[show abstract][hide abstract] ABSTRACT: Tumor-induced immunosuppression remains a significant obstacle that limits the efficacy of biological therapy for renal cell carcinoma. Here we evaluate the role of CD33 myeloid-derived suppressor cells (MDSC) in the regulation of T-cell responses in renal cell carcinoma patients. We also examine effect of all-trans-retinoic acid (ATRA) on MDSC-mediated immune suppression.
CD33-positive myeloid cells were isolated from the peripheral blood of renal cell carcinoma patients with magnetic beads and tested in vitro for their ability to inhibit T-cell responses. T-cell function was evaluated using ELISPOT and CTL assays.
MDSC isolated from renal cell carcinoma patients, but not from healthy donors, were capable of suppressing antigen-specific T-cell responses in vitro through the secretion of reactive oxygen species and nitric oxide upon interaction with CTL. MDSC-mediated immune suppression and IFN-gamma down-regulation was reversible in vitro by exposing cells to the reactive oxygen species inhibitors. Moreover, ATRA was capable of abrogating MDSC-mediated immunosuppression and improving T-cell function by direct differentiation into antigen-presenting cell precursors.
These results may have significant implications regarding the future design of active immunotherapy protocols that may include differentiation agents as part of a multimodal approach to renal cell carcinoma immunotherapy.
Clinical Cancer Research 01/2009; 14(24):8270-8. · 7.84 Impact Factor
[show abstract][hide abstract] ABSTRACT: Metastatic renal cell carcinoma (RCC) associates with overproduction of vascular endothelial growth factor (VEGF) due to the mutation/inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene. Herein we demonstrate that implantation of human RCC tumor cells into athymic nude mice promotes the appearance of VEGF receptor 1 (VEGFR1)/CD11b double-positive myeloid cells in peripheral blood. Avastin-mediated VEGF neutralization was capable of significantly reducing the numbers of circulating VEGFR1+ myeloid cells. Conversely, up-regulation of VEGFR1 by myeloid cells could also be achieved in vitro by coculturing bone marrow cells with RCC-conditioned medium or by short-term exposure of naive myeloid cells to oxidative stress. Treatment of myeloid cells with H2O2, lipid peroxidation product 4-hydroxy-2(E)-nonenal, or an inhibitor of thioredoxin reductase all resulted in increased expression of VEGFR1. Furthermore, after exposure to oxidative stress, myeloid cells acquire immunosuppressive features and become capable of inhibiting T cell proliferation. Data suggest that tumor-induced oxidative stress may promote both VEGFR1 up-regulation and immunosuppressive function in bone marrow-derived myeloid cells. Analysis of tumor tissue and peripheral blood from patients with metastatic RCC revealed that VEGFR1+ cells can be also found in cancer patients. Restoration of immunocompetence in metastatic RCC patients by pharmacological elimination of VEGFR1+ cells may have a significant impact on the therapeutic efficacy of cancer vaccines or other immune-based therapies.
The Journal of Immunology 08/2008; 181(1):346-53. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Overexpression of lysyl oxidase (LOX) is associated with the invasive potential of metastatic breast and head and neck cancer (HNC) cells and reduced metastasis-free and overall survival. Recently, we have demonstrated up-regulation of a new member of the LOX family, lysyl oxidase-like 4 (LOXL4), in invasive HNC revealed a significant correlation between LOXL4 expression and local lymph node metastases and higher tumour stages. The objective of this study was to examine whether cellular LOXL4 may provide an effective target for cell-meditated immunotherapy in invasive tumours associated with LOXL4 overexpression. As a feasibility study we expressed LOXL4 mRNA in immature dendritic cells derived from human peripheral blood mononuclear cells (PBMC). LOXL4 protein expression was ascertained using Western blotting and immunocytochemistry with polyclonal rabbit anti-LOXL4 antibody. The successfully transfected immature dendritic cells (DCs) were induced to mature with GM-CSF, IL-4, IL-1beta, TNF-alpha, IL-6, and PGE2, and then used to stimulate T cell enriched non-adherent fraction of PBMC. LOXL4 specific T cell stimulation induced cytotoxic T lymphocyte (CTL) response was monitored using IFN-gamma secretion from the non-adherent PBMC fraction exposed to mature, LOXL4 transfected DCs acting as the antigen presenting target cells. LOXL4-DC stimulated T cells produced higher IFN-gamma secretion compared to unstimulated T cells and T cells stimulated with untransfected DCs, in the presence of the pan-DR-epitope (PADRE). These initial results demonstrated the potential for LOXL4-transfected DCs to serve as efficient tumour vaccine and support their suitability as a vaccination strategy applicable to cancer patients with tumour specific up-regulation of LOXL4.
