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Keywords:

  • Tubulo-vesicular structures;
  • MIIC compartments;
  • Maturation;
  • Cancer immunotherapy;
  • Cord blood

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Dendritic cells (DCs) are important for the induction of primary T-cell responses and may serve as “biologic adjuvants” in therapeutic protocols. However, given the “plasticity” of this antigen-presenting cell, it remains unclear which DC type (source, subtype, and stage of differentiation) should be applied clinically. To provide additional insight in this selection process, we have, for the first time, analyzed the in vitro differentiation of CD34+ precursor-derived and monocyte-derived DCs for ultrastructure, phenotype, and function. The ultrastructural intracytoplasmic differentiation of DCs correlated with increasing T-cell stimulatory activity of these cells. “Early-stage”-DCs proliferate, exhibit high levels of soluble antigen uptake, and moderate T-cell stimulatory capacity, and are characterized by centrally located nuclei and numerous enlarged mitochondria. “Intermediate-stage”-DCs are enlarged cells with enhanced T-cell stimulatory activity and pronounced cytoplasmic protein synthesis machinery. “Late-stage” (LS)-DCs exhibit a mature secretory cell phenotype and low proliferative index. They express high levels of the HLA-DR, CD40L, B7-1, and B7-2 molecules and CD83, a specific marker of mature DCs, and appear maximally stimulatory to T cells. Ultrastructurally, LS-DCs feature an accentric nucleus, an enlarged cytoplasm, containing numerous secretory storage vesicles, along with a fully developed Golgi complex. LS-DCs exhibited numerous multivesicular and multilaminar structures containing major histocompatibility complex class II molecules, consistent with the MIIC (peptide-loading) compartment. In extended studies, cultured CD14+ monocyte-derived DCs displayed a similar, but accelerated, temporal differentiation staging pattern.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Dendritic cells (DCs) play crucial roles in the activation and potentiation of antigen-specific T-cell responses. DCs originate in bone marrow and are distributed throughout body tissues. They perform a sentinel function, responding to such danger signals as trauma, inflammation, bacterial products, and cytokines [1]. Resting DCs capture and process soluble or particulate antigens in late endosomal and lysosomal compartments that are rich in major histocompatibility complex (MHC) class II molecules [2,, 3], and upon activation, migrate to secondary lymphoid organs. During this trafficking process, DCs traverse through lymphatic or blood vessel endothelium and complete their maturation [4]. Mature DCs upregulate their expression of peptide/MHC class I and class II complexes that can be recognized by antigen-specific T cells. In addition to cognate T-cell receptor ligation of DC-expressed MHC complexes, T-cell activation is facilitated by signaling mediated via intercellular adhesion molecules, CD40/CD40 ligand interaction, and the costimulating molecules, B7-1 (CD80) and B7-2 (CD86) [5].

Stem-cell-derived- or monocyte-derived DCs can be sustained ex vivo with GM-CSF and other cytokines and can be matured in vitro by bacteria, viruses, fungi [6–, 8], bacterial products, such as lipopolysaccharide (LPS) [9,, 10], inflammatory stimuli [11], and cytokines, including interferons, interleukin-1 (IL-1), tumor necrosis factor alpha (TNF-α) and its superfamily, RANTS [12–, 16], and most often, by CD40 ligand, which plays an important role in DC/T-cell interaction [17,, 18]. Maturation stimulates increased expression of HLA-DR, CD40, and costimulatory molecules and secretion of cytokines. However, ultrastructural correlates of DCs have not been extensively studied.

In the current work, we are the first to demonstrate three stages of in vitro differentiation for CD34+ stem-cell-derived DCs, ranging from immature early-stage (ES)-DCs, observed at 2 weeks, “intermediate-stage” (IS)-DCs, observed after 2-3 weeks, and mature, late-stage (LS)-DCs, observed after 3-4 weeks of culture. LS-DCs exhibited the highest degree of cell-surface expression of HLA-DR and costimulatory molecules, MHC class II-loading and secretory compartments and antigen-specific Th1-type T-cell stimulatory capacity (proliferation and interferon gamma [IFN-γ] secretion). Of potential biological importance, we show, for the first time, the accumulation of multivesicular structures in the cytoplasm of LS- DCs that may play an important role in the promotion of T-cell immunity. Three analogous differentiation stages were also observed, with an accelerated kinetic, in cultured CD14+ monocyte-derived DCs.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Isolation of Both CD34+ Cells from Umbilical Cord Blood and CD14+ Cells from Peripheral Blood

