Peter Buttgereit

Interactions Between Dendritic Cells and Cytokine-induced Killer Cells Lead to an Activation of Both Populations

Dendritic cells (DCs) are major antigen-presenting cells. They are capable of capturing and processing tumor antigens, expressing lymphocyte costimulatory molecules, and secreting cytokines to initiate immune responses. Here, the authors tested the effect of cytokine-induced killer (CIK) cells, a population that includes CD3 + CD56 + cells (natural killer T cells), with regard to their capacity to immunomodulate DCs. Cytokine-induced killer cells were cocultured with autologous DCs generated from peripheral blood mononuclear cells. Expression of markers typical for both populations was measured using flow cytometry, and secretion of interleukin (IL)-12 was determined using enzyme-linked immunosorbent assays. Cytotoxicity assays were performed to investigate the role of IL-12 and the importance of cell–cell interactions. Considering this, receptors for IL-12 and CD40 were blocked and cocultures were performed with cell culture inserts. Coculture of CIK cells led to a significant increase of DC-specific, costimulatory, and antigen-presenting molecules in DC cultures. In addition, coculture resulted in a dramatically increase of IL-12 secretion by DCs and to a significant increase in cytotoxic activity of CIK cells toward carcinoma cells. Blockage of IL-12 uptake decreased the cytolytic activity of CIK cells. Cytokine secretion was shown to be important for activation of CIK cells, and also cellular interactions between DCs and effector cells caused a higher cytolytic capacity. Interactions between DCs and CIK cells caused changes in the surface molecule expression of both populations, led to an increase of IL-12 secretion, and rendered an improved cytotoxic activity. The natural killer T cell subpopulation seems to be responsible for this effect. Therefore, coculture of DCs with CIK cells may have a major impact on immunotherapeutic protocols for patients with cancer.

Novel non-viral method for transfection of primary leukemia cells and cell lines.

BackgroundTumor cells such as leukemia and lymphoma cells are possible targets for gene therapy. However, previously leukemia and lymphoma cells have been demonstrated to be resistant to most of non-viral gene transfer methods.MethodsThe aim of this study was to analyze various methods for transfection of primary leukemia cells and leukemia cell lines and to improve the efficiency of gene delivery. Here, we evaluated a novel electroporation based technique called nucleofection. This novel technique uses a combination of special electrical parameters and specific solutions to deliver the DNA directly to the cell nucleus under mild conditions.ResultsUsing this technique for gene transfer up to 75% of primary cells derived from three acute myeloid leukemia (AML) patients and K562 cells were transfected with the green flourescent protein (GFP) reporter gene with low cytotoxicity. In addition, 49(+/- 9.7%) of HL60 leukemia cells showed expression of GFP.ConclusionThe non-viral transfection method described here may have an impact on the use of primary leukemia cells and leukemia cell lines in cancer gene therapy.

Efficient gene transfer into lymphoma cells using adenoviral vectors combined with lipofection

Tumor cells, such as lymphoma cells, are possible targets for gene therapy. In general, gene therapeutic approaches require efficient gene transfer to host cells and sufficient transgene expression. However, lymphoma cells previously have been demonstrated to be resistant to most of the currently available gene transfer methods. The aim of this study was to analyze various methods for transfection of lymphoma cells and to improve the efficiency of gene delivery. In accordance with previously published reports, lymphoma cells were demonstrated to be resistant to lipofection and electroporation. In contrast, we present an improved adenoviral protocol leading to highly efficient gene transfer to lymphoma cell lines derived from B cells as well as primary lymphoma cells being achieved with an adenoviral vector system encoding the β-galactosidase protein. At a multiplicity of infection of 200, up to 100% of Daudi cells and Raji cells and 70% of OCI-Ly8-LAM53 cells could be transfected. Even at high adenoviral concentrations, no marked toxicity was observed, and the growth characteristics of the lymphoma cell lines were not impaired. The transfection rates in primary cells derived from six patients with non-Hodgkin’s lymphoma were 30–65%, respectively. Transfection efficiency could be further increased by addition of cationic liposomes to adenoviral gene transfer. Furthermore, we examined the expression of the Coxsackie-adenoviral receptor (CAR) and the integrin receptors on the lymphoma cell surface. Flow cytometric analysis showed that 88% of Daudi cells, 69% of Raji cells, and 6% of OCI-Ly8-LAM53 cells expressed CAR on the cell surface. According to our data, adenoviral infection of lymphoma cells seems to be mediated by CAR. In contrast, integrin receptors are unlikely to play a major role, because lymphoma cells were negative for αvβ3-integrins and negative for αvβ5-integrins. In conclusion, this study demonstrates that B-lymphoma cell lines and primary lymphoma cells can be efficiently transfected using an adenoviral vector system. By adding cationic liposomes, the efficiency of adenoviral gene transfer to primary tumor cells could be further improved. This protocol may have an impact on the use of lymphoma cells in cancer gene therapy.

