Experimental attempt to produce mRNA transfected dendritic cells derived from enriched CD34+ blood progenitor cells (original) (raw)
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Generation of Dendritic Cells from Fresh and Frozen Cord Blood CD34+Cells
Cryobiology, 1998
Dendritic cells (DCs) are professional antigen-presenting cells that are required for the initiation of the immune response. DCs have been shown to be generated from CD34 ϩ pluripotent hematopoietic progenitor cells in the bone marrow and cord blood (CB), but relatively little is known about the effect of cryopreservation on functional maturation of DCs from hematopoietic stem cells. In this work we report the generation of DCs from cryopreserved CB CD34 ϩ cells. CB CD34 ϩ cells were cryopreserved at Ϫ80°C for 2 days. Cryopreserved CB CD34 ϩ cells as well as freshly isolated CB CD34 ϩ cells cultured with granulocyte-macrophage colonystimulating factor (GM-CSF)/stem cell factor (SCF)/tumor necrosis factor-␣ (TNF-␣) for 14 days gave rise to CD1a ϩ /CD4 ϩ /CD11c ϩ /CD14 Ϫ /CD40 ϩ /CD80 ϩ /CD83 ϩ /CD86 ϩ /HLA-DR ϩ cells with dendritic morphology. DCs derived from cryopreserved CB CD34 ϩ cells showed a similar endocytic capacity for fluorescein isothiocyanate-labeled dextran and lucifer yellow when compared with DCs derived from freshly isolated CB CD34 ϩ cells. Flow cytometric analysis revealed that two CC chemokine receptors (CCRs), CCR-1 and CCR-3, were expressed on the cell surface of DCs derived from both cryopreserved and freshly isolated CB CD34 ϩ cells, and these DCs exhibited similar chemotactic migratory capacities in response to regulated on activation normal T-cell expressed and secreted. DCs derived from cryopreserved as well as freshly isolated CB CD34 ϩ cells were more efficient than peripheral blood mononuclear cells in the primary allogeneic T-cell response. These results indicate that frozen CB CD34 ϩ cells cultured with GM-CSF/TNF-␣/SCF gave rise to dendritic cells which were morphologically, phenotypically and functionally similar to DCs derived from fresh CB CD34 ϩ cells.
European Journal of Immunology, 1996
Peripheral blood CD34' hematopoietic progenitor cells (PBPC) mediate hematopoietic reconstitution in cancer patients after autologous transplantation and can be expanded ex vivo in the presence of colony-stimulating factors. This study shows that functionally active antigen-presenting cells (APC) for soluble proteins are generated and expanded in these PBPC cultures. CD34' cells were cultured ex vivo in medium containing stem cell factor, interleukin-1P (IL-lP), IL-3, IL-6, and erythropoietin (EPO). The cells from these cultures developed into very potent APC of tetanus toxid and purified derivative of tuberculin for autologous T cells in vitro. The antigen-presenting capacity of these cells was maintained for at least 38 days of culture. These APC resembled immature cells of the myelomonocytic cell lineage by surface marker, immunocytochemistry and ultrastructural analysis. Such APC might be able to present antigens from certain tumors to the immune system. Abbreviations: DC: Dendritic cell PBPC: Peripheral blood hematopoietic progenitor cell SCF Stem cell factor EPO: Erythropoietin
Journal of Immunological Methods, 2004
Human dendritic cells (DCs) generated in culture from either monocytes or CD34+ hematopoietic progenitor cells (CD34-HPCs) have been used in cancer immunotherapy protocols with encouraging results. Yet an optimal strategy for the delivery of antigen(s) to DCs still remains to be established. Recent studies demonstrated the feasibility of mRNA transfection to load monocyte-derived DCs. It is not known, however, whether DCs derived by culturing CD34-HPC with GM-CSF and TNF-alpha for 9 days (CD34-DCs) can be efficiently transduced with mRNA. Here we show that clinical-grade CD34-DCs generated after 8 days of culture can be transfected with mRNA without significant alteration of cell viability. About 90% of cells transfected with GFP-RNA express GFP 24 h post-transfection. Remarkably, transfected CD34-DCs retain high levels of GFP expression for at least 14 days. CD34-DCs transfected with Flu-MP RNA were highly efficient in inducing the proliferation of Flu-MP-specific CD8+ T cells as measured by tetramer staining. Furthermore, the stimulated CD8+ T cells produced IFN-gamma upon antigenic stimulation and were able to kill targets pulsed with Flu-MP peptide. Both DC subsets in CD34-DCs, CD1a+-DC (Langerhans cells) and CD14+-DC (interstitial DC), were equally transfected with GFP-RNA, and yielded Flu-specific cytotoxic T cells upon transfection with Flu-MP RNA. Thus, RNA can be used to deliver antigens to two distinct myeloid DC subsets in CD34-DC cultures.
