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T Blankenstein

Publications and source records attributed to T Blankenstein.

At least 37 records · Page 2Linked to original sources
PubMed1998

Interleukin 4 gene-defective mice reconstituted with wild-type bone marrow fail to produce normal immunoglobulin E levels.

The ability to reconstitute interleukin (IL)-4-/- mice with bone marrow of IL-4+/+ mice was investigated. The absence of the IL-4-/- gene in donor or recipient cells did not impair the reconstitution. All immunoglobulin (Ig) subsets occurred at normal serum levels except for IgE and to some extent IgG1. IgE production did not recover in the reconstituted mice over prolonged time. However, these mice were competent for IgE production, because a single intrasplenic injection of IL-4 restored IgE levels, which then remained constant. Wild-type mice reconstituted with wild-type bone marrow constantly had IgE serum levels comparable to untreated animals. In wild-type mice reconstituted with IL-4-/- bone marrow, IgE levels dropped gradually and disappeared by week 12. We make three unrelated but nonetheless important conclusions: (a) (immunoregulation) the tightly regulated IL-4 gene should be expressed constantly in low amounts (and with apparent absence of antigen stimulation) to keep the normal threshold of IgE; (b) (ontogeny of the immune system) an early unidentified source of IL-4 must be postulated which is lost in adult mice; and (c) (bone marrow transfer/gene therapy) under certain circumstances, the genotype of the recipient influences the reconstitution.

Animals↗
PubMed1998

Double suicide gene (cytosine deaminase and herpes simplex virus thymidine kinase) but not single gene transfer allows reliable elimination of tumor cells in vivo.

Suicide genes such as cytosine deaminase (CD) and herpes simplex virus thymidine kinase (TK) encode products that convert nontoxic substances (prodrugs) into toxic metabolites. Suicide gene transfer is currently being used in cancer therapy or can be used as a safety modality. To analyze the reliability of suicide genes as a safety modality for a vaccination study with viable cytokine/B7 gene-modified tumor cells, the individual and combined efficacy of the two suicide genes was compared for in vitro and in vivo cell killing of a murine mammary adenocarcinoma cell line (TS/A). To adapt the system to an in vivo gene delivery situation, bulk cultures cotransfected with the CD and TK gene were used instead of selected clones. In vitro, both CD and TK conferred sensitivity to the respective prodrug but the combined cytotoxic effects of both gene products were always superior. For in vivo analysis BALB/c mice were injected subcutaneously with CD- and TK-modified TS/A cells, treated with prodrugs, and tumor size was evaluated for a period of 100 days. In the in vivo situation the combination of both enzyme/prodrug systems was again most effective. The highest single concentration of 5-FC (500 mg/kg) or GCV (100 mg/kg) was not able to fully protect the animals from developing tumors, whereas a combination of 5-FC (250 mg/kg) and GCV (50 mg/kg) resulted in complete tumor eradication. In nude mice treated in the same way, most CD/TK tumors could not be eliminated. Furthermore, BALB/c mice cured of TS/A-CD/TK tumors developed a systemic tumor immunity against challenge with parental TS/A cells. These findings indicate that reliable tumor elimination by the suicide genes depends on T cells. The cooperative effect of both suicide genes was confirmed in vitro with the human renal cell carcinoma line RCC26. We conclude that TK and CD together, but neither gene alone, act as a safety mechanism for the elimination of tumor cells in a reliable fashion and suggest that a rapid and quantitative antigen release by effective TK- and CD-mediated tumor destruction is necessary for T cell immunity to develop.

Adenocarcinoma↗
PubMed1998

Strong immunogenic potential of a B7 retroviral expression vector: generation of HLA-B7-restricted CTL response against selectable marker genes.