Generation of activated and antigen-specific T cells with cytotoxic activity after co-culture with dendritic cells

Abstract. Co-culturing of immunological effector cells with antigen-pulsed DC leads to an increase of cytotoxic activity against antigen-expressing tumour cells. Using this approach, we could detect up to 2.8% antigen-specific CTLs after co-culture with antigen-pulsed DC. However, the required high effector cell numbers remain a major obstacle in immunotherapy. In this study, we show an approach for generating activated and antigen-specific effector cells that enables us to decrease effector to target cell ratios. We used an interferon-γ secretion assay to enrich activated effector cells after co-culture with antigen-pulsed dendritic cells (DC). Purified immunological effector cells lysed 58.3% of antigen-expressing tumour cells at an effector to target ratio of 1:1. Furthermore, using MHC-IgG complexes, we enriched effector cells expressing antigen-specific T-cell receptor after co-culture with DC. Performing ELISpot, flow cytometry and TCR analysis, we could show a significant increase of activated and specific TCR-expressing effector cells after co-culture with DC.

Increase of the immunostimulatory effect of dendritic cells by pulsing with CA 19-9 protein.

Previously, a relative resistance of solid tumor cells to immunologic effector cells was shown in vitro. This resistance could be one reason for the clinical phenomenon of resistance of patients with colon carcinoma or other solid tumors to immunologic therapeutic approaches. In this study, dendritic cells (DCs) pulsed with CA 19-9 protein were tested for their immunostimulatory capacity of immunologic effector cells against cells derived from colon and pancreatic carcinoma. Dendritic cell cultures coexpressed CMRF-44 and CD1a, markers typical of DCs, in 31.5% ± 5.3% after 13 days of culture. Coculture of NK-like T lymphocytes with DCs led to a significant increase in cytotoxic activity, as measured using a lactate dehydrogenase release assay. Cytotoxic activity could be further increased using DCs pulsed with CA 19-9 protein. The effect of CA 19-9 on increasing the cytotoxic effect of NK-like T lymphocytes was dose dependent. Similarly, cocultivation of DCs with NK-like T cells derived from patients with metastatic pancreatic cancer and elevated CA 19-9 serum levels led to a significant increase in cytotoxic activity. In conclusion, DCs pulsed with CA 19-9 protein can increase the cytotoxic activity of immunologic effector cells against colon carcinoma and pancreatic cancer cells. Dendritic cells pulsed with CA 19-9 protein may have an important effect on immunotherapeutic protocols for patients with cancer.

Effects of adenoviral wild-type p53 gene transfer in p53-mutated lymphoma cells.

