Yield Of Dendritic Cells Research Articles

In vivo maturation and migration of dendritic cells.

The hallmark of immunity is antigen (Ag) recognition. From early life until death we face countless Ags; many are deleterious and come from the outside world. However, lymphocytes, the mediators of specific acquired immunity, are usually not exposed where foreign antigens are encountered. Thus a system must ferry these Ags from the periphery into inner tissues where lymphocytes comfortably reside or traffic. Clearly, translocating Ags from the periphery to the lymphoid niches is a pivotal function whereby the dendritic cell (DC) system initiates immune responses. DCs, however, are not passive Ag couriers; a dynamic process not fully understood is triggered by Ag deposition. Strategically poised at the boundaries between the inner and the outside world, in a way bridging innate and acquired immunity, DCs sample, trap, engulf, and digest Ags of very diverse origin, in what is called Ag processing. Antigenic fragments are then regurgitated, exposed at the cell surface through one of three molecular pathways [Major histocompatibility complex (MHC) CI and CII, and CD1)] responsible for an efficacious Ag presentation. Concomitantly, DC bear an arsenal of powerful costimulatory molecules [CD40; CD80; CD86; DC-specific/intercellular adhesion molecule type 3-grabbing, non-integrin (DC-SIGN)] and the potential to produce critical cytokines [chemokines, interleukin-12 (IL-12), etc.], thus ensuring the initiation and the fate of acquired immunity. The DC system is particularly efficient, not just translocating, but also transforming antigens sampled at the earlier local milieu into an immunologically accesible language, becoming an excellent reporter of the previous antigenic past, translating the conundrum posed by viruses, bacteria, proteins, etc., into T-cell receptor (TCR) ligands; a short-peptide vocabulary that T cells especially now understand. A highly dynamic process is thus triggered by encountering Ags; while these are carried and processed to be appropriately presented, DCs in turn experience a variety of changes: migratory, phenotypical, functional; all encompassed in the term maturation. Essentially, DC maturation means to change from an antigen-capturing to an antigen-presenting, T-cell-priming mode, a process whereby DCs convert antigens into efficacious immunogens, express the necessary cytokines and costimulatory molecules, thus appropriately initiating the specific, acquired clonal immunity.

Development of a clinical-scale method for generation of dendritic cells from PBMC for use in cancer immunotherapy

There is growing interest in the use of dendritic cells (DCs) for treatment of malignancy and infectious disease. Our goal was to develop a clinical scale method to prepare autologous DCs for cancer clinical trials. PBMC were collected from normal donors or cancer patients by automated leukapheresis, purified by counterflow centrifugal elutriation and placed into culture in polystyrene flasks at 1 x 10(6) cells/mL for 5-7 days at 37 degrees C, with 5% CO(2), with IL-4 and GM-CSF. Conditions investigated included media formulation, supplementation with heat in activated allogeneic AB serum or autologous plasma and time to harvest (Day 5 or Day 7). DCs were evaluated for morphology, quantitative yield, viability, phenotype and function, including mixed leukocyte response and recall response to tetanus toxoid and influenza virus. DCs with a typical immature phenotype (CD14-negative, CD1a-positive, mannose receptor-positive, CD80-positive, CD83-negative) were generated most consistently in RPMI 1640 supplemented with 10% allogeneic AB serum or 10% autologous plasma. Cell yield was higher at Day 5 than Day 7, without detectable differences in phenotype or function. In pediatric sarcoma patients, autologous DCs had enhanced function compared with monocytes from which they were generated. In this patient group, starting with 8.0 +/- 3.7 x 10(8) fresh or cryopreserved autologous monocytes, DC yield was 2.1 +/- 1.0 x 10(8) cells, or 29% of the starting monocyte number. In the optimized clinical-scale method, purified peripheral monocytes are cultured for 5 days in flasks at 1 x 10(6) cells/mL in RPMI 1640, 10% allogeneic AB serum or autologous plasma, IL-4 and GM-CSF. This method avoids the use of FBS and results in immature DCs suitable for clinical trials.