The stimulation of a specific immune response is an attractive goal in cancer therapy. Gene transfer of co-stimulatory molecules and/or cytokine genes into tumor cells and the injection of these genetically modified cells leads to tumor rejection by syngeneic hosts and the induction of tumor immunity. However, the development of host immune response could be either due to the introduced immunomodulatory genes or due to vector components. In this study, human renal cell carcinoma cell lines were modified by a retrovirus to express the co-stimulatory molecule B7-1 together with the hygromycin/thymidine kinase fusion protein (HygTk) as positive and negative selection markers. These B7-1-transduced renal cell carcinoma cell lines were able significantly to activate allogeneic T cell proliferation. The cytolytic activity of these T cells was determined by employing several transduced and nontransduced renal cell carcinoma cell lines as targets. Evidence for a strong vector-specific T cell reactivity induced by the Hyg/Tk protein was obtained in autologous renal cell carcinoma systems. Antibody blocking experiments as well as peptide binding assays demonstrated an HLA-B7-restricted T cell response directed against both the Hyg and the Tk genes. Thus, the vector itself may mask the generation of immune reactivity against tumor antigens and may even detract from it. Vectors with immunogenic potential may be useful for tumor vaccination via cross priming in vivo, whereas antivector reactivities would be detrimental in situations where gene defects are being corrected and where long term expression of a therapeutic protein is required.

Antigens, Neoplasm↗
PubMed1998

Neutrophils but not eosinophils are involved in growth suppression of IL-4-secreting tumors.

Local expression of IL-4 by gene-modified tumor cells increases their immunogenicity by inducing an inflammatory response that is dominated by eosinophils. Eosinophils have been implicated as antitumor effector cells because the application of a granulocyte-depleting Ab inhibited rejection of IL-4 transfected tumors. This Ab did not discriminate between eosinophils and neutrophils and, therefore, this experiment could not exclude neutrophils as primary effector cells, whereas eosinophils were innocent bystander cells in IL-4 transfected tumors. We analyzed tumor growth suppression and granulocyte infiltration in IL-5-deficient (IL-5(-/-)) mice that had a deficiency of eosinophils, using two tumor lines (B16-F10 and MCA205) transfected to secrete IL-4. IL-4-expressing tumors were at least as efficiently rejected in IL-5(-/-) mice as in wild-type mice, despite an almost complete absence of tumor-infiltrating eosinophils. However, neutrophils were present in undiminished amounts and their depletion partially restored tumor growth. Furthermore, the growth of IL-5-secreting tumors was not impaired in either wild-type or IL-5(-/-) mice, even though it induced eosinophilia in both mouse strains. These findings demonstrate that eosinophils can be induced in IL-5(-/-) mice by exogenous IL-5 and argue against a compensatory effect of neutrophils in the absence of eosinophils. We conclude that 1) infiltration of IL-4 transfected tumors by eosinophils is completely IL-5 dependent, 2) eosinophils have no tumoricidal activity, and 3) neutrophils are responsible, at least in part, for tumor suppression.

Animals↗
PubMed1998

B cells inhibit induction of T cell-dependent tumor immunity.

Cytotoxic T lymphocyte (CTL) mediated tumor immunity against major histocompatibility antigen (MHC) class I-positive but class II-negative tumors often requires help from CD4+ T cells. These CD4 cells are activated by MHC class II-positive cells that present tumor derived antigens. Considering that different antigen presenting cells, such as B cells, macrophages and dendritic cells compete for antigen and influence the outcome of an immune response, we analyzed tumor immunity in B cell-deficient mice. These mice appear normal with regard to T cell immunity and tolerance to some pure foreign antigens. We show here that the low immunogenicity of tumors is caused by B cells whose presence in the priming phase results in disabled CD4+ T cell help for CTL mediated tumor immunity. Instead, in the presence of B cells, a non-protective humoral immune response is induced. Our results may explain the enigmatic observation that tumor-reactive antibodies occur frequently in cancer patients.

Adenocarcinoma↗
PubMed1998

Retroviral interleukin-7 gene transfer into human dendritic cells enhances T cell activation.