The present study assessed the role of adenoviral vector–mediated wild-type p53 gene transfer in B lymphoma cells. Deficiency of p53-mediated cell death is common in human cancer contributing to both tumorigenesis and chemoresistance. Lymphoma cells are being considered as suitable targets for gene therapy protocols. Recently, we reported an adenoviral protocol leading to highly efficient gene transfer to B lymphoma cells. All lymphoma cell lines ( n =5) tested here showed mutations in the p53 gene locus. The aim of this work was to transduce lymphoma cells with the wild-type p53 gene. Using this protocol, 88% of Raji, 75% of Daudi, and 45% of OCI-Ly8-LAM53 cells were transfected with the reporter gene green fluorescent protein at a multiplicity of infection of 200. The expression of green fluorescent protein in CA46 and BL41 cells was 27% and 42%, respectively. At this multiplicity of infection, growth characteristics of lymphoma cell lines were not changed significantly. In contrast, cells transduced with wild-type p53 gene showed an inhibition of proliferation as well as an increase in apoptosis. Cell loss by apoptosis after p53 gene transfer was up to 40% as compared to transduction with an irrelevant vector. In addition, we determined the effects of DNA damage produced by the DNA topoisomerase II inhibitor etoposide on wild-type p53 transfected lymphoma cells. In Ad-p53–transfected Raji cells, treatment with the drug resulted in a marked increase of cell loss in comparison to Ad-β-Gal–transfected cells (45% vs. 77%). Interestingly, performing cytotoxicity studies, we could show an increased sensitivity of Raji and Daudi cells against immunological effector cells. In conclusion, transduction of wild-type p53 into lymphoma cells expressing mutated p53 was efficient and led to inhibition of proliferation and increase in apoptotic rate in some cell lines dependent on p53 mutation. This protocol should have an impact on the use of lymphoma cells in cancer gene therapy protocols. Cancer Gene Therapy (2001) 8, 430–439

Gene therapy of lymphoma.

Gene therapy offers new and promising treatment for patients with hematological malignancies. Tumor cells--lymphoma cells, for example--are possible targets for gene therapy. In general, gene therapeutic approaches require efficient gene transfer into host cells and sufficient transgene expression. Although many methods of gene transfer into mammalian cells exist, most do not allow efficient DNA transfer into primary lymphocytes. In contrast to gene transfer into tumor cells and many other cell types, which can be successfully performed using a variety of methods, the efficient expression of foreign DNA in lymphoma cells presents unique problems and challenges, requiring a careful selection of the mode of gene transfer. In this review, we discuss the current strategies for gene therapy in the treatment of lymphoma. We also summarize the current gene transfer methods for lymphoma cells and efficiency of transgene expression.

Targeting of natural killer-like T immunologic effector cells against leukemia and lymphoma cells by reverse antibody-dependent cellular cytotoxicity.

Recently, highly efficient natural killer-like T immunologic effector cells called cytokine-induced killer (CIK) cells have been described. Most interestingly, CIK cells have been shown to eradicate established human lymphoma cells in a severe combined immunodeficient (SCID) mouse xenograft model in vivo. The current study was aimed at increasing the sensitivity of leukemia and lymphoma cells to CIK cells. In particular, the authors wanted to target CIK cells to leukemia and lymphoma cells via reverse antibody-dependent cellular cytotoxicity. Binding of an anti-CD3 monoclonal antibody to CIK cell cultures derived from patients with lymphoma was shown using flow cytometric analysis. For the target side, several B-cell lines were found to express CD19 on the cell surface. There was an impressive increase in sensitivity to CIK-mediated lysis of various lymphoma and leukemia cell lines by preincubation of the targets with a monoclonal antibody against CD3. This increase could be partially blocked by preincubation with anti-CD16 (Fc receptor III) and anti-CD32 (Fc receptor II) antibodies. These data suggest that the increase in cytotoxic activity is caused by Fc receptor-mediated antibody binding. Cytotoxic activity could be further increased by adding an anti-CD28 antibody in addition to anti-CD3. Finally, there was a further increase in sensitivity to CIK-mediated lysis of CD19+ malignant cells using the bispecific OKT3xHD37 antibody with specificity against CD3 and CD19. Interestingly, preincubation of malignant cells with an anti-CD3 monoclonal antibody followed by addition of the bispecific OKT3xHD37 antibody led to a further increase of cytotoxic sensitivity compared with the addition of the bispecific antibody alone. In conclusion, these data suggest that cytotoxic activity of immunologic effector cells can be increased not only by using the bispecific antibody OKT3xHD37 in vitro but also by preincubation of CD19+ leukemia and lymphoma cells with a monoclonal antibody against CD3. In addition, the immunostimulatory effect of the bispecific antibody OKT3xHD37 can be further increased by adding a monoclonal antibody against CD3.