Normal human umbilical cord blood was obtained as discarded material from the Obstetrics and Gynecology Department of the Allegheny General Hospital (Pittsburgh, PA). Buffy coats of normal donors were obtained from the Central Blood Bank of Pittsburgh, PA without donor identification. Fresh cord blood samples were diluted 1:4 with Ca++- and Mg++-free phosphate-buffered solution ([PBS] GIBCO BRL; Great Island, NY; http://www.invitrogen.com), and 25 ml of cell suspension were loaded onto 15 ml of LymphoPrep solution (1.077 g/ml; Nycomed Pharma AS; Oslo, Norway; http://www.nycomed.com). Mononuclear cells (MNCs) were isolated from the interface after centrifugation (400 g, 25 minutes, 20°C) and washed twice in PBS. Both CD34+ and CD14+ cells were separated using MACS magnetic cell sorting (Miltenyi Biotec, Inc.; Sunnyvale, CA; http://www.miltenyibiotec.com). MNCs were labeled either with QBEND 10, anti-CD34, or anti-CD14 antibody 24UK4 attached to the superparamagnetic microparticles. Bead-bound cells were positively selected on MiniMacs separation columns in a highly magnetized field. The percentage of CD34+ cells was determined by fluorescence-activated cell sorting (FACScan) analysis and varied between 87% and 96%. The cell viability exceeded 95% in all experiments.

Cytokines and Long-Term DC Cultures

DCs (1-5 × 105 cells/flask) were cultured in 12.5 cm2 tissue culture flasks (Falcon, Becton Dickinson; Franklin Lakes, NJ; http://www.bd.com) in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS), penicillin (100 U/ml), streptomycin (100 μg/ml), and 2 mM L-glutamine (GIBCO-BRL). The following cytokines were used: recombinant human GM-CSF (10 ng/ml; provided by Immunex Corp.; Seattle, WA); stem cell factor ([SCF] 10 ng/ml; Amgen; Thousand Oaks, CA; http://www.amgen.com); TNF-α (2-5 ng/ml); and IL-4 (2 ng/ml) (both from R&D Systems, Minneapolis, MN; http://www.rndsystems.com). The following combination of cytokines was used for generation of CD34+-derived DCs: GM-CSF + SCF + IL-4 + TNF-α, with SCF used only during the first week of culture. CD14+ monocytes were cultured for 6-7 days with GM-CSF + IL-4, and then, TNF-α was added for further culture at a concentration of 5 ng/ml. Cell cultures were incubated at 37°C in a humidified atmosphere containing 5% CO2. One-half of the culture volume was replaced with fresh medium and cytokines every 2-3 days. Cells were harvested for flow cytometric analysis after 7-60 days of culture.

Flow Cytometry Analysis

The following mouse anti-human monoclonal antibodies, conjugated with fluorescein isothiocyanate or phycoerythrin, were used for the direct immunostaining: CD1a, CD86, CD80, CD14, HLA-DR, CD40L, CD34 (all from PharMingen; San Diego, CA; http://www.bdbiosciences.com/pharmingen), and CD83 (Immunotech; Marseille, France). Isotype-matched controls were used for all experiments. Cultured cells were stained for 30 minutes at 4°C, washed twice, and fixed in 1% paraformaldehyde, followed by FACScan analysis (Becton Dickinson FACScan, Lysis II Software Package). Data were analyzed using window software (Joe Trotter; Scripps Research Institute; San Diego, CA; http://www.scripps.edu).

Mixed Leukocyte Reaction (MLR)

DCs were collected after 1-8 weeks in culture (as indicated), irradiated (30 Gy), and used as stimulator cells in MLR assays. Allogeneic CD3+ lymphocytes were isolated from peripheral blood mononuclear cells (PBMCs) using T-cell separation columns (R&D Systems), and CD4+ and CD8+ T cells were separated by MACS (Miltenyi) and used as responder cells. Graded numbers (i.e., 102-103) of stimulator DCs were then added to 105 responder T cells/well in triplicate determinations performed in 96-well round-bottom culture plates (Falcon). After being cultured for 4-5 days, 3H-thymidine (1 μCi/well; NEN; Boston, MA; specific activity 79.10 Ci/mmol) was added and radioactive incorporation evaluated after an additional 18 hours of culture. Results are reported as mean counts per minute (cpm) ± standard deviation (SD).