Tumor vaccination with dendritic cells (DC) presenting tumor antigens to T cells is a promising approach in immunotherapy. The aim of this study was to enhance T cell stimulatory ability of human DC by retroviral expression of the interleukin-7 (IL-7) gene. IL-7 has been shown to provide a potent costimulatory signal for the proliferation of T cells and the generation of cytotoxic T cells (CTL). DC were generated from human peripheral blood mononuclear cells (PBMC). DC were analyzed by light- and electron-microscopy, immunophenotype (CD1a+, CD14-, CD80+, CD86+, HLA-DR+) and functional assays. According to these criteria, 75-85% of the cells were DC. The cells did not produce measurable amounts of IL-7 spontaneously nor did they express the IL-7 receptor. A retroviral IL-7 expression vector was constructed. Retroviral infection was performed with either the LXSN-hIL-7 vector of its variant LXSN. Using the LXSN-hIL-7 vector, IL-7 production of 2296 pg/10(6) cells/24 h could be achieved on average. Transduction of DC was confirmed by RT-PCR in a CD1a-enriched cell fraction. Transduction efficiency by a control virus coding for beta-galactosidase was about 30%. In autologous mixed lymphocyte reaction (MLR), IL-7 transduced DC augmented T cell proliferation by a factor of two compared with unmodified or mock-transfected DC, and in allogeneic MLR there was a 2.7-fold increase in T cell proliferation. The increase in T cell proliferation could be correlated to IL-7 secretion by DC. Dendritic cells that have been simultaneously peptide-loaded and gene-modified to secrete IL-7 are a potential tool to amplify activation of tumor-specific T cells.

Cells, Cultured↗
PubMed1997

Costimulatory signals through B7.1/CD28 prevent T cell apoptosis during target cell lysis.

Expression of B7 on tumor cells can circumvent T cell tolerance and lead to the generation of tumor cell-specific T cell immunity. The effect of B7 expression on the generation of protective antitumor immunity has been attributed primarily to 1) more efficient T cell activation and 2) better generation of tumor-specific killer T cells. We have investigated the role of costimulation through B7.1 and its receptor, the CD28 molecule, in the generation of allogeneic human CTLs against MCF-7 breast cancer cells. In this setting, we describe how activated CTLs undergo activation-induced cell death upon killing the target cell. Instead of proliferation and clonal expansion, the majority of the CTLs underwent apoptotic cell death. CTL apoptosis could be blocked by 50% when binding of the Fas ligand to its receptor, the CD95 (APO-1/Fas) molecule, was prevented. Fas ligand was detected in the activated T cells, but not in MCF-7 or a panel of other breast cancer cell lines. This excludes an active role for MCF-7 during CTL death and indicates that the CTL apoptosis is due to an autocrine production of the Fas ligand by CTLs. Costimulation of CTLs by retrovirally B7.1-transfected MCF-7 drastically reduced the sensitivity of the CTLs to apoptosis during target contact. Thus, in tumor cell vaccination, B7.1 might play a major role in preventing T cell death by altering T cell susceptibility for apoptosis.

Apoptosis↗
PubMed1997

Down-regulation of the expression and function of the transporter associated with antigen processing in murine tumor cell lines expressing IL-10.

The MHC class I molecules present antigenic peptides to CTL. The peptides are delivered to the secretory pathway by TAP, which is formed by the association of MHC-encoded TAP1 and TAP2 gene products. Tumor cells incubated or transfected with IL-10 had decreased but peptide-inducible expression of MHC class I, decreased sensitivity to MHC class I-restricted CTL, and increased NK sensitivity. We here demonstrate that IL-10 expression in the murine lymphoma RMA inhibits the TAP-dependent translocation of peptides to the endoplasmic reticulum, resulting in accumulation of immature MHC class I molecules in the endoplasmic reticulum and subsequently low expression of cell surface MHC class I molecules. This finding is explained by a down-regulation of expression of TAP1 and TAP2, observed in IL-10-transfected RMA cells as well as in IL-10-transfected P815 mastocytoma cells. In the J558L plasmacytoma cell line, constitutively expressing high levels of IL-10, increased TAP-dependent translocation of peptides and expression of cell surface MHC class I could be induced by IL-10 antisense expression. IL-10 is the first example to demonstrate that a cytokine can decrease the expression and function of the TAP1/2 molecular complex and, in more general terms, the first example of a cytokine with an inhibitory effect on MHC class I-mediated Ag presentation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Transient expression of SV 40 large T antigen by Cre/LoxP-mediated site-specific deletion in primary human tumor cells.

