Catherine De Greef

Lentivirally transduced dendritic cells as a tool for cancer immunotherapy

Dendritic cells (DC) are the professional antigen‐presenting cells of the immune system, fully equipped to prime naive T cells and thus essential components for cancer immunotherapy.

Generation of large numbers of dendritic cells in a closed system using Cell Factories

There is a growing interest in using dendritic cells (DC) for vaccine approaches in the treatment of cancer and infectious diseases. This requires a reproducible method for the generation of large numbers of DC in a closed culture system suitable for clinical use and conforming to the current guidelines of good manufacturing practices. We designed a system in which the DC were generated in a closed system from adherent monocytes using Cell Factories (DC-CF). Monocytes were enriched from apheresis products by adherence and then cultured in the presence of AB serum or autologous plasma and GM-CSF and IL-4 for 6 days. The DC generated in Cell Factories were extensively compared to research-grade DC generated in conventional tissue culture flasks (DC-TCF). At day 6, the immature DC were harvested and the yield, the viability, the immunophenotype and the functional characteristics of the DC were compared.DC-CF and DC-TCF showed similar viability and purity and scored equally when tested for stability, dextran and latex bead uptake, in MLR and in the activation of influenza-specific memory cells after electroporation with influenza matrix protein 1 (IMP1) mRNA. These data indicated that large numbers of functional clinical-grade DC could be generated from adherent cells in a closed system using Cell Factories.

THE SERUM RESISTANCE-ASSOCIATED (SRA) GENE OF TRYPANOSOMA BRUCEI RHODESIENSE ENCODES A VARIANT SURFACE GLYCOPROTEIN-LIKE PROTEIN

In the Trypanosoma brucei species, T. b. rhodesiense and T. b. gambiense represent the human infective host range variants, while T. b. brucei is lysed upon exposure to a cytotoxic factor in normal human serum. T. b. rhodesiense can occur in a serum-resistant and a serum-sensitive form. The resistance towards normal human serum was shown to be a labile character and to be determined by the environment in which the parasites live. We have clearly demonstrated the presence of RNA transcripts unique to the resistant forms of T. b. rhodesiense. These transcripts encode a protein with VSG characteristics. The DNA fragment isolated previously, which hybridises with the resistance-specific mRNA sequence, appears to be a pseudogene belonging to the same gene family.

A gene expressed only in serum-resistant variants of Trypanosoma brucei Rhodesiense

The human infective African trypanosomes are host range variants of Trypanosoma brucei which are resistant to a lytic component in primate serum. T. b. rhodesiense occurs both as a form sensitive to lysis by normal human serum and as a form resistant to this lysis. Switching from one phenotype to the other has been observed in both directions. In the cloned T. b. rhodesiense ETAR1-repertoire we have detected 1.5-kb mRNAs only present in the resistant forms. In T. b. gambiense, which always occurs as a normal human serum-resistant form, no such transcript could be detected, indicating that another mechanism of resistance is involved here. Starting from an independent non-cloned T. b. rhodesiense population isolated from an infected patient, both resistant and sensitive trypanosomes have been prepared. Northern blot analysis of the total RNA prepared from these populations has revealed again the differential occurrence of the resistance-specific transcript, indicating that we are dealing with a general phenomenon associated with serum resistance in T. b. rhodesiense. As expected, Southern blot analyses have demonstrated that both serum-resistant and serum-sensitive forms of T. b. rhodesiense contain the gene coding for this transcript.