Effects of recombinant adenovirus-mediated expression of IL-2 and IL-12 in human B lymphoma cells on co-cultured PBMC

BackgroundModulation of the immune system by genetically modified lymphoma cell vaccines is of potential therapeutic value in the treatment of B cell lymphoma. However, the anti-tumor effect of any single immunogene transfer has so far been limited. Combination treatment of recombinant IL-2 and IL-12 has been reported to be synergistic for inducing anti-tumor responses in solid tumors but the potential of IL-2/IL-12 gene modified B cell lymphoma cells has not been explored yet.MethodsUsing three different human B cell lymphoma cell lines and primary samples from patients with B cell neoplasms, expression levels of the coxsackie B-adenovirus receptor (CAR) and alpha (v) integrins were analyzed by fluorescence-activated cell sorter (FACS). Adenoviral transduction efficiencies were determined by GFP expression analysis and IL-2 and IL-12 cytokine production was quantified by enzyme-linked immunosorbent (ELISA) assays. Proliferative activities of peripheral blood mononuclear cells (PBMC) stimulated with either cytokine derived from supernatants of transduced lymphoma cells were measured by cell proliferation (MTT) assays. An EuTDA cytotoxicity assay was used to compare cytotoxic activities of IL-2 and/or IL-12 stimulated PBMC against unmodified lymphoma cells.ResultsWe found that B cell lymphoma cell lines could be transduced with much higher efficiency than primary tumor samples, which appeared to correlate with the expression of CAR. Adenoviral-expressed IL-2 and IL-12 similarly led to dose-dependent increases in proliferation rates of PBMC obtained from healthy donors. IL-2 and/or IL-12 transduced lymphoma cells were co-cultured with PBMC, which were assayed for their cytolytic activity against unmodified lymphoma cells. We found that IL-2 stimulated PBMC elicited a significant anti-tumor effect but not the combined effect of IL-2/IL-12 or IL-12 alone.ConclusionThis study demonstrates that the generation of recombinant adenovirus modified lymphoma cell vaccines based on lymphoma cell lines expressing IL-2 and IL-12 cytokine genes is technically feasible, induces increases in proliferation rates and cytotoxic activity of co-cultured PBMC, and warrants further development for the treatment of lymphoma patients in the future.

Induction of Apoptosis in B Lymphoma Cells by Activation with CD40L

Accessible online at: www.karger.com/journals/aha CD40 is one of the key molecules involved in the survival, growth and differentiation of B lymphocytes [1]. It is expressed on immature and mature B cells, dendritic cells, thymic epithelial cells and some carcinoma cells. Cross-linking of the CD40 receptor on Bcl-2-negative germinal center cells upregulates their Bcl-2 expression leading to prevention of apoptosis. The ligand CD40L (CD154) is mainly expressed in activated CD4-positive T cells and is one of the main costimulatury signals by which specific antigen-reactive T cells offer help to B cells presenting that antigen in the context of MHC class II [2]. Ligation of CD40 by its ligand, CD40L, results in inhibition of apoptosis, proliferation, differentiation and expressing of activation antigens including Fas [3]. The activation of CD40 molecules on normal B cells spontaneously induces CD95 expression on the cell surface. The survival or apoptosis of B cells in such situations seems to depend on the stage of activation, cytolytic factors and the degree of signal expression [4]. Activation of CD40 evokes a cytotoxic T cell response that eradicates lymphoma [5, 6]. Since activation of CD40 can evoke a cytotoxic T cell response that eradicates lymphoma, we tested the effect of activation of B lymphoma cells with fibroblasts transfected with CD40L. Cocultivation of B lymphoma cells with transfected fibroblasts led to a significant increase in CD40L and CD80 costimulatory molecule expression on the OCI-Ly8-LAM53 B lymphoma cell line. Similarly, several B lymphoma cell lines showed induction of apoptosis and decrease in proliferation rate.