A 'bottle-neck' for construction of autologous genetically engineered tumor vaccines and characterization of tumor antigens consists in the difficulty of establishing cell lines from human tumor material. We have constructed two retroviruses allowing transient expression of Simian virus 40 large T as an immortalizing agent. The first vector contains the genes for hygromycin and Herpes Simplex Virus thymidinkinase (TK), for positive and negative selection and the gene encoding large T. They are flanked by LoxP sites, the substrate of the bacteriophage recombinase Cre. The second retrovirus contains the genes for the Cre recombinase and puromycin as selection marker. By sequential infection of NIH3T3 cells with the two viruses, we have shown that the newly expressed large T gene can be deleted in a large proportion (> or =90%) of cells by site-specific recombination. Because the deletion included the TK gene, selection with gancyclovir against cells not having undergone recombination was possible. By infection with the large T retrovirus, cell lines could be easily established from mouse primary kidney cells, human fibroblasts, and cells derived from different surgical specimens of breast or colon cancer patients. One breast carcinoma cell line was further analyzed and shown to be of epithelial origin by characteristic markers (cytokeratins, mucin). This cell line grew continuously in culture for more than a year without any indication of a cell crisis. Infection with the cre-puro retrovirus and GCV selection resulted in complete excision of the large T gene as judged from antibody staining. Remarkably, these cells changed morphology and stopped proliferation comparable to the cells obtained from biopsy demonstrating the requirement of large T for growth. Therefore, this approach may facilitate molecular and cellular characterization of human tumors and other cell types where cell culturing is the limiting step, and gene therapy approaches involving autologous tumor cells.

3T3 Cells↗
PubMed1997

Interleukin-10 prevents dendritic cell accumulation and vaccination with granulocyte-macrophage colony-stimulating factor gene-modified tumor cells.

A wide variety of human tumors express IL-10 for reasons poorly understood. We have analyzed the effect of spontaneous IL-10 expression by a mouse tumor (J558L) on its immunoparalyzing effect. Because "cross-priming" of T cells by host Ag-presenting cells for MHC class I-restricted tumor Ags is a major pathway for induction of tumor immunity and that is enhanced by granulocyte-macrophage (GM) CSF, we expressed this cytokine in J558L cells. GM-CSF-secreting cells were not effective when used for immunization against challenge with the parental tumor. Inhibition of IL-10 expression through an IL-10 antisense retrovirus restored the vaccine efficacy of GM-CSF-producing J558L cells, demonstrating a direct role of IL-10 in paralyzing the GM-CSF-induced antitumor immune response. Since the tumor used for challenge produced IL-10, we conclude that IL-10 interfered primarily with the initiation but not the effector phase of the immune response. Immunohistochemical analysis of the vaccine site showed a GM-CSF-induced accumulation of dendritic cells (DC) (MHC class II+ and DEC-205+) in the absence of IL-10. In the presence of IL-10, DC accumulation was completely inhibited. Together, our results demonstrate an antagonistic effect of IL-10 with respect to GM-CSF-induced DC accumulation and tumor immunity and suggest a new mechanism by which tumors escape immune recognition: namely by preventing APC from obtaining access to tumor Ags.

Animals↗
PubMed1997

Influence of gene-modified (IL-7, IL-4, and B7) tumor cell vaccines on tumor antigen presentation.

Tumor cells genetically modified to coexpress certain cytokines (such as IL-7 or IL-4) and B7.1 have increased immunogenicity. Since tumor Ags can be presented either directly by tumor cells or indirectly by host APC (cross-priming), we asked whether B7.1 and IL-7 or IL-4 complemented each other by improving preferentially one or both pathways of Ag presentation. We used TS/A (H-2d) tumor cells and their IL-7, B7, and IL-7/B7 transfectants, and MCA205 (H-2b) tumor cells and their IL-4 and B7 transfectants. beta-galactosidase (beta-gal) was chosen as surrogate tumor Ag. beta-gal has different predominant MHC class I epitopes in H-2d and H-2b mice. Immunization of (H-2b x d)F1 mice with TS/A/beta-gal transfectants showed that both IL-7 and B7.1 and, as control, granulocyte-macrophage CSF augmented cross-priming and rejection of a challenge with MCA205/beta-gal (H-2b). Similarly, immunization with MCA205/beta-gal B7.1 or IL-4 transfectants enhanced cross-priming and rejection of a challenge with TS/A/beta-gal. beta-gal-specific rejection was confirmed by CTL assay. However, direct Ag presentation by tumor cells was enhanced only by B7.1, and not IL-7. For this study, H-2b nu/nu mice reconstituted with F1 lymphocytes were immunized with H-2d TS/A/beta-gal transfectants and challenged with TS/A/beta-gal. In conclusion, indirect Ag presentation was augmented by B7, IL-7, and IL-4, while direct Ag presentation was improved only by B7.