Enzyme-Linked Immuno Spot (ELISPOT) Assays

ELISPOT assays for IFN-γ secretion were performed as previously described [19]. MACS-purified CD4+ and CD8+ (positively selected) T cells were isolated from normal donor PBMCs using the manufacturer's instructions (Miltenyi). All determinations were performed in triplicate and analyzed using the Zeiss AutoImager (Jena, Germany).

Determination of Uptake and Processing of Soluble Protein

The ability of DCs to endocytose and catalyze the dye-quenched green bovine serum albumin (DQ-BSA) fluorescent substrate BSA (Molecular Probes; Eugene, OR; http://www.probes.com) was analyzed. Green DQ-BSA requires enzymatic cleavage in an acidic intracellular compartment to generate a highly fluorescent product that can be monitored by flow cytometry [20]. DCs (105) were incubated with DQ-BSA (10 μg/ml in RPMI-1640 containing 10% fetal bovine serum) for 1 hour at 37°C, and controls were incubated on ice to inhibit the metabolic uptake of the reagent. Cells were washed twice with PBS containing 0.1% NaN3 and 0.1% BSA (Sigma; St. Louis, MO; http://www.sigmaaldrich.com) prior to analysis by flow cytometry (FACScan; Becton Dickinson).

Electron Microscopy

DCs were placed in cold (4°C) 3% glutaraldehyde overnight. The next day, the samples were postfixed in 1% osmium tetroxide for 90 minutes, then dehydrated through graded ethanol and propylene oxide. The sample was then infiltrated with one change of 50% propylene oxide, 50% epoxy resin for 2 hours, and then pure epoxy resin for two additional hours before embedding. One-micron sections were cut and stained with methylene blue for light microscopic examination. Thin sections were cut on a Reichert Om-U2 ultratome, stained with uranyl acetate and lead citrate, and examined in a Philips CM-10 electron microscope at 60 kv.

Statistical Analysis

The results obtained from multiple experiments were expressed as the mean ± SD. Statistical significance between groups was determined using a Student's t-test analysis. For all analyses, p < 0.05 was considered significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Cultured CD34+ Precursor-Derived DCs Undergo Temporal Alterations in Morphology Allowing for the Discrimination of Three Distinct Stages of Differentiation

CD34+ precursor cells were isolated from cord blood and cultured in cytokine-containing media for up to 8 weeks. DC size and morphology changed dramatically over the course of culture. Short-term (10-15 days) and long-term (28-57 days) cultured DCs were fixed and embedded in epoxy resin for electron microscopy. Thick methylene-blue-stained sections were examined by light microscopy (Figs. 1A, 1B), and cell diameters were determined using an ocular micrometer. The day 14 DC diameters ranged from 10-20 μm (mean = 15.5 ± 3.1 μm), while by 8 weeks, DC diameters were greater at 17.5-30 μm (mean = 21.5 ± 2.8 μm). In total, 100 DCs were evaluated at each time point.

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Figure Figure 1.. CD34-derived DCs enlarge during in vitro maturation.Fourteen- and 57-day-old DCs were fixed with 3% glutaraldehyde and processed for electron microscopy studies. One-micron sections were cut and stained with methylene blue for light microscopic examinations. A) Small-, medium-sized, and large cells having dendritic shape and fragmented nucleus are shown in the 14-day DC populations. B) Large- and giant-sized cells with cytoplasmic processes and numerous vacuoles were typical in LS-DC cultures.

