Generation Of Human Dendritic Cells Research Articles

An animal component-free, serum-free culture method for generation of human dendritic cells

Abstract Dendritic cells (DCs) are central regulators of adaptive immune responses that potentially have major applications in the immunotherapy of cancer and autoimmune disorders. DCs are rare in peripheral blood (PB) and are therefore often generated in vitro from PB monocytes. We developed an animal component-free (ACF) and serum-free medium, ImmunoCult-ACF DC, that supports the generation of DCs from monocytes in culture. CD14+ monocytes were isolated using EasySep immunomagnetic separation and cultured for 5 days in ImmunoCult-ACF DC medium and cytokines (GM-CSF and IL-4) to promote their differentiation into CD14−CD83− immature DCs. The immature DCs were then stimulated for 2 days with the ImmunoCult-DC Maturation Supplement, a combination of cytokines and pro-inflammatory mediators, to promote their maturation to CD14− CD83+ DCs (93 ± 5% CD83+, n=23). The yield of mature DCs was 42 ± 23% (n=23), similar to the yield in a control serum-free medium (49 ± 24%, n=16). Mature DCs produced high levels of IL-12, on average 371 pg/mL (range: 27–1756, n=9 in cultures initiated with 106 monocytes). Mature DCs loaded with CMV, EBV and Flu virus peptides efficiently stimulated the proliferation of autologous CD8+ T cells in 7 day co-cultures. An 18-fold increase in T cell numbers (range: 5–31, n=4) was observed when compared to control culture conditions containing only T cells without mature DCs. In conclusion, functional DCs can be efficiently generated by differentiation of monocytes in a completely animal (including human) component-free and serum-free medium. This medium and culture method will enable further research into the development and application of DCs for cellular therapy.

Dendritic cells enhance the activity of human MUC1-stimulated mononuclear cells against breast cancer

Dendritic cells (DCs) are among the most potent antigen-presenting cells (APCs), stimulating peripheral blood mononuclear cells (PBMCs) to generate antigen-specific cytotoxic T lymphocytes (CTLs). The objectives of this study were to determine if interleukin (IL)-4 is beneficial or detrimental for the generation of human DCs in vitro and to understand whether DCs generated in vitro in the presence or absence of IL-4 stimulate the killing of adenocarcinoma cells by CTLs in vivo. Mucin 1 (MUC1), a glycoprotein found on the surface of adenocarcinoma cells was used to load DCs. MUC1-loaded DCs generated in the absence of IL-4 were superior to their counterparts produced with IL-4 in stimulating PBMCs to kill human breast cancer MCF-7 cells in vitro. A corollary in vivo protection experiment was performed by injecting immunodeficient NOD-SCID mice with MCF-7 cells s.c. and MUC1-loaded CTLs, PBMCs, or DCs generated in the absence of IL-4, i.p. Mice that received CTLs and MUC1-loaded DCs on days 0, 2, 4, 9, 14 and 19 were completely protected against the development of MCF-7-derived tumors, while other schedules conferred lower protection. Therefore, tumor antigen-loaded DCs enhance the efficacy of adoptive CTL transfer, and should thus be considered for combinatorial immunotherapeutic regimens.

A novel and simple method for generation of human dendritic cells from unfractionated peripheral blood mononuclear cells within 2 days: its application for induction of HIV-1-reactive CD4+ T cells in the hu-PBL SCID mice