Potent Inhibition of Dendritic Cell Differentiation and Maturation by Vitamin D Analogs

We show that the immunosuppressive effects of 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) are due, in part, to inhibition of the T cell stimulatory functions of dendritic cells (DCs). Addition of 10−12 and 10−8 M 1α,25(OH)2D3 to murine DC cultures resulted in a concentration-dependent reduction in levels of class II MHC and the co-stimulatory ligands B7-1, B7-2, and CD40 without affecting the number of DCs generated. Higher concentrations of 1α,25(OH)2D3 reduced DC yield. The capacity of DCs to induce proliferation of purified allogeneic T cells was reduced by 1α,25(OH)2D3. The vitamin D3 analog, 1α,25(OH)2-16-ene-23-yne-26,27-hexafluoro-19-nor-D3, exerted identical effects at 100-fold lower concentrations. Inhibition of DC maturation and stimulatory function was absent in cultures from mice genetically lacking vitamin D receptors (VDR). Vitamin D analogs effectively reduce DC function via VDR-dependent pathways.

CD13/N-aminopeptidase is involved in the development of dendritic cells and macrophages from cord blood CD34+ cells

Expression of CD13/N-aminopeptidase may reflect cell activation and growth. We examined its role regarding cell growth in cultures of cord blood CD34(+) cells with stem cell factor/Flt-3 ligand/granulocyte-macrophage colony-stimulating factor/tumor necrosis factor-alpha. Indeed, 82% +/- 6% of cells from culture day 5 were CD13(hi), 25% +/- 8% of which were still Lin-. About 50% of CD13(hi)Lin- cells, which comprise progenitors of dendritic cells (DC), monocytes/macrophages and granulocytes, and 30% of CD13(lo)Lin- cells were CD34(+). Sorted CD34(+)CD13(hi)Lin- cells, cultured further for 7 days with the same cytokines, expanded 31-fold and CD34(-)CD13(hi)Lin- cells 7-fold, but CD34(+)CD13(lo)Lin- and CD34(-)CD13(lo)Lin- cells did not grow. Thus, cell growth correlated with CD13 expression, all the more so that cells were CD34(+). Actinonin, the most potent N-aminopeptidase inhibitor, was used to engage CD13 on sorted CD13(hi)Lin- cells and on culture day-7 bulk cells. In both cases, this resulted in reversible cell growth arrest, with 30% to 60% fewer cells in the G2/S-M phase than in controls. Interestingly, similar effects were noted with CD13 monoclonal antibody TUK1, which does not inhibit N-aminopeptidase activity, but not with N-aminopeptidase-blocking antibodies WM15 and F23. All cycling cells appeared susceptible to actinonin, which induced cell apoptosis at the same time as Bcl-2 was downregulated and caspase-3 activity increased, but finally percentages and yields of DC and macrophage precursors were affected more than those of granulocytic cells. Thus, through engagement of N-aminopeptidase enzymatic site but possibly also of an independent determinant, CD13 plays a role in the growth of DC/macrophage progenitors and precursors. (Blood. 2000;95:453-460)

Immunomodulatory dendritic cells generated from nonfractionated bulk peripheral blood mononuclear cell cultures induce growth of cytotoxic T cells against renal cell carcinoma.