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Electron microscopy was subsequently performed on DCs harvested at different time points of culture. DCs harvested from short-term cultures (i.e., ES-DCs, about 15 days) appeared as rapidly proliferating cells [21] and exhibited an immature morphology. ES-DCs were medium-sized villous cells that contained numerous cytoplasmic hypertrophied mitochondria with reduced cristae (typical for proliferating cells), scattered ribosomes, single profiles of rough endoplasmic reticulum (RER), immature secretion vacuoles, and Golgi complexes. Multivesicular bodies (lysosomal structures) and infrequent multilaminar structures also were detected (Figs. 2A, 2B). IS-DCs (i.e., DCs cultured for approximately 2-3 weeks) appeared to be involved with intense protein synthesis. They exhibited deeply indented achromatin-rich nuclei with a thin rim of heterochromatin located on one side of the cell and enlarged cytoplasms with prominent cisterns of RER. There were also numerous lysosomal vacuoles, multilaminar structures, and hypertrophied Golgi sacks. (Figs. 2C, 2D). LS-DCs (from approximately 3 or 4 to 8 weeks) appeared to be morphologically “mature” secretory cells. These cells were characterized by enlarged dendriform cytoplasms and convoluted or fragmented nuclei located close to the cell membrane. The cytoplasms contained enlarged mitochondria, well-developed Golgi, secretory granules, and vacuoles. Multivesicular and multilaminar structures consistent with MHC class II-containing compartments, previously termed MIIC [22,, 23], were prominent. Numerous vacuoles of smooth endoplasmic reticulum, coated vesicles, and accumulations of tubulo-vesicular structures (TVS) were also differentially observed in LS-DCs (Fig. 3A-C). Birbeck granules were detected in a small fraction of cultured DCs at any time point evaluated. Overall, these electron microscopic studies demonstrate the progressive development of both MHC class II-containing and secretory compartments in CD34+ precursor-derived DCs during extended in vitro culture.

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Figure Figure 2.. The ultrastructure of early-stage (ES) and intermediate-stage (IS) DCs.A) Short-term cultured, ES-DCs (15 day) were villous cells with an indented nucleus (N) (× 4,600). B) The cytoplasm had numerous scattered ribosomes (R), and profiles of granular and agranular endoplasmic reticulum (ER). Mitochondria (M) were hypertrophied (× 29,500). C, D) IS-DCs (23-day) have numerous cisterns of rough endoplasmic reticulum (RER) and enlarged mitochondria ( × 4,800; 28,500).

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Figure Figure 3.. The ultrastructure of late-stage (LS) DCs.A) Eight-week-old (i.e., 57-day) DCs were large cells with polarized nuclei (N), hypertrophied Golgi apparatus (Ga), enlarged mitochondria (M), multivesicular and multilaminar structures (indicated by thick short arrows), and complexes of tubulo-vesicular structures (TVS) (indicated by thin long arrows) (× 4,600). B) TVS complex (× 16,700). C) Multivesicular and multilaminar structures of MHC class II compartments (× 29,900).

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Long-Term Culture of CD34+ Precursor-Derived DCs Results in the Upregulation in Expression of CD83, HLA-DR, CD40, and CD86 Molecules, and is Associated with Enhanced T-cell Stimulatory Capacity

The morphometric analyses of these cultured DCs suggested that, over time, these cells would acquire an increasingly stimulatory phenotype capable of activating antigen-specific T cells. If this were indeed the case, one would predict that LS-DCs would exhibit greater expression of key MHC and costimulatory molecules associated with T-cell activation than ES-DCs or IS-DCs. In addition, maturation markers, such as CD83, would be predicted to increase in a time-dependent manner on these cultured DCs. As depicted in Figure 4, ES-DCs (i.e., at day 14) uniformly expressed moderate levels of the “immature” DC marker, CD1a (i.e., with 45%-66% of cells staining positive), and a minor subset expressed the “mature” DC marker, CD83 (i.e., with 4%-12% of cells staining positive). By 3 weeks of culture, the frequency of IS-DCs expressing CD1a was greater at 75%-91%, while CD83 expression was still low (12%-32%). “Mature” DC markers appeared after 3-4 weeks of culture, CD1a expression was lower at 25%-48%, while CD83 was dimly expressed by 68%-94% of cells. During this same interval (i.e., weeks 2-4), the percentage of cells expressing the costimulatory molecule, B7-2 (CD86) rose from 18%-35% to 82%-95%. Expressions of HLA-DR and CD40 were 43%-55% and 19%-45%, respectively, at day 14, while both were greater, at 83%-97% and 87%-95%, respectively, at 3-4 weeks. These data support the phenotypic maturation of CD34+ precursor-derived DCs over time in long-term cultures.

