCs of different subtypes, 10 renal cell adenoma biopsies, 32 matched metastases of different localization and autologous normal kidney epithelium were immunohistochemically analyzed for the expression of HLA class I antigens, low molecular weight protein (LMP)2 and LMP7, the transporter associated with antigen processing subunit (TAP1) and beta 2-microglobulin (beta 2-m). RESULTS: Normal kidney tissue showed strong cytoplasmic staining intensity for LMP2, LMP7 and TAP1, whereas beta 2-m and HLA class I heavy chains were detected on the cell surface. A low frequency of HLA class I HC and beta 2-m down-regulation was found in RCC. In contrast, primary RCC and metastases showed a high frequency of a total lack of heterogeneous TAP1, LMP2 and LMP7 expression, which was often coordinately regulated. APM component deficiencies were associated with RCC subtypes but not with tumor grading and staging. CONCLUSIONS: HLA class I APM component abnormalities appear to represent an immune escape mechanism of RCC. This finding emphasizes the need to evaluate the integrity and expression of these molecules in patients with RCC, especially in those selected for treatment with T-cell based immunotherapy.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2004

Regulation of beta1 integrin expression by PKCepsilon in renal cancer cells.

Polarized cell movement represents an essential prerequisite for the progression and metastasis of malignant diseases. Protein kinase C (PKC) which physically associates with integrins has been implicated in the promotion of a migratory cell phenotype. In order to identify a direct link between PKC and integrins in renal cell carcinoma (RCC) the influence of PKC isoforms on integrin expression and possible consequences on proliferation and cell migration was analyzed in RCC cells. The constitutive expression of the PKC isoforms alpha, betaI, betaII, gamma, delta, epsilon, eta, theta, xi, lambda and micro was determined in the RCC cell line CCF-RC1. In addition, the influence of PKC inhibitors RO31-8220, GF109203X and GO6976 on the beta1, beta2 and beta3 integrin expression and cell proliferation of RCC cells was investigated by flow cytometry and by BrdU incorporation, respectively. Furthermore, the motility of CCF-RC1 cells was assessed through chamber chemotaxis analysis. All PKC isoforms tested were expressed in CCF-RC1 cells with the exception of PKClambda and theta. The PKC inhibitor RO31-8220 reduced beta1 integrin expression by 92% and inhibited proliferation by 42% of untreated cells, whereas cell migration remained uninfluenced by RO31-8220. GF109203X and GO6976 reduced beta1 integrin expression to approximately 50% of untreated cells. In contrast, beta2 and beta3 integrins were only weakly affected by RO31-8220, GF109203X and GO6976 treatment. The most significant influence on beta1 integrin expression was obtained by the PKC inhibitor RO31-8220. This leads to the assumption that PKCepsilon is involved in the regulation of beta1 integrin expression. Downregulation of beta1 integrins by RO31-8220 was associated with reduced proliferation, but did not influence migration. These findings provide a conceptual basis for treatment of renal cell carcinoma by interfering with tumor cell proliferation.

CD18 Antigens↗

PubMed2003

Switching off HER-2/neu in a tetracycline-controlled mouse tumor model leads to apoptosis and tumor-size-dependent remission.

Overexpression of the receptor tyrosine kinase HER-2/neu is associated with poor prognosis in patients with breast and ovarian cancer. Recent excitement has surrounded the therapeutic effects of HER-2-blocking therapy strategies and has rekindled interest on the molecular mechanisms of HER-2/neu in tumor biology. To study the role of HER-2/neu overexpression in vivo, we used a murine fibroblast cell line (NIH3T3-her2) conditionally expressing human HER-2/neu under control of a tetracycline-responsive promoter. Expression of HER-2 could be down-regulated below detection limit (>625-fold dilution) by exposure of NIH3T3-her2 cells to anhydrotetracycline (ATc). Subcutaneous injection of NIH3T3-her2 cells into nude mice resulted in rapid tumor growth. Mice with mean tumor volumes of 0.2, 0.8, 1.9, and 14.9 cm(3) were treated daily with 10 mg/kg ATc to switch off HER-2/neu expression, producing reductions in tumor size of 100, 98.1, 81.4, and 74.2%, respectively, by 7 days after onset of ATc administration (P = 0.005, Kruskal-Wallis test). Different long-term effects of HER-2 down-regulation were observed when mice with small (0.2 cm(3); n = 7), intermediate (0.8-1.2 cm(3); n = 10) and large (> or =1.9 cm(3); n = 11) tumors received ATc for up to 40 days. Complete remission was observed for 100, 40, and 18% of the small-, intermediate-, and large-sized tumors, respectively (P = 0.003). However, after 20-45 days of ATc administration, recurrent tumor growth was observed for all mice, even in those with previous complete remissions. The time periods for which mean tumor volume could be suppressed to volumes <0.1 cm(3) under ATc administration were 34, 22, 8, and 0 days for tumors with initial volumes of 0.2, 0.8, 1.9 and 14.9 cm(3), respectively (P = 0.005, Kruskal-Wallis test). Interestingly, HER-2 remained below the detection limit in recurrent tumor tissue, suggesting that initially HER-2-dependent tumors switched to HER-2 independence. The "second hits" leading to HER-2-independent tumor growth have not yet been identified. The rapid regression of tumors after down-regulation of HER-2 was explained by two independent mechanisms: (a) a block in cell cycle progression, as evidenced by a decrease in Ki-67 antigen expression from 40% before ATc treatment to 8.3% after 7 days of ATc treatment; and (b) induction of apoptosis as demonstrated by caspase-3 activation and by the terminal deoxynucleotidyltransferase (Tdt)-mediated nick end labeling assay (TUNEL). In conclusion, we have shown that switching off HER-2 may disturb the sensitive balance between cell proliferation and cell death, leading to apoptosis and tumor remission. Tumor remission was dependent on the volume of the tumors before down-regulation of HER-2/neu.