Dendritic cells (DCs) loaded with tumor antigens have the potential to become a powerful tool for clinical cancer treatment. Recently, the authors showed that a tumor-specific immune response can be elicited in culture via stimulation with autologous renal tumor lysate (Tuly)-loaded DCs that were generated from cytokine-cultured adherent peripheral blood mononuclear cells (PBMCs). Here, the authors show that immunomodulatory DCs can be generated directly from nonfractionated bulk PBMC cultures. Kinetic studies of DC differentiation and maturation in PBMC cultures were performed by monitoring the acquisition of DC-associated molecules using fluorescence-activated cell sorting analysis to determine the percentage of positive immunostained cells and the mean relative linear fluorescence intensity (MRLFI). Compared with conventional adherent CD14+ cultures, which have mostly natural killer, T, and B cells removed before cytokine culture, bulk PBMC cultures exhibited an early loss of CD14+ cells (day 0 = 78.8%, day 2 = 29.6% versus day 0 = 74%, day 2 = 75%) with an increase in yield of mature DCs (DC19- CD83+) (day 5 = 17%, day 6 = 21%, day 7 = 22% versus day 5 = 11%, day 6 = 15%, day 7 = 23%). Although a comparable percentage of DCs expressing CD86+ (B7-2), CD40+, and HLA-DR+ were detected in both cultures, higher expression levels were detected in DCs derived from bulk culture (CD86 = MRLFI 3665.1 versus 2662.1 on day 6; CD40 = MRLFI 1786 versus 681.2 on day 6; HLA-DR = MRLFI 6018.2 versus 3444.9 on day 2). Cytokines involved in DC maturation were determined by polymerase chain reaction demonstrating interleukin-6 (IL-6), IL-12, interferon-gamma, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-alpha mRNA expression by bulk culture cells during the entire 9-day culture period. This same cytokine mRNA profile was not found in the conventional adherent DC culture. Autologous renal Tuly (30 micrograms protein/10(7) PBMCs) enhanced human leukocyte antigen expression by DCs (class I = 7367.6 versus 4085.4 MRFLI; class II = 8277.2 versus 6175.7 MRFLI) and upregulated cytokine mRNAs levels. Concurrently, CD3+ CD56-, CD3+ CD25+, and CD3+ TCR+ cell populations increased and cytotoxicity against autologous renal cell carcinoma tumor target was induced. Specific cytotoxicity was augmented when cultures were boosted continuously with IL-2 (20 U/mL biological response modifier program) plus Tuly stimulation. These results suggest that nonadherent PBMCs may participate in enhancing DC maturation. Besides the simplicity of this culture technique, bulk DC cultures potentially may be used with the same efficiency as conventional purified DCs. Furthermore, bulk culture-derived DCs may be used directly in vivo as a tumor vaccine, or for further ex vivo expansion of co-cultured cytotoxic T cells to be used for adoptive immunotherapy.

Fetal Calf Serum-Free Generation of Functionally Active Murine Dendritic Cells Suitable for In Vivo Therapeutic Approaches

Standard protocols to generate mouse dendritic cells (DC) generally use culture medium supplemented with fetal calf serum; however, reinjection in vivo of DC cultured in fetal calf serum results in priming to xenogeneic proteins that clearly limits the use of such DC. We therefore established a fetal calf serum-free culture system for the generation of murine DC from bone marrow precursors. DC can be generated fetal calf serum-free using RPMI supplemented with 1.5% syngeneic mouse serum. Although the yield of DC grown under fetal calf serum-free conditions was somewhat lower than that of the standard culture, large numbers of DC could be generated without the exposure to xenogeneic proteins. The yield of fetal calf serum-free cultured DC was further enhanced by addition of the proinflammatory cytokines TNF-alpha and IL-1beta with the combination resulting in up to 10% more DC. Phenotypically, CD11c + DC cultured fetal calf serum-free homogenously coexpressed the DC-specific molecule DEC-205 as well as the costimulatory molecules CD40, CD80, and CD86. In contrast, only a subpopulation of the CD11c + DC cultured in fetal calf serum-containing medium coexpressed these molecules. Functionally, fetal calf serum-free DC showed strong stimulatory capacity for naïve allogeneic CD4 + and CD8 + T cells. Importantly, fetal calf serum-free DC showed spontaneous in vivo migratory activity. Moreover, 5 x 105 subcutaneously injected TNBS-conjugated fetal calf serum-free DC were able to mediate contact sensitivity. Furthermore, the intravenous or subcutaneous injection of a single dose of 5 x 105 OVA-pulsed fetal calf serum-free DC resulted in the induction of an OVA-specific immune response in naïve TCR transgenic animals. Thus DC cultured under fetal calf serum-free conditions are suitable instruments for in vivo therapeutic approaches, especially in autoimmune models. DC vaccines/dendritic cell development/fetal calf serum-free culture conditions for DC/in vivo therapeutic DC approaches.

Immunobiology of DC in NOD mice.