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Figure Figure 4.. Greater expression of CD83, HLA-DR, and costimulatory molecules in LS-DC cultures than in ES-DC and IS-DC cultures.Cord blood CD34+ cells were cultured with GM-CSF + IL-4 + TNF-α for 2-4 weeks, with SCF added during the first week of culture. Depicted are flow phenotypes for: A) Day 14 ES-DCs; B) Day 21 IS-DCs; and C) Day 28 LS-DCs. These data are from one representative experiment of a total of seven performed.

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Cultured CD14+ Monocyte-Derived DCs (MDCs) Exhibit Three Similar Differentiation Stages in Vitro

CD14+ monocytes were isolated from the peripheral blood of normal healthy volunteers using specific MACS beads and cultured in the same basal media and cytokines used for the CD34+ progenitor-derived DC experiments reported above. DCs were harvested from 7-25 days in culture and analyzed for their morphology and phenotype, as well as for their functional ability to process soluble antigens and to stimulate the activation of specific T cells. As depicted in Figure 5, day 7 MDCs expressed a CD1a+, CD83, CD86dim+, CD40dim+, HLA-DR+ phenotype (like that of the ES-DCs) that transitioned through a CD1adim+, CD83+, CD86+, CD40+, HLA-DR+ phenotype on day 10 (like that of the IS-DCs), ultimately attaining a CD1a, CD83+, CD86+, CD40+, HLA-DR+ phenotype (like that of the LS-DCs) by day 14-21.

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Figure Figure 5.. CD14+ monocyte-derived DCs are phenotypically mature by day 21 of in vitro culture.Depicted are the phenotype of cultured day 7 (A), day 14 (B), and day 21 (C) DCs derived from CD14+ precursors. These DCs progressively lost expression of the CD1a marker while increasing their expression of both CD83 and CD86. These data are from one representative experiment of a total of three performed.

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Processing of Soluble Antigen (DQ-BSA) by CD34 Precursor-Derived and CD14-Derived DCs

In order to assess the comparative ability of these cultures to process soluble antigens, we evaluated the uptake and catalytic activation of the DQ-BSA substrate by our DC preparations [20]. As shown in Figure 6, CD34+ stem-cell-derived DCs demonstrated the highest level of antigen-uptake/processing activity in ES populations (i.e., day 14 of culture), which was substantially lower at later time points in IS-DC and LS-DC populations (i.e., days 21 and 28 of culture, respectively). CD14+ monocyte-derived DCs exhibited a similar loss in DQ-BSA uptake and enzymatic activation over time, with the highest processing potential noted for the early day 7 DC populations.

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Figure Figure 6.. Antigen uptake and processing are most pronounced in early-stage (ES)-DCs.DCs derived from either CD34+ or CD14+ precursors were harvested at the indicated time points of culture and assessed for their ability to uptake and process the profluoregenic DQ-BSA substrate as monitored by flow cytometry. CD34+ stem-cell-derived ES- (day 14), IS- (day 21), and LS- (day 28) DC populations, or day 7, 14, and 21 CD14+ monocyte-derived DCs were evaluated. Data are reported as percent positive cells, with the mean fluorescence intensity provided in parentheses in each instance. These data were derived from one representative experiment of three performed.

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LS-DCs Preferentially Promote the Proliferation as Well as the Production and Secretion of IFN-γ from Allospecific CD4+ and CD8+ T Cells

Normal donor CD4+ and CD8+ T cells were isolated from PBMCs by positive selection, using magnetic bead separation techniques, and then used as responder cells in overnight (i.e., 20-hour) IFN-γ ELISPOT assays. Cultured CD34+ precursor-derived ES-DCs (day 10), IS-DCs (days 19, 24), or LS-DCs (days 31, 40, 44) from an allogeneic donor (to the T cells) were harvested and used as stimulators in IFN-γ ELISPOT and proliferation assays (Fig. 7). The results depicted in Figure 7A suggest that, while IS-DCs (day 19) can promote alloreactive T-cell production of IFN-γ, they are rather weak in this capacity compared with LS-DCs (day 40). Similarly, LS-DCs (days 31 and 44) were superior to ES-DCs (day 24) in stimulating the proliferation of allospecific T cells in primary MLR assays (Fig. 7B).

