Azacytidine impairs NK cell reactivity while decitabine augments NK cell responsiveness toward stimulation.

Azacytidine and decitabine are approved for treatment of acute myeloid leukemias and myelodysplastic syndromes. While clinical responses are attributed to epigenetic effects and induction of apoptosis in malignant cells, these azanucleosides also affect antitumor immune responses. NK cells as components of innate immunity may confine development and progression of cancer. Numerous therapeutic strategies presently aim to reinforce NK reactivity against hematopoietic malignancies. We here comparatively analyzed the effect of the two clinically available azanucleosides and report that NK cytotoxicity and IFN-γ production are significantly impaired by pharmacological concentrations of azacytidine but enhanced by decitabine. This was not due to alterations in the target cells but caused by direct effects on NK cells depending on the chemical modifications by which azanucleosides differ from their physiological analogues. Although azacytidine impaired mRNA synthesis and induced apoptosis in NK cells, decitabine did not per se alter NK cell viability or reactivity but enhanced responsiveness to activating stimuli by inducing transcription of genes involved in NK reactivity. Tantalizingly, these effects were independent of incorporation of the azanucleosides into DNA during cell division. While azacytidine impairs NK antitumor immunity, decitabine augments NK reactivity by yet unidentified mechanisms and may thus serve well in therapeutic strategies combining its effects on malignant cells with its ability to enhance NK functions.

Matrilysin (MMP-7) is a novel broadly expressed tumor antigen recognized by antigen-specific T cells.

PURPOSE: A prerequisite for the development of vaccination strategies is the identification and characterization of relevant tumor-associated antigen. Using microarray and reverse transcription-PCR analysis, we found matrix metalloproteinase (MMP)-7 to be extensively up-regulated in renal cell carcinomas and expressed in a broad variety of malignant cells. MMP-7 can promote cancer invasion and angiogenesis by proteolytic cleavage of extracellular matrix and basement membrane proteins, thus making it a promising target in the context of immunotherapies. EXPERIMENTAL DESIGN: To analyze the possible use of MMP-7 as a tumor-associated antigen, specific CTLs were induced using monocyte-derived dendritic cells electroporated with MMP-7-mRNA. In addition, to better characterize the fine specificity of these CTLs, MMP-7 MHC class I ligands were isolated and characterized in renal cell carcinoma tissue, which overexpressed MMP-7, by mass spectrometry-based peptide sequencing. Using this approach, we identified a novel HLA-A3-binding antigenic MMP-7 peptide. CTLs generated from healthy donors by in vitro priming with dendritic cells, pulsed with the novel peptide, were used as effectors in (51)Cr-release assays. RESULTS: The induced CTLs elicited an antigen-specific and HLA-restricted cytolytic activity against tumor cells endogenously expressing the MMP-7 protein. Furthermore, we were able to induce MMP-7-specific CTLs using peripheral blood mononuclear cells from a patient with acute lymphoblastic leukemia capable of recognizing the autologous leukemic blasts while sparing nonmalignant cells. CONCLUSIONS: Our study describes the identification of a novel broadly expressed T-cell epitope derived from the MMP-7 protein that represents an interesting candidate to be applied in immunotherapies of human malignancies targeting both tumor cells and neovascularization.

The use of dendritic cells in cancer immunotherapy.

Cancer immunotherapy aims at eliciting an immune response directed against tumor antigens to help fight off residual tumor cells and thereby improve survival and quality of life of cancer patients. Different immunotherapeutic approaches share the use of dendritic cells (DCs) to present tumor-associated antigens to T-lymphocytes. Ex vivo generated DCs can be loaded with antigens and re-infused to the patients, or they can be used for ex vivo expansion of antitumor lymphocytes. Alternatively, methods exist to target antigens to DCs in vivo without need for ex vivo cell manipulations. The clinical studies have shown that DC administration to patients is safe and induces antigen-specific immunity. However, it seldom elicits objective clinical responses in patients with advanced-stage malignancies. Novel insights into DC and lymphocyte regulation are expected to lead to more effective vaccines in the near future. Meanwhile, efforts are directed at identifying the most appropriate clinical targets for active specific immunotherapies. Data suggests that vaccinations may indeed be beneficial when given in the adjuvant setting rather than to treat metastatic cancers. These issues are discussed here together with an overview of the DC-based antitumor immunotherapy studies.

Severe diarrhoea caused by Aeromonas veronii biovar sobria in a patient with metastasised GIST.

This report describes the isolation of Aeromonas veronii biovar sobria as the causative enteropathogen of diarrhoea in an oncological patient after failure of detection of other infectious agents. The case points out the severe and long course of the infection, the diagnostic dilemma, and the prompt recovery after antibiotic treatment.