NOD mice spontaneously develop diabetes between 15 and 20 weeks of age, which is preceded by insulitis characterized by the infiltration of lymphocytes. Dendritic cells (DC) are among the first cells to infiltrate the islet and they have been implicated in the pathogenesis of the disease. Our work has been concerned with the detailed characterization of four distinct DC populations in NOD mice: two derived from bone marrow (BM) cells cultured in either granulocyte-macrophage colony-stimulating factor (GM-CSF) plus interleukin-4 (IL-4) or GM-CSF alone and two from the spleen of Flt3 ligand (Flt3L) -treated mice, isolated on the basis of CD8alpha expression. Phenotypic and functional differences between these DC subsets in NOD mice have been identified. In addition, we obtained a lower yield of NOD BM-derived DC and they expressed higher levels of cell-surface CD40 and IL-12 p40 mRNA than BM-derived DC from the diabetes-resistant strain, B10.BR. We have also investigated the ability of these DC populations to modulate the development and progression of diabetes in NOD mice.

Functional and Phenotypic Analysis of Thymic CD34+CD1a− Progenitor-Derived Dendritic Cells: Predominance of CD1a+ Differentiation Pathway

Whether thymic dendritic cells (DC) are phenotypically and functionally distinct from the monocyte lineage DC is an important question. Human thymic progenitors differentiate into T, NK, and DC. The latter induce clonal deletion of autoreactive thymocytes and therefore might be different from their monocyte-derived counterparts. The cytokines needed for the differentiation of DC from thymic progenitors were also questioned, particularly the need for GM-CSF. We show that various cytokine combinations with or without GM-CSF generated DC from CD34+CD1a- but not from CD34+CD1a+ thymocytes. CD34+ thymic cells generated far fewer DC than their counterparts from the cord blood. The requirement for IL-7 was strict whereas GM-CSF was dispensable but nonetheless improved the yield of DC. CD14+ monocytic intermediates were not detected in these cultures unless macrophage-CSF (M-CSF) was added. Cultures in M-CSF generated CD14-CD1a+ DC precursors but also CD14+CD1a- cells. When sorted and recultured in GM-CSF, CD14+ cells down-regulated CD14 and up-regulated CD1a. TNF-alpha accelerated the differentiation of progenitors into DC and augmented MHC class II transport to the membrane, resulting in improved capacity to induce MLR. The trafficking of MHC class II molecules was studied by metabolic labeling and immunoprecipitation. MHC class II molecules were transported to the membrane in association with invariant chain isoforms in CD14+ (monocyte)-derived and in CD1a+ thymic-derived DC but not in monocytes. Thus, thymic progenitors can differentiate into DC along a preferential CD1a+ pathway but have conserved a CD14+ maturation capacity under M-CSF. Finally, CD1a+-derived thymic DC and monocyte-derived DC share very close Ag-processing machinery.

Generation of dendritic cells from patients with chronic myelogenous leukemia.

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) specialized to internalize, process, and present antigen. They have the capacity to stimulate the primary immune response of resting T-cells. We generated DCs from the adherent cell fraction of peripheral blood, as well as from purified CD34+ cells from CML patients. Characterizing DCs from ten CML patients by flow cytometry, we found that these cells are highly positive for HLA-DR, CD1a, CD23, and CD80 and negative for CD14, CD15, and CD16. The yield of DCs ranged from 19.5 to 68%. In addition, we used a functional test of FITC-dextran uptake to verify that early DCs take up large particles (0.5-3 microm) by macropinocytosis while monocytes do not. FITC-dextran uptake was detected by flow cytometry, showing that DCs had accumulated these fluorescent particles. Electron-microscopic analysis showed no major morphological differences between normal and CML-derived DCs. Furthermore, cultured DCs were isolated by FAC sorting for CD1a and HLA-DR expression. In these highly purified cells the Ph chromosome was detected by interphase fluorescence in situ hybridization (FISH) and by fluorescence immunophenotyping and interphase cytogenetics as a tool for the investigation of neoplasms (FICTION); 30-85% of DCs generated were Ph-chromosome positive. It might therefore be possible not only to prime T-cells with bcr/abl-specific synthetic peptides, but also to stimulate T-cells directly with Ph-positive DCs. Use of DCs might serve as a novel therapeutic approach in CML patients, due to their ability to induce highly specific T-cell responses in an autologous system.