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Figure Figure 7.. Mature LS-DCs exhibit the greatest capacity to stimulate specific T-cell proliferation and cytokine release.A) CD34+ precursor-derived DCs cultured for 19 days (♦ IS-DCs) or 40 days (□ LS-DCs) were used as allostimulators for peripheral-blood-purified CD8+ T cells in 20-hour IFN-γ ELISPOT assays at the indicated DC/T cell ratios. B) CD34+ precursor-derived DCs were harvested at days 10 (ES-DCs), 24 (IS-DCs), 31, and 44 (LS-DCs), irradiated (30 Gy), and used as stimulator cells at a T cell/DC ratio of 1,000 in alloproliferative MLR assays. These data were derived from one representative experiment of a total of five performed.

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Like the CD34+ precursor-derived DCs, MDCs increased their size and granularity throughout the culture period (data not shown), and their T-cell stimulatory capacity, as depicted in Figure 8 and summarized in Table 1. MDCs cultured for 10 days (i.e., early-stage) or 21 days (i.e., late-stage) were used to stimulate isolated allogeneic CD4+ and CD8+ responder T cells in MLR proliferation and IFN-γ ELISPOT assays. As shown in Fig. 8A and 8C, day 21 MDCs promoted superior proliferation of both CD4+ and CD8+ alloresponder T cells in 3H-thymidine incorporation assays, with CD4+ T cells providing a greater proliferation index than CD8+ T cells in response to DCs in all cases. In the IFN-γ ELISPOT analyses, CD8+ T cells were the superior producers of IFN-γ (versus CD4+ T cells), with late-stage MDCs proving to be superior stimulators of allo-CD8+ T cells (versus early-stage MDCs). However, in contrast to alloantigens that do not require “processing,” late-stage MDCs lose their capacity to process soluble antigens, such as the DQ-BSA substrate (Fig. 6, Table 1). Hence, for exogenous antigens, early-stage MDCs may prove the superior antigen-presenting cell (APC) in promoting epitope-specific T cells. In aggregate, these data suggest that MDCs undergo a similar pattern of developmental progression as that observed for CD34+ precursor-derived DCs, although the time required for the ES-DC to LS-DC transition is substantially accelerated (Table 1).

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Figure Figure 8.. LS CD14+ monocyte-derived DCs promote greater T cell proliferation and IFN-γ production from allogeneic T-cell responders than earlier stage DCs.MLR proliferation (A, C) and the number of IFN-γ ELISPOTS produced (B, D) per 105 T cells/well in response to allogeneic DCs are indicated. DCs are either early-stage (day 10, A, B) or late-stage (day 21, C, D) CD14+ monocyte-derived DCs. Purified CD4+ (♦) and CD8+ (▪) T cells were separated from responder PBMCs for analysis in both assays that are described in greater detail inMaterials and Methods. The presented data were derived from one representative experiment of three performed.

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Table Table 1.. Stages of dendritic cell differentiation
Characteristics of dendritic cellsEarly-stage (ES)Intermediate-stage (IS)Late-stage (LS)
Function   
Proliferation of DCs+ + ++ +±
Protein synthesis in DCs++ + ++
Phagocytosis by DCs+ + ++ +±
T-cell stimulation by DCs++ ++ + +
DC phenotypeimmaturematuremature
CD1a+ + ++ +±
CD83±+ ++ + +
HLA-DR+ ++ + ++ + +
CD86+ ++ + ++ + +
CD40L+ ++ + ++ + +
Intracellular organoids   
Rough endoplasmic reticulum++ + ++
Smooth endoplasmic reticulum++ ++ + +
Hyperthrophied Golgi vacuoles++ ++ + +
MIIC compartments++ ++ + +
Multivesicular structures±++ + +
Hyperthrophied mitochondria+ + ++ ++ +
Multilaminar structures++ ++ + +
Birbeck granules+ + + (in some cells)±_
Cell size15.5 ± 3.1 μm 21.5 ± 2.8 μm
Days to stage   
CD34-derived DCs10-1521-2830-60
CD14-derived DCs6-1012-1821-30