Animals↗

PubMed2003

Cloning and functional analyses of the mouse tapasin promoter.

The expression of tapasin is critical for an optimized MHC class I assembly and stable MHC class I surface expression. Thus, impaired MHC class I antigen expression of tumors can be attributable to tapasin downregulation. In order to understand the molecular mechanisms of deficient tapasin expression, the mouse tapasin promoter region and its 5'-flanking sequences were characterized. The mouse tapasin promoter lacks the TATA box and its transcription is initiated at multiple sites within a 51-nucleotide stretch. Sequence analyses revealed transcription factor binding motifs for NF-kappaB, GATA, E2F, p300, AP1, SP1 and IRF-1/2. Detailed analysis of deletion mutants and elimination of transcription factor binding motifs demonstrated an important role of NF-kappaB at position -468 for its basal activity, whereas E2F at position -229 represses constitutive promoter activity. Furthermore, the IRF-1/2 binding site is required for gamma interferon inducibility of the tapasin promoter in vitro, but also negatively interferes with its constitutive activity. Thus, characterization of the tapasin promoter represents the molecular basis for the understanding of the heterogeneous expression levels of tapasin under physiological and pathophysiological conditions.

Animals↗

PubMed2003

A critical requirement of interferon gamma-mediated angiostasis for tumor rejection by CD8+ T cells.

It is thought that tumor rejection by CD8(+) T-cell effectors is primarily mediated by direct killing. We show that rejection of different tumors (fibrosarcoma, ras-transformed fibroblasts, colon carcinoma, and plasmacytoma) by CD8(+) T cells is always preceded by inhibition of tumor-induced angiogenesis. Angiostasis and tumor rejection were observed in perforin but not in IFN-gamma-deficient mice. Furthermore, adoptive transfer of tumor-specific CD8(+) T cells from IFN-gamma-competent mice inhibited angiogenesis of lung metastases in comparison to those from IFN-gamma gene-deficient mice. Taken together with our previous findings, we conclude that IFN-gamma-dependent antiangiogenesis is a general mechanism involved in tumor rejection by CD4(+) and CD8(+) T-cell effectors.

3T3 Cells↗

PubMed2003

Functional role of human leukocyte antigen-G up-regulation in renal cell carcinoma.

The nonclassical HLA-G molecule exhibits a limited tissue distribution and exerts multiple immune regulatory functions. Recent studies indicate that HLA-G expression plays a key role in the induction of immune tolerance and may represent a novel immune escape mechanism of tumor cells. Despite a high frequency of tumor-infiltrating T lymphocytes in renal cell carcinoma (RCC) lesions, outgrowth of tumor cells occurs that might be attributable to abrogation-efficient antitumor responses. To delineate the potential role of HLA-G in RCC immunology, the HLA-G expression pattern and its functional consequences on immune responses were analyzed in cell lines and lesions derived from primary RCC lesions. A heterogeneous constitutive and IFN-gamma-inducible HLA-G mRNA and protein expression was found in 12.5% of RCC cell lines but not in autologous normal kidney cells. Western blot analysis of 37 primary RCC lesions revealed HLA-G protein expression in 27% of RCC lesions. Functional studies performed with alloreactive natural and lymphokine-activated killer cells as well as antigen-specific CD8(+) T-cell populations demonstrated that HLA-G expression inhibits lysis of RCC cells by these different immune effector cells, whereas HLA-G(-) normal kidney cells were recognized. Furthermore, the HLA-G-mediated counteraction of immune response could be restored by antibody blocking experiments. Thus, aberrant HLA-G expression is found at a relatively high frequency in RCC and might participate in evasion of these tumor cells from immunosurveillance.