Proliferation in Monocyte-Derived Dendritic Cell Cultures Is Caused by Progenitor Cells Capable of Myeloid Differentiation

Dendritic cells (DC) can be generated by culture of adherent peripheral blood (PB) cells in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). There is controversy as to whether these DC arise from proliferating precursors or simply from differentiation of monocytes. DC were generated from myeloid-enriched PB non-T cells or sorted monocytes. DC generated from either population functioned as potent antigen-presenting cells. Uptake of [3H]-thymidine was observed in DC cultured from myeloid-enriched non-T cells. Addition of lipopolysaccharide or tumor necrosis factor-α led to maturation of the DC, but did not inhibit proliferation. Ki67+ cells were observed in cytospins of these DC, and by double staining were CD3−CD19−CD11c−CD40−and myeloperoxidase+, suggesting that they were myeloid progenitor cells. Analysis of the starting population by flow cytometry demonstrated small numbers of CD34+CD33−CD14− progenitor cells, and numerous granulocyte-macrophage colony-forming units were generated in standard assays. Thus, production of DC in vitro from adherent PB cells also enriches for progenitor cells that are capable of proliferation after exposure to GM-CSF. Of clinical importance, the yield of DC derived in the presence of GM-CSF and IL-4 cannot be expanded beyond the number of starting monocytes.© 1998 by The American Society of Hematology.

Proliferation in Monocyte-Derived Dendritic Cell Cultures Is Caused by Progenitor Cells Capable of Myeloid Differentiation

Dendritic cells (DC) can be generated by culture of adherent peripheral blood (PB) cells in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). There is controversy as to whether these DC arise from proliferating precursors or simply from differentiation of monocytes. DC were generated from myeloid-enriched PB non-T cells or sorted monocytes. DC generated from either population functioned as potent antigen-presenting cells. Uptake of [3H]-thymidine was observed in DC cultured from myeloid-enriched non-T cells. Addition of lipopolysaccharide or tumor necrosis factor- led to maturation of the DC, but did not inhibit proliferation. Ki67+ cells were observed in cytospins of these DC, and by double staining were CD3−CD19−CD11c−CD40−and myeloperoxidase+, suggesting that they were myeloid progenitor cells. Analysis of the starting population by flow cytometry demonstrated small numbers of CD34+CD33−CD14− progenitor cells, and numerous granulocyte-macrophage colony-forming units were generated in standard assays. Thus, production of DC in vitro from adherent PB cells also enriches for progenitor cells that are capable of proliferation after exposure to GM-CSF. Of clinical importance, the yield of DC derived in the presence of GM-CSF and IL-4 cannot be expanded beyond the number of starting monocytes. © 1998 by The American Society of Hematology.

The Role of Tumor Necrosis Factor α in Modulating the Quantity of Peripheral Blood-Derived, Cytokine-Driven Human Dendritic Cells and Its Role in Enhancing the Quality of Dendritic Cell Function in Presenting Soluble Antigens to CD4+ T Cells In Vitro

Because dendritic cells (DC) are critically involved in both initiating primary and boosting secondary host immune responses, attention has focused on the use of DC in vaccine strategies to enhance reactivity to tumor-associated antigens. We have reported previously the induction of major histocompatibility complex class II-specific T-cell responses after stimulation with tumor antigen-pulsed DC in vitro. The identification of in vitro conditions that would generate large numbers of DC with more potent antigen-presenting cell (APC) capacity would be an important step in the further development of clinical cancer vaccine approaches in humans. We have focused attention on identifying certain exogenous cytokines added to DC cultures that would lead to augmented human DC number and function. DC progenitors from peripheral blood mononuclear cells (PBMC) were enriched by adherence to plastic, and the adherent cells were then cultured in serum-free XVIVO-15 medium (SFM) for 7 days with added granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). At day 7, cultures contained cells that displayed the typical phenotypic and morphologic characteristics of DC. Importantly, we have found that the further addition of tumor necrosis factor α (TNFα) at day 7 resulted in a twofold higher yield of DC compared with non–TNFα-containing DC cultures at day 14. Moreover, 14-day cultured DC generated in the presence of TNFα (when added at day 7) demonstrated marked enhancement in their capacity to stimulate a primary allogeneic mixed leukocyte reaction (8-fold increase in stimulation index [SI]) as well as to present soluble tetanus toxoid and candida albicans (10- to 100-fold increases in SI) to purified CD4+ T cells. These defined conditions allowed for significantly fewer DC and lower concentrations of soluble antigen to be used for the pulsing of DC to efficiently trigger specific T-cell proliferative responses in vitro. When compared with non–TNFα-supplemented cultures, these DC also displayed an increased surface expression of CD83 as well as the costimulatory molecules, CD80 and CD86. Removal of TNFα from the DC cultures after 2 or 4 days reduced its enhancing effect on DC yield, phenotype, and function. Thus, the continuous presence of TNFα over a 7-day period was necessary to achieve the maximum enhancing effect observed. Collectively, our findings point out the importance of exogenous TNFα added to cultures of cytokine-driven human DC under serum-free conditions, which resulted in an enhanced number and function of these APC. On the basis of these results, we plan to initiate clinical vaccine trials in patients that use tumor-pulsed DC generated under these defined conditions.