Because dendritic cells (DCs) play a critical role in the regulation of adaptive immune responses, they have been ideal candidates for cell-based immunotherapy of cancers and infections in humans. Generally, monocyte-derived DCs (MDDCs) were generated from purified monocytes by multiple steps of time-consuming physical manipulations for an extended period cultivation. In this study, we developed a novel, simple and rapid method for the generation of type-1 helper T cell (Th1)-stimulating human DCs directly from bulk peripheral blood mononuclear cells (PBMCs). PBMCs were cultivated in the presence of 20 ng/ml of granulocyte-macrophage colony-stimulating factor, 20 ng/ml of interleukin-4 (IL-4) and 1,000 U/ml of interferon-β for 24 h followed by 24 h maturation with a cytokine cocktail containing 10 ng/ml of tumor necrosis factor-α (TNF-α), 10 ng/ml of IL-1β and 1 μg/ml of prostaglandin E2. The phenotype and biological activity of these new DCs for induction of allogeneic T cell proliferation and cytokine production were comparable to those of the MDDCs. Importantly, these new DCs pulsed with inactivated HIV-1 could generated HIV-1-reactive CD4+ T cell responses in humanized mice reconstituted with autologous PBMCs from HIV-1-negative donors. This simple and quick method for generation of functional DCs will be useful for future studies on DC-mediated immunotherapies.

Isolation and generation of human dendritic cells.

Dendritic cells are highly specialized antigen-presenting cells (APC), which may be isolated or generated from human blood mononuclear cells. Although mature blood dendritic cells normally represent ∼0.2% of human blood mononuclear cells, their frequency can be greatly increased using the cell enrichment methods described in this unit. More highly purified dendritic cell preparations can be obtained from these populations by sorting of fluorescence-labeled cells. Alternatively, dendritic cells can be generated from monocytes by culture with the appropriate cytokines, as described here. In addition, a negative selection approach is provided that may be employed to generate cell preparations that have been depleted of dendritic cells to be used for comparison in functional studies.

Generation of Human Dendritic Cells That Simultaneously Secrete IL-12 and Have Migratory Capacity by Adenoviral Gene Transfer of hCD40L in Combination With IFN-γ

Dendritic cells (DCs) are professional antigen presenting cells and have key functions in the initiation of immune responses. Hence, antigen-loaded DCs have become important tools for active-specific immunotherapy. In addition to defining strategies for antigen loading, effective T-cell activation by DCs will depend on vaccination protocols that facilitate DC migration to secondary lymphoid tissues and expression of costimulatory molecules and cytokines. Adenoviral gene transfer has been successfully implemented for genetic antigen loading of DCs. In this study, we exploit an adenoviral vector encoding human CD40 ligand (CD40L), Ad5hCD40L, to establish DCs that feature both migration potential and prolonged secretion of the key T-helper 1 cytokine interleukin-12p70 (IL-12p70). Transduction of human monocyte-derived DCs with Ad5hCD40L resulted in efficient CD40L expression, which was detected only intracellularly, and in secretion of IL-12p70. Addition of recombinant interferon (IFN)-gamma shortly after DC transduction substantially increased IL-12p70 secretion. Maturation of DCs was achieved with a standard cytokine maturation cocktail (MC) containing prostaglandin E2 which, however, abolished IL-12p70 secretion by Ad5hCD40L-transduced cells in the absence of IFN-gamma. Only DCs treated with Ad5hCD40L, MC, and IFN-gamma migrated efficiently towards CCL19 and continued to secrete IL-12p70. Finally, DCs transduced with both Ad5hCD40L and an adenoviral vector encoding the melanoma antigen MelanA/MART-1 and treated with MC and IFN-gamma efficiently primed naive autologous CD8+ T cells into antigen-specific cytotoxic T lymphocyte. This strategy to generate DCs that exert both migration capacity and prolonged IL-12p70 secretion after intracellular CD40L expression and IFN-gamma treatment has the potential to further improve current DC vaccination protocols.

A novel approach for the generation of human dendritic cells from blood monocytes in the absence of exogenous factors