Adult↗

PubMed2003

Identification of metabolic enzymes in renal cell carcinoma utilizing PROTEOMEX analyses.

PROTEOMEX, an approach which combines conventional proteome analysis with serological screening, is a powerful tool to separate proteins and identify immunogenic components in malignant diseases. By applying this approach, we characterized nine metabolic enzymes which were differentially expressed in renal cell carcinoma (RCC) cell lines and compared their expression profiles to that of normal kidney epithelium cells. Four of these proteins, superoxide dismutase (SODC), triosephosphatase isomerase (TPIS), thioredoxin (THIO) and ubiquitin carboxyl-terminal hydrolase (UBL1) were further analysed for both their constitutive and interferon (IFN)-gamma inducible protein expression pattern in cell lines or tissue specimens derived from RCC or normal kidney epithelium using Western blot analysis and immunohistochemistry, respectively. With the exception of the RCC cell line MZ1940RC, which completely lacks the expression of UBL1, a heterogeneous and variable expression pattern of the different metabolic enzymes was detected in RCC and normal renal epithelium. The highest differences in the expression levels were found for THIO in the RCC cell lines, which was 2-fold upregulated when compared to autologous normal kidney epithelium. Moreover, IFN-gamma treatment did not influence the constitutive expression of these metabolic enzymes. Thus, PROTEOMEX represents a valuable approach for the identification of metabolic enzymes which might be used as markers for the diagnosis of RCC.

Carcinoma, Renal Cell↗

PubMed2003

Impaired transporter associated with antigen processing (TAP) function attributable to a single amino acid alteration in the peptide TAP subunit TAP1.

The heterodimeric peptide transporter TAP belongs to the ABC transporter family. Sequence comparisons with the P-glycoprotein and cystic fibrosis transmembrane conductance regulator and the functional properties of selective amino acids in these ABC transporters postulated that the glutamic acid at position 263 and the phenylalanine at position 265 of the TAP1 subunit could affect peptide transporter function. To define the role of both amino acids, TAP1 mutants containing a deletion or a substitution to alanine at position 263 or 265 were generated and stably expressed in murine and human TAP1(-/-) cells. The different TAP1 mutants were characterized in terms of expression and function of TAP, MHC class I surface expression, immune recognition, and species-specific differences. The phenotype of murine and human cells expressing human TAP1 mutants with a deletion or substitution of Glu(263) was comparable to that of TAP1(-/-) cells. In contrast, murine and human TAP1 mutant cells containing a deletion or mutation of Phe(265) of the TAP1 subunit exhibit wild-type TAP function. This was associated with high levels of MHC class I surface expression and recognition by specific CTL, which was comparable to that of wild-type TAP1-transfected control cells. Thus, biochemical and functional evidence is presented that the Glu(263) of the TAP1 protein, but not the Phe(265), is critical for proper TAP function.

3T3 Cells↗

PubMed2003

Functional cysteine-less subunits of the transporter associated with antigen processing (TAP1 and TAP2) by de novo gene assembly.

Within the adaptive immune system the transporter associated with antigen processing (TAP) plays a pivotal role in loading of peptides onto major histocompatibility (MHC) class I molecules. As a central tool to investigate the structure and function of the TAP complex, we created cysteine-less human TAP subunits by de novo gene synthesis, replacing all 19 cysteines in TAP1 and TAP2. After expression in TAP-deficient human fibroblasts, cysteine-less TAP1 and TAP2 are functional with respect to adenosine triphosphate (ATP)-dependent peptide transport and inhibition by ICP47 from herpes simplex virus. Cysteine-less TAP1 and TAP2 restore maturation and intracellular trafficking of MHC class I molecules to the cell surface.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2003

Identification of markers for the selection of patients undergoing renal cell carcinoma-specific immunotherapy.

Renal cell carcinoma (RCC) represents the most common malignant tumor in the kidney and is resistant to conventional therapies. The diagnosis of RCC is often delayed leading to progression and metastatic spread of the disease. Thus, validated markers for the early detection of the disease as well as selection of patients undergoing specific therapy is urgently needed. Using treatment with the monoclonal antibody (mAb) G250 as a model, proteome-based strategies were implemented for the identification of markers which may allow the discrimination between responders and nonresponders prior to application of G250-mediated immunotherapy. Flow cytometry revealed G250 surface expression in approximately 40% of RCC cell lines, but not in the normal kidney epithelium cell lines. G250 expression levels significantly varied thereby distinguishing between low, medium and high G250 expressing cell lines. Comparisons of two-dimensional gel electrophoresis expression profiles of untreated RCC cell lines versus RCC cell lines treated with a mAb directed against G250 and the characterization of differentially expressed proteins by mass spectrometry and/or Edman sequencing led to the identification of proteins such as chaperones, antigen processing components, transporters, metabolic enzymes, cytoskeletal proteins and unknown proteins. Moreover, some of these differentially expressed proteins matched with immunoreactive proteins previously identified by proteome analysis combined with immunoblotting using sera from healthy donors and RCC patients, a technique called PROTEOMEX. Immunohistochemical analysis of a panel of surgically removed RCC lesions and corresponding normal kidney epithelium confirmed the heterogeneous expression pattern found by proteome-based technologies. In conclusion, conventional proteome analysis as well as PROTEOMEX could be successfully employed for the identification of markers which may allow the selection of patients prior to specific immunotherapy.