The Role of Tumor Necrosis Factor α in Modulating the Quantity of Peripheral Blood-Derived, Cytokine-Driven Human Dendritic Cells and Its Role in Enhancing the Quality of Dendritic Cell Function in Presenting Soluble Antigens to CD4+ T Cells In Vitro

AbstractBecause dendritic cells (DC) are critically involved in both initiating primary and boosting secondary host immune responses, attention has focused on the use of DC in vaccine strategies to enhance reactivity to tumor-associated antigens. We have reported previously the induction of major histocompatibility complex class II-specific T-cell responses after stimulation with tumor antigen-pulsed DC in vitro. The identification of in vitro conditions that would generate large numbers of DC with more potent antigen-presenting cell (APC) capacity would be an important step in the further development of clinical cancer vaccine approaches in humans. We have focused attention on identifying certain exogenous cytokines added to DC cultures that would lead to augmented human DC number and function. DC progenitors from peripheral blood mononuclear cells (PBMC) were enriched by adherence to plastic, and the adherent cells were then cultured in serum-free XVIVO-15 medium (SFM) for 7 days with added granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). At day 7, cultures contained cells that displayed the typical phenotypic and morphologic characteristics of DC. Importantly, we have found that the further addition of tumor necrosis factor α (TNFα) at day 7 resulted in a twofold higher yield of DC compared with non–TNFα-containing DC cultures at day 14. Moreover, 14-day cultured DC generated in the presence of TNFα (when added at day 7) demonstrated marked enhancement in their capacity to stimulate a primary allogeneic mixed leukocyte reaction (8-fold increase in stimulation index [SI]) as well as to present soluble tetanus toxoid and candida albicans (10- to 100-fold increases in SI) to purified CD4+ T cells. These defined conditions allowed for significantly fewer DC and lower concentrations of soluble antigen to be used for the pulsing of DC to efficiently trigger specific T-cell proliferative responses in vitro. When compared with non–TNFα-supplemented cultures, these DC also displayed an increased surface expression of CD83 as well as the costimulatory molecules, CD80 and CD86. Removal of TNFα from the DC cultures after 2 or 4 days reduced its enhancing effect on DC yield, phenotype, and function. Thus, the continuous presence of TNFα over a 7-day period was necessary to achieve the maximum enhancing effect observed. Collectively, our findings point out the importance of exogenous TNFα added to cultures of cytokine-driven human DC under serum-free conditions, which resulted in an enhanced number and function of these APC. On the basis of these results, we plan to initiate clinical vaccine trials in patients that use tumor-pulsed DC generated under these defined conditions.

Generation and functional characterization of human dendritic cells derived from CD34 cells mobilized into peripheral blood: comparison with bone marrow CD34+ cells.