Human dendritic cells (DCs) for research and clinical applications are typically derived from purified blood monocytes that are cultured in a cocktail of cytokines for a week or more. Because it has been suggested that these cytokine-derived DCs may be deficient in some important immunological functions and might not accurately represent antigen presenting cell (APC) populations found under normal conditions in vivo, there is an interest in developing methods that permit the derivation of DCs in a more physiologically relevant manner in vitro. Here, we describe a simple and reliable technique for generating large numbers of highly purified DCs that is based on a one-way migration of blood monocytes through a layer of human umbilical vein endothelial cells (HUVECs) that are cultured to confluency in the upper chamber of a Transwell device. The resultant APCs, harvested from the lower Transwell chamber, resemble other cultured DC populations in their expression of major histocompatibility (MHC) and costimulatory molecules, ability to phagocytose protein antigens and capacity to trigger primary antigen-specific T cell responses. This technique offers several advantages over the standard method of in vitro cytokine-driven DC development, including: (1) the rapidity of this approach, as DC differentiation occurs in only 2 days, (2) the differentiation process itself, which is more akin to the development of DCs under physiologic conditions and (3) the cost-effectiveness of the system, since no monocyte pre-selection is required and DC development occurs in the absence of expensive recombinant cytokines.

A Novel Approach for the Generation of Human Dendritic Cells from Blood Monocytes in the Absence of Exogenous Factors

Human dendritic cells (DCs) for research and clinical applications are typically derived from purified blood monocytes cultured in a mixture of cytokines for a week or more. Because it has been suggested that these cytokine‐derived DCs may be deficient in some important immunological functions attributed to antigen presenting cell (APC) populations found under normal conditions in vivo, there is an interest in developing more physiologically relevant methods of DC generation in vitro. Here, we describe a simple, reliable technique for generating large numbers of highly purified DCs based on a one‐way migration of blood monocytes through a confluent layer of human umbilical vein endothelial cells grown in the upper chamber of a Transwell device. The resultant APCs, harvested from the lower Transwell chamber, resemble other cultured DC populations in their expression of MHC and costimulatory molecules, ability to phagocytose protein antigens and capacity to elicit primary antigen‐specific T cell responses. This technique offers many advantages over the standard method of in vitro cytokine‐driven DC development: (1) DC differentiation occurs in only two days, (2) the differentiation process is more akin to the development of DCs under physiologic conditions and (3) lack of monocyte pre‐selection and recombinant cytokine addition makes the system cost‐effective.

GMCSF in the absence of other cytokines sustains human dendritic cell precursors with T cell regulatory activity and capacity to differentiate into functional dendritic cells

Dendritic cell precursors, from human peripheral blood, express epitopes reactive with monoclonal antibodies specific for the empty conformation of HLA-DR1. Expression is substantially up-regulated during GMCSF-induced differentiation to immature dendritic cells, but is strongly down-regulated by IL-4. In the conventional protocol for in vitro generation of human dendritic cells from monocyte precursors, both GMCSF and IL-4 are used together, with IL-4 thought to have an effect on preventing macrophage outgrowth but not substantially altering the dendritic cell maturation pathway, whereas conventional protocols for generation of murine dendritic cells use GMCSF alone. We characterized human monocytes cultured in the presence of GMCSF and in the absence of IL-4, and found that the resultant cultures are stable for long periods in vitro, and have T cell stimulatory and immunodulatory activity characteristic of immature dendritic cells. Their response to maturation stimuli is weak in the absence of IL-4 but these cells retain the ability to differentiate into fully functional mature dendritic cells upon IL-4 treatment. We suggest that these cells may provide a useful model to investigate tolerogenic function as a developmental feature of DC and to understand molecular events involved in IL-4 priming for maturation.

