In vitro and in vivo analysis of human cell-based immunotherapies for acute myeloid leukemia

Background: Antigen-loaded dendritic cells (DC) are capable of priming naïve T cells and therefore represent an attractive adjuvant for vaccine development in anti-tumor immunotherapy. Numerous protocols have been described to date using different maturation cocktails and time periods for the induction of mature DC (mDC) in vitro. For clinical application, the use of mDC that can be generated in only three days saves on the costs of cytokines needed for large scale vaccine cell production and provides a method to produce cells within a standard work-week schedule in a GMP facility. Methods: In this study, we addressed the properties of antigen uptake, processing and presentation by monocytederived DC prepared in three days (3d mDC) compared with conventional DC prepared in seven days (7d mDC), which represent the most common form of DC used for vaccines to date. Results: Although they showed a reduced capacity for spontaneous antigen uptake, 3d mDC displayed higher capacity for stimulation of T cells after loading with an extended synthetic peptide that requires processing for MHC binding, indicating they were more efficient at antigen processing than 7d DC. We found, however, that 3d DC were less efficient at expressing protein after introduction of in vitro transcribed (ivt)RNA by electroporation, based on published procedures. This deficit was overcome by altering electroporation parameters, which led to improved protein expression and capacity for T cell stimulation using low amounts of ivtRNA. Conclusions: This new procedure allows 3d mDC to replace 7d mDC for use in DC-based vaccines that utilize long peptides, proteins or ivtRNA as sources of specific antigen. Background The benefit of dendritic cells (DC) as adjuvants to induce tumor-specific cytotoxic T cells as well as helper T cells has been demonstrated in animal experiments and initial human trials [1,2]. In different tumor vaccines that were successfully applied in mice, mature DC (mDC) were used that were loaded with tumor antigens, supplied in various forms, including tumor extracts, short peptides or antigen-encoding RNA [3,4]. Several clinical trials using DC as tumor-vaccines have also been performed, where an increased T cell response against tumor-associated antigens could be observed [5]. DC are the most potent antigen-presenting cells for the stimulation of naïve T cells [6]. Immature DC (iDC) patrol peripheral tissues and take up antigens via macropinocytosis, phagocytosis or receptor-mediated endocytosis. After uptake of antigen, iDC process and present antigen-derived peptides on their MHC molecules. Since DC have the ability for cross-presentation, exogenous antigens can be presented on MHC-II as well as on MHC-I molecules [7]. Presentation of antigens by iDC leads to T cell anergy, deletion of T cells or the induction of IL-10-secreting T regulatory cells [8,9]. Following antigen uptake, iDC convert to a mature phenotype, characterized by the upregulation of different cell surface molecules, such as CD40, CD80 and CD83 [10]. These mDC also show higher expression of the chemokine-receptor CCR7, which plays an important role for DC homing to lymph nodes [11]. Upon arrival in the * Correspondence: [email protected] Helmholtz Zentrum München, German Research Center for Environmental Health, Institute of Molecular Immunology, Marchioninistr. 25, 81377 München, Germany Bürdek et al. Journal of Translational Medicine 2010, 8:90 http://www.translational-medicine.com/content/8/1/90 © 2010 Bürdek et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. lymph nodes, antigen-loaded mDC are able to prime naïve T cells, which then exit the lymph nodes after antigen-encounter. The primed effector T cells can recognize and eliminate specific target cells in the periphery. Different protocols for the generation of DC have been described to date. In vitro, DC can be developed from CD34 precursor cells or CD14 monocytes [10,12]. Monocytes can be enriched from peripheral blood mononuclear cells (PBMC) via plate adherence, by the use of anti-CD14 antibodies or by elutriation of leukapheresis products. iDC are usually induced by stimulation with GM-CSF and IL-4 [13,14]. It has also been shown that IL-4 could be replaced by IL-15, leading to the differentiation of monocytes into cells with properties of Langerhans