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

An analysis of CD34+ precursor-derived DCs cultured in the presence of low doses of TNF-α supports the novel discrimination of at least three distinct temporal stages of DC differentiation in vitro, based on morphology, phenotype, and functional parameters. The first discriminatory stage of DC differentiation (i.e., ES-DCs) is characterized by medium-sized villous cells with round hypertrophied mitochondria, typically observed in proliferating cells. Scattered ribosomes and single profiles of RER suggest a low protein synthesis capability. The presence of few multivesicular and multilaminar structures consistent with MHC class II-loading compartments, along with low expression of costimulating molecules may explain the limited ability of these cells to promote strong T-cell responses (Fig. 9A, Table 1). Indeed, this type of DC may be predisposed to proliferation [21], rather than to APC effector function.

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Figure Figure 9.. Ultrastructural schema of CD34+ precursor-derived DC differentiation during long-term culture.A) Early-stage (ES)-DCs were medium-sized villous cells with light cytoplasm, hypertrophied mitochondria (M), few multivesicular and multilaminar structures, scattered ribosomes, and profiles of endoplasmic reticulum (ER). The nucleus was located in the centrum of the cell. ES-DCs were highly proliferative and phagocytic cells. B) Intermediate-stage (IS)-DCs were characterized by extensive protein synthesis machinery. The cytoplasms of these cells were enlarged and filled with cisterns of rough endoplasmic reticulum (RER). The nuclei were polarized to one side of the cell. IS-DCs had greater numbers of multivesicular and multilaminar structures (specific MHC class II compartments, i.e., MIIC), and more extensive Golgi complexes (Ga). The phagocytic activity of intermediate DCs was lower than that of ES-DCs. C) Late-stage (LS, mature) DCs exhibited the morphology of highly secretory cells. The cytoplasms of these cells was hypertrophied and contained fully developed Golgi complexes (Ga), smooth endoplasmic reticulum, numerous MIICs, and arrays of tubulo-vesicular structures (TVS), likely associated with secretory processes. Mature DCs were weakly or nonphagocytic. For additional functional and phenotypic discrimination of ES-, IS-, and LS-DCs refer to Table1.

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IS-DCs (Fig. 9B, Table 1), in marked contrast, exhibit signs of intensive protein synthesis. Their cytoplasms are filled with cisterns of RER, vacuoles, and enlarged mitochondria. Notably, the cytoplasms of IS-DCs also contain hypertrophied Golgi complexes and numerous MIIC-like structures.

LS-DCs (Fig. 9C, Table 1) contain numerous vacuoles of smooth endoplasmic reticulum, complexes of TVS, secretion vacuoles, and hypertrophied Golgi sacks. As is typical of highly secretory cells, the nuclei of LS-DCs are located to one side of the cell. The cytoplasms are filled with both multivesicular and multilaminar structures that appear to be intracellular endosomal/lysosomal MHC class II-positive compartments, which likely play a role in antigen processing and the formation of peptide-MHC II complexes [22–, 32]. Multivesicular structures have been demonstrated to be a prerequisite for the formation of multilaminar structures, since endocytosed antigens are first observed in multivesicular compartments, and then subsequently, can be localized in multilaminar structures [25]. Multi-laminar structures, such as MIICs, are intracellular compartments that represent the most abundant storehouse of intracellular MHC class II molecules [22] and are detected in such diverse APCs as human B lymphoblastoid cells [23], DCs, Langerhans cells, and macrophages [25–, 28]. MIIC structures are designed to process antigenic peptides derived from extracellular sources and to then load them into MHC class II molecular complexes. MIICs are acidic compartments that contain proteases as well as the HLA-DM molecules required for appropriate loading of peptides into the mature MHC class II complex [29,, 30].

LS-DCs also contained greater numbers of class II vesicles (CIIVs) that may represent those intracellular structures responsible for the transportation of MHC class II/peptide complexes to the plasma membrane. CIIVs are lysosome-negative vesicles that contain MHC class I and II as well as costimulatory CD86 molecules. CIIVs have been suggested to serve as accumulation points for T-cell receptor ligands after antigen processing has occurred in mature DCs [31]. In contrast, immature DCs are unable to assemble MHC class II peptide complexes until a maturation stimulus is applied [32].