Antibodies, Monoclonal↗

PubMed2003

Altered expression of nonclassical HLA class Ib antigens in human renal cell carcinoma and its association with impaired immune response.

An optimal antitumoral immune response requires the activation of both CD8(+) and CD4(+) T lymphocytes by the peptide antigen presentation via the human leukocyte antigen (HLA) class I and class II molecules, respectively. Downregulation or loss of HLA molecules has been found in human renal cell carcinoma (RCC) and provides a strategy of these tumors to evade T-cell mediated immunosurveillance. In addition, a tumor-specific upregulation of HLA-G has been recently described in RCC, which also leads to an impaired immune response. We here summarize the frequency of the constitutive and/or interferon-gamma (IFN-gamma) inducible expression of nonclassical HLA class Ib antigens in RCC cell lines, surgically removed RCC lesions and normal kidney epithelium, the molecular characteristics of HLA-G expression, and its role in immune recognition.

Antibodies, Monoclonal↗

PubMed2003

The role of HLA-G for protection of human renal cell-carcinoma cells from immune-mediated lysis: implications for immunotherapies.

HLA-G as a non-classical MHC class I molecule exhibits a limited tissue distribution and exerts multiple immune regulatory functions including the induction of immune tolerance. In addition, HLA-G has been detected in some tumors of different histology and therefore may represent a novel immune escape mechanism of tumor cells. Despite the immunogenicity of renal cell carcinoma (RCC), outgrowth of tumor cells occurs which might be attributable to abrogation of efficient anti-tumor responses. We here review the potential role of HLA-G in RCC immunology, the HLA-G expression pattern and its functional consequences on immune responses. A heterogenous constitutive and interferon-gamma inducible HLA-G mRNA and protein expression was found in RCC cell lines and tumor lesions, but not in autologous normal kidney epithelium. HLA-G transcription and protein expression was detected at a high frequency in primary RCC lesions and RCC cell lines. Functional studies performed with alloreactive natural and lymphokine activated killer cells as well as antigen-specific CD8+ T cell populations demonstrated that HLA-G expression inhibits lysis of RCC cells by these different immune effector cells, whereas HLA-G- normal kidney cells were recognized. Thus, aberrant HLA-G expression might participate in evasion of these tumor cells from immunosurveillance.

Carcinoma, Renal Cell↗

PubMed2003

HLA-G and MIC expression in tumors and their role in anti-tumor immunity.

Non-classical MHC class Ib molecules have attracted growing interest in recent years, especially because they interact with non-T-cell inhibitory or triggering receptors expressed on natural killer (NK) and T cells, suggesting that they have a role in immune recognition. Abnormalities in MHC class Ib expression are frequently found in human tumors of various histologies and might be associated with poor clinical outcome despite the local accumulation of immune competent cells. Available data suggest that the balance between activating and suppressing signals significantly influences the efficacy of the immune response and consequently of tumor progression.

Female↗

PubMed2003

Tumors as elusive targets of T-cell-based active immunotherapy.

The understanding of tumor-host interactions remains elusive despite significant progress in the identification of tumor antigens (TAs) recognized by autologous T cells. In particular, most human tumors do not regress and continue to grow in spite of spontaneous or immunization-induced immune responses demonstrated in circulating lymphocytes. Indeed, systemic immune responses might insufficiently address the complexity of tumor-host interactions because of factors, such as (1) the lack of productive T-cell receptor (TCR) engagement with epitope owing to qualitative and/or quantitative defects in the generation and maintenance of the immune response, (2) insufficient costimulation provided by the host, (3) the lack of localization of the immune response in target tissues and (4) the complexity of tumor-host interactions within the tumor microenvironment caused by temporal changes in tumor phenotypes and an array of immune mediators expressed in the tumor microenvironment. Here, we will review current knowledge of the different 'levels' of immune response that might be necessary for immunotherapy to be effective in the treatment of cancer. Furthermore, we will discuss the information still required in order to understand the mechanism(s) governing tumor rejection by the immune system in response to TA-specific immunization.

Animals↗