Adenocarcinoma↗
PubMed1997

Lack of correlation between rejection of tumor cells co-expressing interleukin-2 and B7.1 and vaccine efficiency.

Genetically modifying tumor cells to express a variety of cytokines such as interleukin-2 (IL-2) or the co-stimulatory molecule B7.1 leads to increased immunogenicity and reduced tumorigenicity of tumors in several models with T cells involved in the process. We have previously reported decreased tumorigenicity of the murine plasmacytoma J558L [major histocompatibility complex (MHC) class I+ and class II-] expressing IL-2 or B7.1. When systemic immunity was analyzed, immunization with either J558-IL2 or J558-B7.1 cells generated moderate protection against unmodified J558L tumor cells, comparable to immunization with a tumor cells/adjuvant Corynebacterium parvum mixture. In this study, we asked whether the co-expression of IL-2 and B7.1 in tumor cells would augment vaccine potency, cytotoxic T lymphocyte (CTL) activity and protective immunity. Rejection of single IL-2 or B7.1 or co-transfected IL-2/B7.1 cells occurred in most syngeneic animals but not in T cell-deficient nude mice, thus confirming that T cells were required for tumor rejection. We knew from previous experiments that CD8+ T cells were responsible for rejection. Surprisingly, immunization with J558-IL2/B7.1 cells followed by challenge with parental J558L caused a reduction in systemic protection as compared to J558-B7.1 or J558-IL2 alone. We examined the mechanism underlying this unexpected result: 6 days after injection of J558-IL2/B7.1 cells, tumor were nearly completely destroyed and were almost devoid of CD8+ cells, while CD8+ cells were increased in both IL-2- and B7.1-transfected tumors. In addition, immunization with J558-IL2/B7.1 tumors had an adverse effect on the generation of CTL. Mice immunized with J558-B7.1 and to a lesser extent J558-IL2 cells mounted a CTL response against J558L cells while, in contrast, no CTL activity could be detected in mice immunized with J558-IL2/B7.1, thus showing a correlation between the absence of CTL activity and the lack of in vivo protection. We demonstrate that "hyperstimulation" of the immune response by genetically modified cancer vaccines can have adverse effects on tumor immunity, even though the mechanism is not yet completely understood.

Animals↗
PubMed1997

Loss of retroviral gene expression in bone marrow reconstituted mice correlates with down-regulation of gene expression in long-term culture initiating cells.

The question of whether a retroviral vector is expressed in vivo after infection of hematopoietic stem cells remains unpredictable. In this study, we show that a switch off of retroviral sequences can be tested in vitro using the long-term bone marrow culture system. The time kinetics of the down-regulation of retroviral expression in vitro and in vivo have been demonstrated to be similar. The correlation of switch off events in both systems allows for evaluation of retroviral expression in vitro provided the silencing is caused by viral sequences. This approach could be useful for testing vectors for gene therapeutical applications of the hematopoietic system.

Animals↗
PubMed1997

Successful retroviral mediated transduction of a reporter gene in human dendritic cells: feasibility of therapy with gene-modified antigen presenting cells.

In this study we have analyzed the feasibility of gene transfer in human dendritic cells (DCs). DCs were generated from T and B cell-depleted peripheral blood mononuclear cells cultured for 7 days in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). The cells showed morphologic and immunophenotypical features typical of DCs, including expression of major histocompatibility complex (MHC) class I and II molecules, CD1a, CD80, CD86, CD13, CD33, CD40, and CD54. The cells showed high stimulatory activity in both allogeneic and autologous mixed lymphocyte reaction (MLR). The bacterial reporter gene lacZ coding for beta-galactosidase (beta-gal) was introduced in DCs by three sequential cycles of infection using a MFG retroviral vector system. After 7 days of culture 35-67% of the cells showed high expression of beta-gal activity, proving successful gene transfer. Stable integration of the lacZ gene was demonstrated by genomic DNA-polymerase chain reaction (PCR) up to 20 days after gene transfer. The percentage of transduction was similar when DCs were further purified by immunomagnetic separation according to CD1a-expression. We conclude that human DCs can be efficiently gene modified, further broadening the spectrum of possible DC-based clinical applications.