Immunologic and clinical responses after vaccinations with peptide-pulsed dendritic cells in metastatic renal cancer patients.

A phase I trial was conducted to evaluate the feasibility, safety, and efficacy of a dendritic cell-based vaccination in patients with metastatic renal cell carcinoma (RCC). Autologous mature dendritic cells derived from peripheral blood monocytes were pulsed with the HLA-A2-binding MUC1 peptides (M1.1 and M1.2). For the activation of CD4(+) T-helper lymphocytes, dendritic cells were further incubated with the PAN-DR-binding peptide PADRE. Dendritic cell vaccinations were done s.c. every 2 weeks for four times and repeated monthly until tumor progression. After five dendritic cell injections, patients additionally received three injections weekly of low-dose interleukin-2 (1 million IE/m(2)). The induction of vaccine-induced T-cell responses was monitored using enzyme-linked immunospot and Cr release assays. Twenty patients were included. The treatment was well tolerated with no severe side effects. In six patients, regression of the metastatic sites was induced after vaccinations with three patients achieving an objective response (one complete response, two partial responses, two mixed responses, and one stable disease). Additional four patients were stable during the treatment for up to 14 months. MUC1 peptide-specific T-cell responses in vivo were detected in the peripheral blood mononuclear cells of the six patients with objective responses. Interestingly, in patients responding to the treatment, T-cell responses to antigens not used for vaccinations, such as adipophilin, telomerase, or oncofetal antigen, could be detected, indicating that epitope spreading might occur. This study shows that MUC1 peptide-pulsed dendritic cells can induce clinical and immunologic responses in patients with metastatic RCC.

Proteasome inhibitor bortezomib modulates TLR4-induced dendritic cell activation.

Evidence from the animal model suggests that proteasome inhibitors may have immunosuppressive properties; however, their effects on the human immune system remain poorly investigated. Here, we show that bortezomib, a proteasome inhibitor with anticancer activity, impairs several immune properties of human monocyte-derived dendritic cells (DCs). Namely, exposure of DCs to bortezomib reduces their phagocytic capacity, as shown by FITC-labeled dextran internalization and mannose-receptor CD206 down-regulation. DCs treated with bortezomib show skewed phenotypic maturation in response to stimuli of bacterial (lipopolysaccharide [LPS]) and endogenous sources (including TNF-alpha and CD40L), as well as reduced cytokine production and immunostimulatory capacity. LPS-induced CCL-2/MCP-1 and CCL5/RANTES secretions by DCs were prevented by DC treatment with bortezomib. Finally, CCR7 up-regulation in DCs exposed to LPS as well as migration toward CCL19/MIP-3beta were strongly impaired. As a suitable mechanism for these effects, bortezomib was found to down-regulate MyD88, an essential adaptor for TLR signaling, and to relieve LPS-induced activation of NF-kappaB, IRF-3, and IRF-8 and of the MAP kinase pathway. In summary, inhibition of DC function may represent a novel mechanism by which proteasome inhibitors exert immunomodulatory effects. These compounds could prove useful for tuning TLR signaling and for the treatment of inflammatory and immune-mediated disorders.

Identification of C-met oncogene as a broadly expressed tumor-associated antigen recognized by cytotoxic T-lymphocytes.

PURPOSE: C-Met proto-oncogene is a receptor tyrosine kinase that mediates the oncogenic activities of the hepatocyte growth factor. Using a DNA chip analysis of tumor samples from patients with renal cell carcinoma and sequencing of peptides bound to the HLA-A*0201 molecules on tumor cells a peptide derived from the c-Met protein was identified recently. EXPERIMENTAL DESIGN: We used this novel HLA-A*0201 peptide for the induction of specific CTLs to analyze the presentation of this epitope by malignant cells. RESULTS: The induced CTL efficiently lysed target cells pulsed with the cognate peptide, as well as HLA-A*0201-matched tumor cell lines in an antigen-specific and HLA-restricted manner. Furthermore, the induced c-Met-specific CTLs recognized autologous dendritic cells (DCs) pulsed with the peptide or transfected with whole-tumor mRNA purified from c-Met-expressing cell lines. We next induced c-Met-specific CTLs using peripheral blood mononuclear cells and DC from an HLA-A*0201-positive patient with plasma cell leukemia to determine the recognition of primary autologous malignant cells. These CTLs lysed malignant plasma cells while sparing nonmalignant B- and T-lymphocytes, monocytes, and DCs. CONCLUSION: Our results demonstrate that c-Met oncogene is a novel tumor rejection antigen recognized by CTL and expressed on a broad variety of epithelial and hematopoietic malignant cells.

Induction of adipophilin-specific cytotoxic T lymphocytes using a novel HLA-A2-binding peptide that mediates tumor cell lysis.