Dendritic cells (DCs) are the most powerful professional antigen-presenting cells (APC), specializing in capturing antigens and stimulating T-cell-dependent immunity. In this study we report the generation and characterization of functional DCs derived from both steady-state bone marrow (BM) and circulating haemopoietic CD34+ cells from 14 individuals undergoing granulocyte colony-stimulating factor (G-CSF) treatment for peripheral blood stem cells (PBSC) mobilization and transplantation. Clonogenic assays in methylcellulose showed an increased frequency and proliferation of colony-forming unit-dendritic cells (CFU-DC) in circulating CD34+ cells, compared to that of BM CD34+ precursors in response to GM-CSF and TNF-alpha with or without SCF and FLT-3L. Moreover, peripheral blood (PB) CD34+ cells generated a significantly higher number of fully functional DCs, as determined by conventional mixed lymphocyte reactions (MLR), than their BM counterparts upon different culture conditions. DCs derived from mobilized stem cells were also capable of processing and presenting soluble antigens to autologous T cells for both primary and secondary immune response. Replacement of the early-acting growth factors SCF and FLT-3L with IL-4 at day 7 of culture of PB CD34+ cells enhanced both the percentage of total CD1a+ cells and CD1a+ CD14- cells and the yield of DCs after 14 d of incubation. In addition, the alloreactivity of IL-4-stimulated DCs was significantly higher than those generated in the absence of IL-4. Furthermore, autologous serum collected during G-CSF treatment was more efficient than fetal calf serum (FCS) or two different serum-free media for large-scale production of DCs. Thus, our comparative studies indicate that G-CSF mobilizes CD34+ DC precursors into PB and circulating CD34+ cells represent the optimal source for the massive generation of DCs. The sequential use of early-acting and intermediatelate-acting colony-stimulating factors (CSFs) as well as the use of autologous serum greatly enhanced the growth of DCs. These data may provide new insights for manipulating immunocompetent cells for cancer therapy.

Pro-inflammatory cytokines and prostaglandins induce maturation of potent immunostimulatory dendritic cells under fetal calf serum-free conditions.

Culture conditions for human dendritic cells (DC) have been developed by several laboratories. Most of these culture methods, however, have used conditions involving fetal calf serum (FCS) to generate DC in the presence of granulocyte-macrophage colony-stimulating factor and interleukin (IL)-4. Recently, alternative culture conditions have been described using an additional stimulation with monocyte-conditioned medium (MCM) and FCS-free media to generate DC. As MCM is a rather undefined cocktail, the yield and quality of DC generated by these cultures varies substantially. We report that a defined cocktail of tumor necrosis factor (TNF)-alpha, IL-1beta and IL-6 equals MCM in its potency to generate DC. Addition of prostaglandin (PG)E2 to the cytokine cocktail further enhanced the yield, maturation, migratory and immunostimulatory capacity of the DC generated. More importantly, culture conditions also influenced the outcome of the T cell response induced. DC cultured with TNF-alpha/IL-1/IL-6 or MCM alone induced CD4+ T cells that release intermediate levels of interferon (IFN)-gamma and no IL-4 or IL-10. Production of IFN-gamma was significantly induced by addition of PGE2, while no effect on production of IL-4 or IL-10 was observed. Even more striking differences were observed for CD8+ T cells. While MCM conditions only induced IFN-gamma(low), IL-4(neg) cells, TNF-alpha/IL-1/IL-6 promoted growth of IFN-gamma(intermediate), IL-4(neg) CD8+ T cells. Addition of PGE2 again only further polarized this pattern enhancing IFN-gamma production by alloreactive CD8+ T cells in both cultures without inducing type 2 cytokines. Taken together, the data indicate that the defined cocktail TNF-alpha/IL-1/IL-6 can substitute for MCM and that addition of PGE2 further enhances the yield and quality of DC generated. TNF-alpha/IL-1, IL-6 + PGE2-cultured DC seem to be optimal for generation of IFN-gamma-producing CD4/CD8+ T cells.

Dendritic cells generated from the blood of patients with multiple myeloma are phenotypically and functionally identical to those similarly produced from healthy donors.

Using a combination of GM-CSF, SCF, flk-2/flt-3 ligand, and IL-4, dendritic cells (DC) have been generated in vitro from the adherent fraction of mononuclear cells isolated from the blood of patients with MM. Analysis of cell yield showed no significant difference in DC yield (numbers or percentage of leucocytes) or total number of leucocytes generated in myeloma cultures compared to similar cultures prepared using mononuclear cells from the blood of healthy donors. The mean number of DC produced after 10d of culture were 8.19 x 10(5) and 9.87 x 10(5) cells (41% and 51% of all leucocytes) for the myeloma and normal cultures respectively. Flow cytometry investigation of phenotypic markers including CD1a, HLA-DR, CD80 (BB1/B7.1) and CD86 (B70/B7.2), and functional status (stimulatory potential in allogeneic mixed leucocyte reactions (MLR)) confirmed the generation of cells phenotypically identified as cultured DC. In addition, these cells were more effective than PBMC at stimulating allogeneic PBMC proliferation. These data demonstrate no difference between DC generated from patients with MM and healthy donors. This study was considered a prerequisite for future investigations directed towards developing effective immunotherapies for myeloma.