2023
Dendritic cells (DC) are antigen-presenting cells between innate and adaptive immune cells and commonly used as immunotherapy. Despite this promising potential, protocols detailing the specifics of the DC production are varied, affecting the potency of dendritic as immunotherapy. There are various factors affecting the production and DC potency, such as sample source, culture period, differentiation and maturation cytokines. Due to the limited number and quality of DC in humans, the monocyte could be isolated and differentiated to mature DC. The purity and viability monocytes shall be maintained to produce a high yield of DC. Negative sorting maintains the potency of DC as a therapeutic agent. Monocytes from umbilical cord blood (UCB) are naïve and can be differentiated to DC easily. Meanwhile, the tumor microenvironment (TME) may inhibit DC maturation from monocyte-derived peripheral blood. Without pro-inflammatory cytokines and a short maturation period, DC remain immature and fails to activate T cells. Long-period culture correlates with decreased DC viability and function. This review outlines several factors which can produce higher cytotoxic T cells and pro-inflammatory cytokines that might help each facility in developing its protocol to ensure the best procedure in DC production. Increasing purity and yield through close and automatic system under GMP production are mandatory to decrease risk of contamination during DC production.
Transfusion, 2009
In vitro generated dendritic cells (DCs) are widely used as adjuvants in cancer immunotherapy. The major sources for DC generation are monocytes and CD34+ cells. CD34+-derived DCs are less frequently used in clinical applications because it requires complex generation methods. Here a simple method for the large-scale generation of mature functional DCs from umbilical cord blood-derived CD34+ cells is described. STUDY DESIGN AND METHODS: CD34+ cells were first expanded with a combination of early acting growth factors in a medium containing autologous plasma. In the second step the DC precursors were further either enriched by plastic adherence or sorted on a cell sorter and differentiated as DCs. DCs generated by both methods were compared for their morphology, phenotype, and different functional variables. RESULTS: This culture system provided a large-scale expansion of CD34+ cells giving a mean fold increase of 615. The majority of the expanded cells were interstitial DC precursors, that is, CD14+-positive cells. In vitro generated immature DCs could be matured into functional DCs by appropriate maturation stimuli. DCs generated by the plastic adherence method had a better cytokine profile and strong mixed leukocyte reaction compared to those generated by cell sorting. CONCLUSION: A two-step culture system provides a large-scale expansion of CD34+ cells with a preferential lineage commitment toward CD14+ cells. Enrichment of these precursors with a simple plastic adherence technique results in generation of large numbers of mature, functional DCs. This method of in vitro DC generation will have applications in cancer immunotherapy. ABBREVIATIONS: DC(s) = dendritic cell(s); Flt-3L = fms-like tyrosine kinase 3 ligand; IMDM = Iscove's modified Dulbecco's medium; MLR = mixed leukocyte reaction; TPO = thrombopoietin; UCB = umbilical cord blood.