Antigen Presentation↗
PubMed1996

Coexpression of interleukin-4 and B7.1 in murine tumor cells leads to improved tumor rejection and vaccine effect compared to single gene transfectants and a classical adjuvant.

To improve the vaccine potency of gene-modified tumor cells, using retroviruses, we have expressed the B7.1 gene in J558L cells and a subline previously transfected with the gene for interleukin-4 (IL-4). Complete longterm tumor eradication occurred in only 73-82% of syngeneic BALB/c mice injected with IL-4 or B7.1 transfectants or tumor cells mixed with the adjuvant Corynebacterium parvum. In contrast, none of the mice injected with J558-IL4/B7.1 cells developed a tumor, thus demonstrating that IL-4 and B7.1 together induced a more potent antitumor immune response compared to either molecule alone. Immunization/challenge experiments demonstrated that IL-4/B7.1 co-transfected cells possessed improved and tumor-specific vaccine potency when compared to single gene transfectants and, more importantly, to a tumor cell/C. parvum mixture. Furthermore, irradiation of vaccine cells almost completely abrogated the vaccine effect. Together, our results mean a step toward an improved tumor cell vaccine that acquires efficacy by the concerted action of IL-4 and B7.1 and the use of viable cells.

Adjuvants, Immunologic↗
PubMed1996

Treatment of experimental encephalomyelitis with a peptide analogue of myelin basic protein.

Following induction of experimental encephalomyelitis with a T-cell clone, L10C1, that is specific for the myelin basic protein epitope p87-99, the inflammatory infiltrate in the central nervous system contains a diverse collection of T cells with heterogeneous receptors. We show here that when clone L10C1 is tolerized in vivo with an analogue of p87-99, established paralysis is reversed, inflammatory infiltrates regress, and the heterogeneous T-cell infiltrate disappears from the brain, with only the T-cell clones that incited disease remaining in the original lesions. We found that antibody raised against interleukin-4 reversed the tolerance induced by the altered peptide ligand. Treatment with this altered peptide ligand selectively silences pathogenic T cells and actively signals for the efflux of other T cells recruited to the site of disease as a result of the production of interleukin-4 and the reduction of tumour-necrosis factor-alpha in the lesion.

Amino Acid Sequence↗
PubMed1996

Attenuation of collagen-induced arthritis in mice by treatment with vector cells engineered to secrete interleukin-13.

The anti-inflammatory effects of the recently identified cytokine interleukin (IL)-13 on collagen-induced arthritis (CIA) was explored and compared to those of IL-4 using systemic administration of these cytokines via two injections of xenogeneic vector cells transfected with a plasmid construct. CIA was induced in DBA/I mice by immunization with native bovine type II collagen (CII). Chinese hamster ovary (CHO) fibroblasts transfected with the mouse IL-13 or IL-4 genes were inoculated subcutaneously on days 10 and 25 post-priming with CII and mice were monitored for signs of arthritis by observers unaware of the status of the animal. Incidence and severity of CIA were significantly reduced in the groups of mice treated with IL-13 and IL-4 gene-transfected CHO cells compared to control groups receiving nontransfected cells. Expression of various cytokines in spleen cells from individual mice was assessed by quantitative reverse transcriptase-polymerase chain reaction at different times after immunization. Our data show that IL-13-induced suppression of CIA coincided with a decreased TNF-alpha mRNA expression in the spleen of treated animals. This may explain at least partially the anti-inflammatory effects of IL-13 in CIA. Thus, our results may have important implications for the clinical use of T helper (Th)1/TH2 modulatory cytokines as therapeutic agents in the treatment of autoimmune diseases.

Animals↗