cells [15-17]. Furthermore, DC can also be induced in the presence of IFN-b and IL-3 [18,19]. The induction of mDC can be initiated by several different stimuli, including microbial components (e.g. LPS as a Toll-like receptor 4 ligand), proinflammatory cytokines, viral-like stimuli [e.g. poly (I:C)] or T cell-derived molecules (e.g. CD40L) [16,18,20-24]. Depending on the composition of the maturation cocktails, mDC show different stimulatory and polarizing capacities on naïve T cells. Most protocols for the generation of mDC require approximately one week of cell culture. As such, Jonuleit and colleagues induced mDC on day five to six of a seven-day culture period by adding a four-component maturation cocktail (hereafter 4C cocktail), containing TNF-a, IL-1b, IL-6 and PGE2 [22], that is commonly used for the induction of DC maturation. It has been shown that mDC could also be generated within two days [25,26]. These “fast DC” were generally able to prime naïve T cells or stimulate effector cells [25,27,28]. The faster development of mDC may better reflect the situation in vivo [29]. In this study, we performed a systematic comparison of 3d and 7d mDC in terms of phenotype, chemokinedirected migration, antigen uptake and subsequent stimulation of cytotoxic T lymphocytes (CTL) after incubation with exogenous peptides or loading with antigen via electroporation. Because different forms of antigen are considered for use in DC-based vaccine development, it was important to demonstrate that mDC prepared in a three-day protocol would have antigen processing capacity comparable to the well known properties of 7d mDC. Materials and methods Peptides, antibodies and reagents The short MART-1/Melan-A26-35 peptide (ELAGIGILTV) (purchased from Metabion, Martinsried, Germany) and the long MART-1/Melan-A peptide (GSGHWDFAWPWGSGLAGIGILTV) (purchased from Biosyntan, Berlin, Germany) were reconstituted in 50% DMSO containing water at a concentration of 1 mg/ml and 20 mg/ml, respectively. Further dilutions were performed in medium. Monoclonal antibodies specific for DC surface molecules were directly labelled and purchased from Becton Dickinson (Heidelberg, Germany). The unlabelled CCR7 (clone 2H4) antibody (Becton Dickinson) and the MART-1/Melan-A antibody (clone A103; Dako Cytomation, Hamburg, Germany) were detected with the additional use of secondary antibodies [Cy5-coupled F(ab’)2-antibody (Dianova, Hamburg, Germany) and biotinylated F(ab’)2-antibody (Becton Dickinson)] and streptavidin-PE (Dianova). FITC-dextran from Sigma-Aldrich (Deisenhofen, Germany) and CCL19 from R&D Systems (Wiesbaden, Germany) were used. IL-1b, IL-4, IL-6 and TNF-a were purchased from R&D Systems, IL-2 from Chiron Behring (Marburg, Germany), GM-CSF (Leukine®) from Berlex (Seattle, USA) and PGE2 from Sigma-Aldrich. Tumor cell lines and CTL The melanoma cell lines Mel-93.04A12 (HLA-A2, Melan-A; gift from P. Schrier, Department of Immunohematology, Leiden University Hospital, Leiden, the Netherlands), Mel A375 (HLA-A2, Melan-A; CRL1619; ATCC) and SK-Mel-29 (HLA-A2; gift from T. Wölfel, Third Department of Medicine, Hematology and Oncology, Johannes Gutenberg University of Mainz, Mainz, Germany) were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 1 mM sodium pyruvate and non-essential amino acids. AK-EBV-B cells (gift from T. Wölfel) were cultured in RPMI 1640, containing 10% fetal calf serum. The HLA-A2-restricted, MART-1/Melan-A26-35 specific CTL A42 (gift from M. C. Panelli, National Institutes of Health, Bethesda, MD) were cultured in RPMI 1640 supplemented with 10% human serum (Lonza, Walkersville, USA), 2 mM L-glutamin, 1 mM sodium pyruvate, 100 IU penicillin/streptomycin, 0.5 μg/ml mycoplasma removal agent (MP Biomedicals, Eschwege, Germany) and 125 IU/ml IL-2. 5 × 10 CTL were restimulated every two weeks using 1 × 10 SK-Mel 29 and 2 × 10 AK-EBV-B (both irradiated with 100 Gy) in 1.5 ml A42 CTL medium per well of a 24-well plate. On the day of restimulation, 500 IU/ml IL-2 were added to the culture. A42 CTL were used for coculture experiments 8 days after restimulation. Generation and culture of 3d DC and 7d DC Monocytes were enriched from heparinized blood by Ficoll density gradient centrifugation and subsequent plate adherence or from a leukapheresis product via elutriation, as described previously [30]. For freezing of Bürdek et al. 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