Efficient genetic modification of murine dendritic cells by electroporation with mRNA

Recently, human dendritic cells (DCs) pulsed with mRNA encoding a broad range of tumor antigens have proven to be potent activators of a primary anti–tumor-specific T-cell response in vitro. The aim of this study was to improve the mRNA pulsing of murine DC. Compared to a standard lipofection protocol and passive pulsing, electroporation was, in our hands, the most efficient method. The optimal conditions to electroporate murine bone marrow–derived DCs with mRNA were determined using enhanced green fluorescent protein and a truncated form of the nerve growth factor receptor. We could obtain high transfection efficiencies around 70–80% with a mean fluorescence intensity of 100–200. A maximal expression level was reached 3 hours after electroporation. A clear dose–response effect was seen depending on the amount of mRNA used. Importantly, the electroporation process did not affect the viability nor the allostimulatory capacity or phenotype of the DC. To study the capacity of mRNA-electroporated DCs to present antigen in the context of MHC classes I and II, we made use of chimeric constructs of ovalbumin. The dose-dependent response effect and the duration of presentation were also determined. Together, these results demonstrate that mRNA electroporation is a useful method to generate genetically modified murine DC, which can be used for preclinical studies testing immunotherapeutic approaches.

Identification of new antigenic peptide presented by HLA-Cw7 and encoded by several MAGE genes using dendritic cells transduced with lentiviruses.

Antigens encoded by MAGE genes are of particular interest for cancer immunotherapy because they are tumor specific and shared by tumors of different histological types. Several clinical trials are in progress with MAGE peptides, proteins, recombinant poxviruses, and dendritic cells (DC) pulsed with peptides or proteins. The use of gene-modified DC would offer the major advantage of a long-lasting expression of the transgene and a large array of antigenic peptides that fit into the different HLA molecules of the patient. In this study, we tested the ability of gene-modified DC to prime rare Ag-specific T cells, and we identified a new antigenic peptide of clinical interest. CD8+ T lymphocytes from an individual without cancer were stimulated with monocyte-derived DC, which were infected with a second-generation lentiviral vector encoding MAGE-3. A CTL clone was isolated that recognized peptide EGDCAPEEK presented by HLA-Cw7 molecules, which are expressed by >40% of Caucasians. Interestingly, this new tumor-specific antigenic peptide corresponds to position 212–220 of MAGE-2, -3, -6, and -12. HLA-Cw7 tumor cell lines expressing one of these MAGE genes were lysed by the CTL, indicating that the peptide is efficiently processed in tumor cells and can therefore be used as target for antitumoral vaccination. The risk of tumor escape due to appearance of Ag-loss variants should be reduced by the fact that the peptide is encoded by several MAGE genes.

Induction of Influenza Matrix Protein 1 and MelanA-specific T lymphocytes in vitro using mRNA-electroporated dendritic cells

Genetically modified dendritic cells (DC) constitute a promising approach in cancer immunotherapy. Viral gene delivery systems have been shown to be very efficient strategies, but safety concerns for their clinical use in immunotherapy remain an important issue. Recently, the technique of mRNA electroporation was described as a very efficient tool for the genetic modification of human monocyte-derived DC. Here, we show that transgene expression can be modulated by varying the amount of mRNA used for electroporation. We document that CD40 ligation leads to a significant production of IL-12 by the electroporated DC, although the level of IL-12 production is somewhat lower than for non- or mock-electroporated DC. Furthermore, we show that the electroporated DC can be frozen and thawed without loss of viability or function and that Influenza virus Matrix Protein 1 mRNA electroporated DC are capable of inducing a memory cytotoxic T lymphocyte response and are more potent in doing so than mRNA-pulsed DC. Similar results were obtained with MelanA/MART-1 mRNA electroporated DC. These results clearly indicate that mRNA-electroporated DC represent powerful candidates for use as tumor vaccines and could constitute an improvement compared with vaccines using peptide-pulsed DC.

Activation of Monocytes via the CD14 Receptor Leads to the Enhanced Lentiviral Transduction of Immature Dendritic Cells