A short method to obtain high yields of dendritic cells from human peripheral blood monocytes

A short method to obtain high yields of dendritic cells from human peripheral blood monocytes

A short method to obtain high yields of dendritic cells from human peripheral blood monocytes

A short method to obtain high yields of dendritic cells from human peripheral blood monocytes

Dendritic cell development in culture from thymic precursor cells in the absence of granulocyte/macrophage colony-stimulating factor.

The earliest lymphoid precursor population in the adult mouse thymus had previously been shown to produce not only T cells, but also dendritic cell (DC) progeny on transfer to irradiated recipients. In this study, culture of these isolated thymic precursors with a mixture of cytokines induced them to proliferate and to differentiate to DC, but not to T lineage cells. At least 70% of the individual precursors had the capacity to form DC. The resultant DC were as effective as normal thymic DC in the functional test of T cell stimulation in mixed leukocyte cultures. The cultured DC also expressed high levels of class I and class II major histocompatibility complex, together with CD11c, DEC-205, CD80, and CD86, markers characteristic of mature DC in general. However, they did not express CD8 alpha or BP-1, markers characteristic of normal thymic DC. The optimized mixture of five to seven cytokines required for DC development from these thymic precursors did not include granulocyte/macrophage colony stimulating factor (GM-CSF), usually required for DC development in culture. The addition of anti-GM-CSF antibody or the use of precursors from GM-CSF-deficient mice did not prevent DC development. Addition of GM-CSF was without effect on DC yield when interleukin (IL) 3 and IL-7 were present, although some stimulation by GM-CSF was noted in their absence. In contrast, DC development was enhanced by addition of the Flt3/Flk2 ligand, in line with the effects of the administration of this cytokine in vivo. The results indicate that the development of a particular lineage of DC, probably those of lymphoid precursor origin, may be independent of the myeloid hormone GM-CSF.

Generation of mature dendritic cells from human blood An improved method with special regard to clinical applicability

Two methods to generate human dendritic cells from hematopoietic precursor cells in peripheral blood have recently been published. One approach utilizes the rare CD34 + precursors and GM-CSF plus TNF-α. The other method makes use of the more abundant CD34 − precursor population and GM-CSF plus IL-4. Here we report a method that is based on the latter approach. However, the GM-CSF and IL-4 treated cells are not stable mature dendritic cells, e.g., the characteristic morphology and nonadherence of dendritic cells is lost if the cytokines are removed. We describe the need for a monocyte-conditioned medium to generate fully mature and stable dendritic cells. This is achieved by adding a 3 day ‘maturation culture’ to the initial 6–7 day culture in the presence of GM-CSF and IL-4. Macrophage-conditioned medium contains the critical maturation factors. Mature dendritic cells are defined by their pronounced display of motile cytoplasmic processes (‘veils’), their high capacity to induce proliferative responses in resting T cells, particularly in naive umbilical cord T cells, their down-regulated antigen processing ability, and their characteristic phenotype: expression of CD83, high levels of MHC molecules and CD86, lack of CD115 and perinuclear dot-like CD68 staining. These features are stable for at least 3 days upon withdrawal of cytokines and conditioned media. IL-4 can be replaced by IL-13. When CD34 + progenitors are depleted from blood, there is only a minor reduction in the yield of dendritic cells by this method. We have adapted the method to consider several variables that are pertinent to clinical use, including a change from fetal calf serum to human plasma and to media approved for clinical use like X-VIVO or AIM-V. 1% plasma and RPMI 1640 are currently optimal. Additional reagents used for cell culture (Ig, cytokines) and cell separation (immunomagnetic beads) are approved for or already used in clinical applications. For 40 ml blood, the yield is 0.8–3.3 × 10 6 mature dendritic cells as defined by the expression of the new dendritic cell-restricted marker CD83. CD83 + cells constitute between 30 and 80% of all cells recovered at the end of the culture period. Yields can be enhanced up to six-fold if the blood donors are pretreated with G-CSF. Stable, mature dendritic cells generated by this method should be a powerful tool for active immunotherapy.