International Journal of Oncology 03/2008; 32(2):317-22. · 2.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: Selective up-regulation of the mRNA of LOXL4, a member of the LOX matrix amine oxidase family, significantly correlated with lymph node metastases and higher tumour stages in head and neck squamous cell carcinomas (HNSCC). To evaluate the diagnostic and prognostic value of the protein we produced an antibody specific for LOXL4 and assessed the expression in 317 human HNSCC specimens. The LOXL4 protein was detected in 92.7% of primary tumours, in 97.8% of lymph node metastases and in affected oral mucosa with high-grade dysplasia, but was absent in various non-neoplastic tissues of the head and neck. TNM categories and overall survival did not link to grades of immunoreactivity. Studies in cultured primary hypopharyngeal HTB-43 carcinoma cells detected perinuclear and cell surface expression of LOXL4, but no nuclear localisation. Therefore, its interactive SRCR-domains and catalytic activity combined with tumour cell specific expression and cell surface associated location indicate multiple functions in tumour cell adhesion and interactions with the extracellular matrix. Our data suggest that LOXL4 is useful both as tumour marker and target in the treatment of HNSCC.
[show abstract][hide abstract] ABSTRACT: The use of electrical charge for electroporation or electrofusion is widely applied to customize dendritic cells (DC) and their immunological properties as anticancer vaccines. The aim of this study was to evaluate the influence of various electrical field strengths on the recovery, viability and physiology of DC. Immature DC were transferred into low-conductive medium and electrically charged within a range of 0-1500 V/cm. Viability was assessed by Trypan Blue dye exclusion or staining with impermeant nucleic acid stains and fluorescence-activated cell sorter analysis. Additionally, apoptosis was determined by flow cytometry after staining with Annexin-V, endocytosis by uptake of fluorescein isothiocyanate-dextran and metabolic activity by a standardized fluorescent live/dead assay. There was a strong correlation between the electrical field strength and the viability and physiology of DC. Field strengths > or =1000 V/cm significantly impaired viability, metabolism and endocytotic activity. Dual fluorescence with 7-7-amino-actinomycin D and Annexin-V demonstrated that loss of viability was predominantly due to necrosis rather than apoptosis. Field strengths < or =500 V/cm allowed to maintain good cell viability and recovery of DC and did not cause alterations of metabolism and endocytosis. Therefore, the frequently used amplification of field strengths to improve the efficacy of electroporation and electrofusion requires critical re-evaluation.
Scandinavian Journal of Immunology 11/2005; 62(4):399-406. · 2.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: Antiangiogenic immunotherapy benefits from targeting antigens expressed on genetically stable endothelial cells and represents a novel modality for cancer treatment. Vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2, also known as flk1 in mouse) mediated VEGF signaling is the key rate-limiting step in angiogenesis. Blockade of the flk1 signaling pathway can significantly inhibit tumor cell-induced angiogenesis and lead to inhibition of tumor metastasis. Interferon-gamma (IFN-gamma) is a pleiotropic cytokine, which plays an important role in cell-mediated immunity. In this study, we tested the hypothesis that immunization of mice with soluble flk1 (sflk1) and IFN-gamma fusion gene-transfected dendritic cells (DC-sflk1-IFN-gamma) would induce a potent CTL response to flk1, leading to an inhibition of tumor-induced angiogenesis and metastasis. Our data show that immunization of mice with sflk1 gene-modified DC (DC-sflk1) could induce a CTL response to flk1, leading to profound inhibition of tumor-cell-induced angiogenesis and metastasis. However, more striking antimetastatic effects were achieved through induction of enhanced CTL response to flk1 and augmented inhibition of angiogenesis when mice were immunized with DC-sflk1-IFN-gamma. In vivo T-cell subset depletion experiments showed that CD8(+) T cells were mainly responsible for this antimetastatic effect. Our data extend the notion that DC-based active antiangiogenic immunotherapy is an effective modality for cancer treatment, and show that the antitumor efficacy of this strategy can be improved by combination with DC-based cytokine immunotherapy.
[show abstract][hide abstract] ABSTRACT: Zusammenfassung Immuntherapien sind aufgrund der häufig geringen Tumorlast nach Primärtherapie und der fehlenden Kreuzresistenz zu etablierten Verfahren eine attraktive Option zur Verbesserung der Therapie gynäkologischer Malignome. Beispiele sind die Etablierung des monoklonalen Antikörpers Trastuzumab beim Mammakarzinom und die erfolgreiche HPV-basierte Impfung zur Prävention des Zervixkarzinoms. Dieser Beitrag stellt die präklinischen und klinischen Erfahrungen mit passiven und aktiven Immunisierungsstrategien beim Ovarial-, Zervix- und Mammakarzinom vor.