Archivum Immunologiae et Therapiae Experimentalis, 2009
Introduction: Dendritic cells (DCs) are required for initiation of the immune response and may therefore be used for the production of cancer vaccines. As mature DCs (mDCs) are the most potent antigen-presenting cells, there is increasing interest in generating them ex vivo. The present study was designed to obtain mDCs from CD34 + hematopoietic progenitors by culturing them in different media. Materials and Methods: Cord blood CD34 + hematopoietic progenitors were expanded for 7 days in FST medium containing fms-related tyrosine kinase 3 ligand (Flt3-L), stem cell factor (SCF), and thrombopoietin (TPO). Then the cells were divided into three parts and cultured for 21 days in different media: FST medium or FST enriched in interleukin (IL)-3 (FST3 medium) or supplemented with IL-7 and IL-13 (FST713 medium). At the end of culture part of the cells was harvested, counted, and analyzed while the other part was matured with proinflammatory cytokines for 2 days. The cells' phenotypes, ability to induce proliferation of allogeneic lymphocytes in the mixed lymphocyte reaction (allo-MLR), chemotaxis, phagocytosis, and O 2 production were determined. Results: The average fold increase of DCs at the end of culture in FST medium was 127, in FST3 1043, and in FST713 71. In comparison with the other media, FST713 medium supported the generation of mDCs that were characterized by higher expression of CD83, costimulatory molecules, and HLA-DR, enhanced ability to induce allo-MLR and migration to macrophage inflammatory protein (MIP) 3β, poor phagocytosis, and O 2 production. Conclusions: This study indicates that FST713 medium allows the generation of limited numbers of more mature DCs, while FST3 medium leads to the production of immature DCs in high numbers.
The Journal of Immunology
Bone marrow-derived dendritic cells (BmDC) are potent APC and can promote antitumor immunity in mice when pulsed with tumor Ag. This study aimed to define the culture conditions and maturation stages of BmDC that enable them to optimally function as APC in vivo. BmDC cultured under various conditions (granulocyte-macrophage CSF (GM-CSF) or GM-CSF plus IL-4 alone or in combination with Flt3 ligand, TNF-␣, LPS, or CD40 ligand (CD40L)) were analyzed morphologically, phenotypically, and functionally and were tested for their ability to promote prophylactic and/or therapeutic antitumor immunity. Each of the culture conditions generated typical BmDC. Whereas cells cultured in GM-CSF alone were functionally immature, cells incubated with CD40L or LPS were mature BmDC, as evident by morphology, capacity to internalize Ag, migration into regional lymph nodes, IL-12 secretion, and alloantigen or peptide Ag presentation in vitro. The remaining cultures exhibited intermediate dendritic cell maturation. The in vivo Ag-presenting capacity of BmDC was compared with respect to induction of both protective tumor immunity and immunotherapy of established tumors, using the poorly immunogenic squamous cell carcinoma, KLN205. In correspondence to their maturation stage, BmDC cultured in the presence of CD40L exhibited the most potent immunostimulatory effects. In general, although not entirely, the capacity of BmDC to induce an antitumor immune response in vivo correlated to their degree of maturation. The present data support the clinical use of mature, rather than immature, tumor Ag-pulsed dendritic cells as cancer vaccines and identifies CD40L as a potent stimulus to enhance their in vivo Ag-presenting capacity.
Production of functional dendritic cells from mouse bone marrow
Currently, immune cell-based therapies, particularly those that utilize dendritic cells (DCs), are a promising therapy approach for cancer treatment. Therefore, DC therapy is the focus of many studies in many laboratories worldwide that are developing novel cancer therapies. This study aimed to develop a reproducible procedure to produce functional DCs from mouse bone marrow for DC therapy research. Bone marrow was collected from mouse femur bones by flushing with buffered saline. These cells were used to isolate mononuclear cells (MNCs) by Ficoll gradient centrifugation. MNCs were cultured in RPMI 1640 medium supplemented with 20 ng/mL of IL-4 and 20 ng/mL of GMCSF to induce maturation of immature DCs. The results showed that this procedure induced cells exhibiting the DC phenotypes, such as the expression of CD40, CD80, and CD86, high phagocytic capacity, strong production of IL-12, and efficient stimulation of T-CD4 lymphocytes. These results suggest that this procedure can be used to produce functional DCs in future studies that use DCs for immune therapy.