DENDRITIC CELL BASED VACCINES: PROGRESS IN IMMUNOTHERAPY STUDIES FOR PROSTATE CANCER

No effective treatment is currently available for metastatic prostate cancer. Dendritic cell (DC) based cancer vaccine research has emerged from the laboratories to human clinical trials. We describe progress in the development of DC based prostate cancer vaccine. The literature was reviewed for major contributions to a growing number of studies that demonstrate the potential of DC based immunotherapeutics for prostate cancer. Background topics relating to DC based immunotherapy theory and practice are also addressed. DCs have been recognized as the most efficient antigen presenting cells that have the capacity to initiate naive T cell response in vitro and in vivo. During their differentiation and maturation pathways, dendritic cells can efficiently capture, process and present antigens for T cell activation. These characteristics make DC an attractive choice as the cellular adjuvant for cancer vaccines. Advances in DC generation, loading, and maturation methodologies have made it possible to generate clinical grade vaccines for various human trials. More than 100 DC vaccine trials, including 7 studies of patients with advanced prostate cancer have been reported to date. These vaccines were generally well tolerated with no significant adverse toxicity reported. Clinical responders have been identified in these studies. The new prospects opened by DC based vaccines for prostate cancer are fascinating. When compared to conventional treatments, DC vaccinations have few side effects. Improvements in patient selection, vaccine delivery strategies, immune monitoring and vaccine manufacturing will be crucial in moving DC based prostate cancer vaccines closer to the clinics.

Large-scale monocyte enrichment coupled with a closed culture system for the generation of human dendritic cells

Conventional methods for generating monocyte-derived dendritic cells (DC) for clinical trials utilize the property of plastic adherence to select monocytes from leukapheresis samples. This method is labor-intensive and has the potential for contamination at various steps. We evaluated a large-scale monocyte enrichment procedure using a cell selector (Isolex 300i®) followed by culture in a sterile bag system (Stericell®) for generation of DC. DC generated in tissue culture flasks after monocyte selection by plastic adherence were compared to those generated in Stericell® bags after monocyte enrichment by negative selection with the Isolex® 300i. DC were matured with lipopolysaccharide and pulsed with a peptide derived from the melanoma antigen gp100. Peptide-pulsed DC cultured by the two techniques were evaluated for phenotype, viability, ability to induce allogeneic and peptide-specific autologous proliferative responses as well as peptide-specific cytotoxic T-cell responses. The mean monocyte yield from leukapheresis collections was 17±2.4%, which increased to 52±11% after Isolex® selection. The DC yield of plated mononuclear cells from flasks or bags was 2.7±0.96% and 4.84±2.65%, respectively. DC cultured by both methods expressed high levels of CD86, CD80, CD40, CD83, CD44, CD11c and CD58, and was comparable in their ability to induce allogeneic and peptide-specific autologous proliferative responses as well as gp100 peptide-specific cytotoxic T-cell responses. These results indicate that potent monocyte-derived DC can be generated in a closed culture bag system after monocyte enrichment by immunomagnetic negative selection. Due to the closed nature of the enrichment and culture systems, the potential for contamination is minimized. This protocol is well suited for culturing large numbers of DC for clinical immunotherapy trials.

Efficient ex vivo generation of human dendritic cells from mobilized CD34+ peripheral blood progenitors.

We tried to efficiently generate human dendritic cells (DCs) from CD34+ peripheral blood hematopoietic progenitor cells mobilized by high-dose chemotherapy and subsequent administration of granulocyte colony-stimulating factor, using a liquid suspension culture system. Among various combinations, the combination of c-kit ligand, flt-3 ligand, c-mpl ligand (TPO), and interleukin (IL)-4 most potently generated the number of CD1a+CD14- DCs in cultures containing granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha (TNF-alpha). The delayed addition of IL-4 on day 6 of culture gave rise to an additional increase in the yield of CD1a+CD14-DCs that were characterized by the expression of HLA-ABC, HLA-DR, CD80, CD86, and CD83. The majority of the sorted CD1a-CD14+ cells derived from 6-day culture of CD34+ cells gave rise to CD1a+CD14- DCs and CD1a-CD14+ macrophages on day 12 of culture in the presence and absence of IL-4, respectively. These findings suggest that IL-4 promotes the differentiation of CD1a- CD14+ cells derived from mobilized CD34+ peripheral blood hematopoietic progenitors to CD1a+ CD14- DCs. The majority of these DCs expressed CD68 but not the Langerhans-associated granule antigen, a finding that suggests they emerge through the monocyte differentiation pathway. The addition of TPO and IL-4 to cultures did not affect the potential of DCs to stimulate the primary allogeneic T-cell response. These findings demonstrated that the combination of c-kit ligand plus flt-3 ligand plus TPO with GM-CSF plus TNF-alpha, followed by IL-4, is useful for ex vivo generation of human DCs from mobilized CD34+ peripheral blood progenitors.