Der Onkologe 01/2005; 11(5):530-535. · 0.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: Hybrid cells generated from dendritic cells (DC) and tumor cells provide tumor-associated antigens (TAA) in a polyvalent mode and therefore they have aroused interest in cancer immunotherapy. The present study was designed to investigate the hybrid cell generation and optimize its implementation for a TAA-target treatment of head and neck squamous cell carcinoma (HNSCC).
Hybrid cells from mature DC and laryngeal carcinoma cell line UTSCC-19A were generated by electrofusion. Fusion efficiency and viability were determined by flow cytometry, light and fluorescence microscopy analyses.
The gradual electrofusion process constituted real human tumor and dendritic cell hybrids characterized by polynuclear cells and double staining as a result of overlay of red (HLA-DR:R-PE) and green (HEA:FITC) fluorescence. Furthermore, analyses have proven viability of fusion results, and factors influencing fusion yield were determined.
Physical fusion of mature dendritic cells with laryngeal carcinoma cells provides a dendritic cell based hybrid cell vaccine as a quantitative prerequisite for anti-cancer vaccination. Specific cytotoxic T-lymphocytes need to be induced before hybrid cell application in clinical studies.
[show abstract][hide abstract] ABSTRACT: Hybrid cells generated from dendritic cells (DC) and tumour cells provide tumour-associated antigens (TAA) in a polyvalent mode. The present study was designed to investigate the hybrid cell generation by dendritic cells and different tumour cell lines to establish an electrofusion protocol with an optimal fusion setting.
Hybrid cells from mature DC and tumour cells were generated by electrofusion. Fusion efficiency was determined by flow cytometry, as well as by light and fluorescence microscopy analyses.
The gradual electrofusion process constituted different human dendritic cell tumour cell hybrids of high diversity depending on electrical and non-electrical parameters. Factors influencing fusion frequency were determined by specific cell staining with mAbs, FACS analysis and trypan blue dye exclusion.
Increased fusion efficiency was associated with reduced viability. The protocol presented in this work might be helpful for future fusion studies as a prerequisite for comparable in vitro and human vaccination trials.
Anticancer research 01/2004; 24(2B):929-34. · 1.71 Impact Factor
[show abstract][hide abstract] ABSTRACT: Survivin is a member of the inhibitors of apoptosis family and is overexpressed in many types of human cancers, making it an attractive target for T cell-based immunotherapeutic strategies. Recently, HLA-A2-binding peptides derived from the survivin protein were identified as capable of inducing specific T cell responses in cancer patients. Here we demonstrate that human survivin-specific CTLs generated from PBMC by stimulation with autologous dendritic cells transfected with survivin-RNA were cytotoxic for a range of hemopoietic malignant cell lines and primary tumor cells isolated from patients with acute myeloid leukemia. We also show that vaccination of mice with survivin-RNA-transfected dendritic cells leads to long term resistance to challenge by a survivin-expressing lymphoma, demonstrating the potential of survivin as a tumor rejection Ag. Our data provide evidence for the use of survivin as a target structure for immunotherapeutic strategies against hematological neoplasms.
The Journal of Immunology 07/2003; 170(11):5391-7. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Autologous dendritic cells transfected with total renal tumor RNA have been shown to be potent stimulators of CTLs and antitumor immunity in vitro. A Phase I trial was conducted to evaluate this strategy for feasibility, safety, and efficacy to induce tumor-specific T-cell responses in subjects with metastatic renal cell carcinoma. Renal tumor RNA-transfected dendritic cells were administered to 10 evaluable study patients with no evidence of dose-limiting toxicity or vaccine-related adverse effects including autoimmunity. In six of seven evaluable subjects, expansion of tumor-specific T cells was detected after immunization. The vaccine-induced T-cell reactivities were directed against a broad set of renal tumor-associated antigens, including telomerase reverse transcriptase, G250, and oncofetal antigen, but not against self-antigens expressed by normal renal tissues. Although most patients underwent secondary therapies after vaccination, tumor-related mortality of the study subjects was unexpectedly low with only 3 of 10 patients dying from disease after a mean follow-up of 19.8 months. These data provide a scientific rationale for continued clinical investigation of this polyvalent vaccine strategy in the treatment of metastatic renal cell carcinoma and, potentially, other cancers.
Cancer Research 06/2003; 63(9):2127-33. · 8.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Autologous dendritic cells (DCs) transfected with mRNA encoding prostate-specific antigen (PSA) are able to stimulate potent, T cell-mediated antitumor immune responses in vitro. A phase I trial was performed to evaluate this strategy for safety, feasibility, and efficacy to induce T cell responses against the self-protein PSA in patients with metastatic prostate cancer. In 13 study subjects, escalating doses of PSA mRNA-transfected DCs were administered with no evidence of dose-limiting toxicity or adverse effects, including autoimmunity. Induction of PSA-specific T cell responses was consistently detected in all patients, suggesting in vivo bioactivity of the vaccine. Vaccination was further associated with a significant decrease in the log slope PSA in six of seven subjects; three patients that could be analyzed exhibited a transient molecular clearance of circulating tumor cells. The demonstration of vaccine safety, successful in vivo induction of PSA-specific immunity, and impact on surrogate clinical endpoints provides a scientific rationale for further clinical investigation of RNA-transfected DCs in the treatment of human cancer.