The progressive formation of MIIC structures in the cytoplasms of LS-DCs (versus ES-DCs) may explain the concurrent observation of greater stimulatory activity of these cells in MLR (proliferation and IFN-γ production) assays. Overall, these morphological data suggest that LS-DCs are principally geared toward the processing and MHC-presentation of antigenic peptides to T cells, and are also capable of secreting elevated levels of important immunoregulatory factors that may be preformed and stored in secretion compartments (i.e., TVS).

The secretory machinery of mature DCs revealed in our morphological studies may prove critical to the delivery of DC-produced cytokines and chemokines, as previously demonstrated by immunocytochemical methods [33]. DC-secreted mediators, identified in Langerhans cells as well as in vitro-derived DCs, include TNF-α, RANTES, IL-6, IL-8, IL-10, IL-12, GM-CSF, macrophage inflammatory protein-1 (MIP-1α and MIP-1β). We are currently evaluating the nature and magnitude of cytokines and chemokines differentially produced by these three stages of in vitro cultured DCs when induced with cognate T-cell stimuli, such as CD40L, and bacterial derivatives, such as lipopolysaccharide and CpG oligonucleotides [34–, 36].

In a somewhat analogous manner, DCs in human superficial lymph nodes have been phenotypically classified by others into three subsets [37]. The first, a CD1a+ immature DC subset, was found mainly in lymph sinuses. The second, a CD83+ dendriform DC subset, was found scattered in normal T-cell zones, and the third, a CD1a+CD83+CD86+ large dendriform DC subset, was found in small numbers in hyperplastic T-cell zones. This description correlates well with our findings of three temporal stages of DC maturation in vitro.

Notably, intermediate- and late-stage CD34+ precursor-derived DCs were far more effective (than early-stage DCs) in their abilities to activate proliferative and IFN-γ cytokine secretion reactivity in alloreactive CD4+ and CD8+ T-cell responders in vitro. This accentuated immunostimulatory phenotype is likely to be, at least partially, due to high expression levels of costimulatory molecules expressed by these DCs. It may also be due to the likely greater secretion capacity and development of numerous MHC class II compartments dedicated to antigen processing and presentation in these more “mature” APCs. The close correspondence between in vitro and native DCs [37] in their immunophenotypic, functional, and morphologic maturation suggests that mature DCs may prove highly effective in promoting specific T-cell anticancer immunity in many immunotherapy protocols.

Notably, we have also discerned three similar stages of monocyte-derived MDC development in vitro, although the temporal kinetic for transition of ES-like MDCs to LS-like MDCs was greatly accelerated over that observed for the CD34+ precursor-derived DCs. Like the ES-DCs, MDCs harvested after 1 week of in vitro culture were able to effectively acquire and process soluble antigens. However, they expressed low levels of MHC and costimulatory molecules, which was reflected in their comparatively poor capacity (versus MDCs cultured for longer periods of time) to stimulate specific T cells. In aggregate, these data suggest a predictable in vitro programming for both CD34+ precursor-derived DCs and MDCs that may prove useful for the selection of the appropriate DC temporal stage for clinical applications (Table 1). Of note, it is highly unlikely that a single DC stage will prove optimally efficacious for all clinical applications. For instance, in vaccine protocols implementing peptides, LS-DCs or day 15-25 MDCs may be preferred due to their heightened immunostimulatory capacity and the lack of required processing of the epitope. On the other hand, if proteins (i.e., recombinant antigens or cell lysates) or cells (i.e., apoptotic or necrotic bodies [38–, 40]) are to be applied as an antigen source, ES-DCs or day 6-8 MDCs may be preferred, due to their enhanced capacity to take up and process extracellular antigens. These antigen-fed DCs may then be further cultured to attain a more stimulatory APC phenotype prior to clinical administration. We are currently evaluating these aspects in preclinical vaccine models.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The authors wish to thank Dr. Andrew Yeager and Jan Mueller-Berghaus for careful review and insightful comments that have aided in the preparation of this manuscript. This work was supported by a National Institutes of Health Program Project grant CA 73743 to W.J.S.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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