In this study, we compared dendritic cells (DCs) differentiated from positively selected monocytes (CD14-DCs) to DCs differentiated from adherence-selected monocytes (adh-DCs) with emphasis on lentiviral transduction. Using a second-generation, triple-helix containing, self-inactivating lentiviral vector at a multiplicity of infection (MOI) of 15, we observed enhanced transduction of CD14-DCs (72.8 +/- 5.3%, mean fluorescence intensity [MFI] = 166 +/- 76) compared to adh-DCs (32.3 +/- 13.1%, MFI = 119 +/- 76, n = 5). More importantly, the efficiency to transduce adh-DCs was significantly increased when monocytes were incubated with antiCD14 antibody coupled beads, anti-CD14 antibodies, or lipopolysaccharide (LPS), reaching transduction efficiencies up to 86.6%, 53.3%, and 80.9%, respectively. We showed that this enhanced transduction was correlated to an activation of the monocytes, characterized by the up regulation of the cytokines interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha and the de novo synthesis of IL-6 and IL-10. However, the enhanced transduction of immature CD14-DCs was not correlated with a progression in the cell cycle from G(0) to G(1). We further showed that CD14-DCs were phenotypically comparable to adh-DCs. Functional analysis revealed that there were no differences in allostimulatory capacity, production of IL-12 p70 on CD40 ligation or expression of IL-1beta, IL-6, IL-8, IL-10, IL-12, and TNF-alpha as evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). Finally, we showed that lentivirally transduced CD14-DCs were equally capable as adh-DCs in stimulating MAGE-A3 antigen-specific CD4(+) and CD8(+) T cells in vitro.

Production of fibronectin and adherence to fibronectin by human myeloma cell lines

SUMMARY. In the present study we examined the production of fibronectin (FN) in 10 human myeloma cell lines (HMCL). By Northern blot analysis we could detect the presence of FN‐mRNA in most of these lines. A majority of the cell lines (LP‐1, OPM1, SKMM‐2, EJM, JJN3 and ARH‐77) hybridized with two probes recognizing total FN while the mRNA of one cell line (LB84‐1) was shown to hybridize also with a probe recognizing the EDA segment of cellular FN. In one cell line (L363) FN‐mRnA could only be detected after PCR amplification. Using an enzyme‐linked immunosorbent assay, we could also demonstrate that HMCL secrete FN in their culture medium. Seven myeloma cell lines that produce FN showed a significant adherence to soluble FN. By blocking experiments, this adhesion was found to be mediated by the VLA‐4 (α4β1) receptor. The production of fibronectin and the expression of a functional receptor for this protein may represent independent features of myeloma cells but may also be functionally linked. Since fibronectin has recently been identified as a crucial co‐factor of IL6 in the regulation of the terminal B cell differentiation, the endogenous FN production may be part of an autocrineline process mediating the autonomous growth of these cell lines. Alternatively, the FN production may also reflect a mechanism that myeloma cells use to communicate with their natural environment, i.e. the bone marrow stroma. In order to examine whether FN can be regarded as a reliable tumour marker for myeloma, serum FN levels were measured in 24 myeloma patients and were found to be increased when compared to normal controls (mean values 40.7 mg/dl versus 29.2 mg/dl; P < 0.01). It was, however, not possible to relate the FN concentration to serum IL6 and β2m levels or bone marrow plasmacytosis.

Human serum-sensitive Trypanosoma brucei rhodesiences: a comparison with serologically identical human serum-resistant clones

Trypanosoma brucei rhodesiense clones, which are susceptible to lysis by normal human serum, were isolated from 3 different human serum-resistant clones originally derived from strain ETat 1.10. Serologically, these pairs of serum-sensitive and serum-resistant clones displayed the same variant surface glycoprotein (VSG) on their surface. Acquisition of human serum sensitivity correlated with susceptibility to lysis by human high density lipoprotein, a trypanocidal factor in normal human serum. Analysis of these paired populations by two-dimensional gel electrophoresis of whole trypanosomes and various subcellular fractions failed to reveal any differences in mobility of VSG and other proteins. Northern blot analysis of mRNAs from serum-sensitive and serum-resistant clones showed no differences when probed with a previously described resistance-specific probe. In addition, the ethanolamine membrane transport system and the overall membrane lipid fluidity did not reveal any detectable biochemical or biophysical differences in membrane properties. If resistance to lysis is indeed mediated by membrane changes at the enzymatic or structural level, the data presented suggest that the gene product(s) responsible for this change in human serum sensitivity may be present in very small quantities.