Journal of Clinical Investigation 03/2002; 109(3):409-17. · 12.81 Impact Factor
[show abstract][hide abstract] ABSTRACT: Although renal cell carcinoma has been shown to respond to immunotherapy, renal cell carcinoma-specific rejection antigens and their corresponding CTL epitopes have rarely been described. The use of dendritic cells (DCs) transfected with mRNA isolated from tumor cells may allow specific immunotherapy even in cancers for which potent rejection antigens have not been identified. Here we show that DCs transfected with RNA isolated from renal cancer tissue are remarkably effective in stimulating tumor-specific T-cell response in vitro but do not cross-react with normal tissue antigens including antigens expressed by renal parenchyma. In contrast, the tumor-specific CTLs lysed allogeneic tumor but not allogeneic normal tissue targets, suggesting the presence of shared albeit unidentified antigens among renal carcinomas. CTL responses against telomerase reverse transcriptase (TERT) accounted in part for the reactivities against allogeneic tumors because renal tumor RNA-transfected DCs stimulated polyclonal CTL responses, which encompassed as a subcomponent a response against TERT. Nonetheless, the tumor-specific CTLs were consistently superior to the CTLs stimulated with TERT RNA-transfected DCs in recognizing and lysing tumor targets, suggesting that tumor-specific CTLs represent a polyclonal response providing more effective antitumor activity than T-cell responses directed against a single antigen in the form of TERT. Tumor RNA-transfected DCs were capable of stimulating T-cell reactivities not only against the primary tumor but also against metastatic tumors, although discrete differences in the antigenic repertoire expressed by these tissues were apparent. Thus, total tumor RNA-transfected DCs may represent a broadly applicable vaccine strategy to induce polyclonal and potentially therapeutic T-cell responses in renal cancer patients.
Cancer Research 05/2001; 61(8):3388-93. · 8.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Polyvalent cancer vaccines targeting the entire antigenic spectrum on tumor cells may represent a superior therapeutic strategy for cancer patients than vaccines solely directed against single Ags. In this study, we show that autologous dendritic cells (DC) transfected with RNA amplified from microdissected tumor cells are capable of stimulating CTL against a broad set of unidentified and critical prostate-specific Ags. Although the polyclonal CTL responses generated with amplified tumor RNA-transfected DC encompassed as a subcomponent a response against prostate-specific Ag (PSA) as well as against telomerase reverse transcriptase, the tumor-specific CTL were consistently more effective than PSA or telomerase reverse transcriptase CTL to lyse tumor targets, suggesting the superiority of the polyclonal response. Although tumor RNA-transfected DC stimulated CTL, which recognized not only tumor but also self-Ags expressed by benign prostate tissue, these cross-reactive CTL were exclusively specific for the PSA, indicating an immunodominant role of PSA in the prostate cancer-specific immune response. Our data suggest that tumor RNA-transfected DC may represent a broadly applicable, potentially clinically effective vaccine strategy for prostate cancer patients, which is not limited by tumor tissue availability for Ag preparation and may minimize the risk of clonal tumor escape.
The Journal of Immunology 04/2001; 166(5):2953-60. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: The polypeptide component of telomerase (TERT) is an attractive candidate for a broadly expressed tumor rejection antigen because telomerase is silent in normal tissues but is reactivated in more than 85% of cancers. Here we show that immunization against TERT induces immunity against tumors of unrelated origin. Immunization of mice with TERT RNA-transfected dendritic cells (DC) stimulated cytotoxic T lymphocytes (CTL), which lysed melanoma and thymoma tumor cells and inhibited the growth of three unrelated tumors in mice of distinct genetic backgrounds. TERT RNA-transfected human DC stimulated TERT-specific CTL in vitro that lysed human tumor cells, including Epstein Barr virus (EBV)-transformed B cells as well as autologous tumor targets from patients with renal and prostate cancer. Tumor RNA-transfected DC were used as surrogate targets in the CTL assays, obviating the difficulties in obtaining tumor cells from cancer patients. In one instance, where a tumor cell line was successfully established in culture from a patient with renal cancer, the patient's tumor cells were efficiently lysed by the CTL. Immunization with tumor RNA was generally more effective than immunization with TERT RNA, suggesting that an optimal immunization protocol may have to include TERT as well as additional tumor antigens.
Nature Medicine 10/2000; 6(9):1011-7. · 22.86 Impact Factor