Long-Term Culture of Human CD34+ Progenitors With FLT3-Ligand, Thrombopoietin, and Stem Cell Factor Induces Extensive Amplification of a CD34−CD14− and a CD34−CD14+ Dendritic Cell Precursor

Current in vitro culture systems allow the generation of human dendritic cells (DCs), but the output of mature cells remains modest. This contrasts with the extensive amplification of hematopoietic progenitors achieved when culturing CD34+ cells with FLT3-ligand and thrombopoietin. To test whether such cultures contained DC precursors, CD34+ cord blood cells were incubated with the above cytokines, inducing on the mean a 250-fold and a 16,600-fold increase in total cell number after 4 and 8 weeks, respectively. The addition of stem cell factor induced a further fivefold increase in proliferation. The majority of the cells produced were CD34−CD1a− CD14+(p14+) and CD34−CD1a−CD14−(p14−) and did not display the morphology, surface markers, or allostimulatory capacity of DC. When cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), both subsets differentiated without further proliferation into immature (CD1a+, CD14−, CD83−) macropinocytic DC. Mature (CD1a+, CD14−, CD83+) DCs with high allostimulatory activity were generated if such cultures were supplemented with tumor necrosis factor- (TNF). In addition, p14− cells generated CD14+ cells with GM-CSF and TNF, which in turn, differentiated into DC when exposed to GM-CSF and IL-4. Similar results were obtained with frozen DC precursors and also when using pooled human serum AB+ instead of bovine serum, emphasizing that this system using CD34+ cells may improve future prospects for immunotherapy.

Long-Term Culture of Human CD34+ Progenitors With FLT3-Ligand, Thrombopoietin, and Stem Cell Factor Induces Extensive Amplification of a CD34−CD14− and a CD34−CD14+ Dendritic Cell Precursor

AbstractCurrent in vitro culture systems allow the generation of human dendritic cells (DCs), but the output of mature cells remains modest. This contrasts with the extensive amplification of hematopoietic progenitors achieved when culturing CD34+ cells with FLT3-ligand and thrombopoietin. To test whether such cultures contained DC precursors, CD34+ cord blood cells were incubated with the above cytokines, inducing on the mean a 250-fold and a 16,600-fold increase in total cell number after 4 and 8 weeks, respectively. The addition of stem cell factor induced a further fivefold increase in proliferation. The majority of the cells produced were CD34−CD1a− CD14+(p14+) and CD34−CD1a−CD14−(p14−) and did not display the morphology, surface markers, or allostimulatory capacity of DC. When cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), both subsets differentiated without further proliferation into immature (CD1a+, CD14−, CD83−) macropinocytic DC. Mature (CD1a+, CD14−, CD83+) DCs with high allostimulatory activity were generated if such cultures were supplemented with tumor necrosis factor-α (TNF). In addition, p14− cells generated CD14+ cells with GM-CSF and TNF, which in turn, differentiated into DC when exposed to GM-CSF and IL-4. Similar results were obtained with frozen DC precursors and also when using pooled human serum AB+ instead of bovine serum, emphasizing that this system using CD34+ cells may improve future prospects for immunotherapy.

Isolation and generation of human dendritic cells.

Dendritic cells are highly specialized antigen-presenting cells (APC), which may be isolated or generated from human blood mononuclear cells. Although mature blood dendritic cells normally represent 0.2% of human blood mononuclear cells, their frequency can be greatly increased using the cell enrichment methods described in this unit. More highly purified dendritic cell preparations can be obtained from these populations by sorting of fluorescence-labeled cells. Alternatively, dendritic cells can be generated from monocytes by culture with the appropriate cytokines, as described here. In addition, a negative selection approach is provided that may be employed to generate cell preparations that have been depleted of dendritic cells to be used for comparison in functional studies.