Identification of tumor-associated antigens and advances in tumor immunology resulted in the development of vaccination strategies to treat patients with malignant diseases. Using a novel approach that combines DNA chip analysis of tumor samples with isolation of peptides on the surface of tumor cells, a HLA-A*0201-binding peptide derived from the adipophilin protein was identified. Adipophilin is involved in lipid storage and was thought to be expressed only in adipocytes, but it can be found in other cell types such as macrophages or tumor cells. In the present study, we analyzed the possible use of this peptide as a T-cell epitope presented by malignant cells. To accomplish this, we induced CTL responses using this HLA-A*0201-binding peptide. The in vitro-induced CTLs efficiently lysed cells pulsed with the adipophilin peptide and HLA-matched tumor cell lines in an antigen-specific and HLA-restricted manner. Finally, the induced CTLs recognized autologous dendritic cells (DCs) pulsed with the antigenic peptide or transfected with tumor RNA purified from an adipophilin-expressing tumor cell line. To further analyze the possible use of this peptide in immunotherapies of human malignancies, we induced adipophilin-specific CTLs using peripheral blood mononuclear cells and DCs from HLA-A*0201-positive patients with chronic lymphatic leukemia and plasma cell leukemia. The in vitro-generated CTLs recognized autologous chronic lymphatic leukemia cells and malignant plasma cells, whereas they spared nonmalignant resting or activated B and T lymphocytes, monocytes, or DCs. Our results demonstrate that this peptide might represent an interesting candidate for the development of cancer vaccines designed to target adipophilin-derived epitopes in a wide range of malignancies.

Induction of chronic lymphocytic leukemia (CLL)-specific CD4- and CD8-mediated T-cell responses using RNA-transfected dendritic cells.

Recently, it was demonstrated that transfection of dendritic cells (DCs) with tumor-derived RNA can elicit effective T-cell responses. This technique does not require the definition of the tumor antigen or HLA haplotype of the patients. We applied this approach to induce HLA class I- and class II-restricted T-cell responses directed against malignant cells from patients with chronic lymphocytic leukemia (B-CLL). Here, we show that DCs generated from monocytes of patients with B-CLL induce leukemia-specific cytotoxic and proliferative T-cell responses on transfection with total RNA isolated from autologous leukemic B lymphocytes. Standard 51Cr-release assays showed specific major histocompatibility complex (MHC) class I-restricted cytotoxic activity against the autologous leukemic B cells and DCs transfected with CLL-RNA, whereas nonmalignant B cells were spared. The specificity of the cytotoxic T-lymphocyte (CTL) response was confirmed using cold target inhibition assays and by blocking HLA class I molecules. Furthermore, we established a protocol for the amplification of whole B-CLL mRNA. The use of DCs transfected with in vitro amplified B-CLL mRNA elicited specific T-cell responses similar to the results obtained with native mRNA. These data suggest that vaccinations using DCs transfected with RNA might be a potent new strategy in the treatment of CLL.

Survivin is a shared tumor-associated antigen expressed in a broad variety of malignancies and recognized by specific cytotoxic T cells.

Survivin, a member of the inhibitor of apoptosis protein family, is expressed in almost all types of malignancies, making this protein a useful tool for the development of broadly applicable vaccination therapies. We used a recently identified HLA-A2 binding peptide and dendritic cells (DCs) from healthy donors to induce survivin-specific cytotoxic T lymphocytes (CTLs) in vitro. These T cells efficiently lysed target cells pulsed with the cognate peptide. Furthermore, survivin-specific CTLs recognized HLA-A2-matched tumor cell lines and primary malignant cells from patients with leukemia in an antigen-specific and HLA-restricted manner as demonstrated with the use of cold target inhibition assays and blocking antibodies. To validate the immunogenicity of survivin we performed the experiments in an autologous setting and used monocyte-derived DCs as targets. Interestingly, we found that DCs up-regulate survivin expression on stimulation with tumor necrosis factor alpha (TNF-alpha). However, these mature DCs were not recognized by survivin-specific CTLs, whereas they lysed autologous mature DCs pulsed with the antigenic peptide or transfected with whole tumor RNA purified from a survivin-expressing cell line. To further analyze the possible use of survivin-specific CTLs in cancer therapies, we induced survivin-specific CTLs using peripheral blood mononuclear cells (PBMNCs) and DCs from a patient with chronic lymphocytic leukemia (CLL). The in vitro-generated T cells efficiently recognized autologous malignant CLL cells, whereas they spared autologous-purified nonmalignant B cells or DCs. Our results demonstrate that survivin epitopes are presented on a broad variety of malignancies and can be applied in vaccination therapies.