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Selective Generation of Different Dendritic Cell Precursors from CD34 Cells by [复制链接]

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发表于 2009-3-5 10:36 |只看该作者 |倒序浏览 |打印
a Instituto de Biolog赤a Celular,Agencia Valenciana de Ciencia y Tecnolog赤a,Valencia, Spain;  a8 B, i2 `5 R) b( }$ w
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b Centro de Transfusi車n de la Comunidad Valenciana,Valencia, Spain;
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c Fundaci車n Hospital General Universitario,Valencia, Spain
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Key Words. Interleukin-6 ? Interleukin-3 ? Myeloid dendritic cell precursors ? Plasmacytoid dendritic cells5 W' k! S: Y  i+ J- c, f
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Correspondence: Mar赤a Dolores Mi?ana, Ph.D., Instituto de Biolog赤a Celular, Agencia Valenciana de Ciencia y Tecnolog赤a, Avda del Cid 65 A, 46014 Valencia, Spain. Telephone: 34-96-3868132; Fax: 34-96-3868109; e-mail: minyana_mdo@gva.es" w$ w  j% P1 a5 U7 a2 ], P
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ABSTRACT
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Dendritic cells (DCs) represent heterogeneous populations of rare antigen (Ag)-presenting cells that play crucial roles in the elicitation of T cell–dependent immunity. DCs originate in bone marrow, and their precursors migrate via the blood stream to almost all organs of the body, where they capture Ag and present it processed to CD4  and CD8  naive T cells, thereby initiating primary cellular immune responses. DCs, in addition to their role in innate immunity, induce and regulate adaptive immune responses . The distinct capacity of DCs to induce immunity versus tolerance or Th1 versus Th2 responses depends on their maturation stage , signals that induce or inhibit DC maturation , and the lineage origin of DCs . Ontogenically, DCs are heterogeneous and derive from lymphoid or myeloid lineages . In humans, a subpopulation of CD34 Lin– CD45RA  expressing CD10 gives rise to T, B, and NK cells as well as DCs . These lymphoid progenitors may home to the thymus and lack myeloid cell markers . These cells, also named plasmacytoid T cells, which correspond to the Lin– CD4 CD11c– blood DC precursors, express high levels of -subunit of interleukin-3 (IL-3) receptor (CD123)  and are IL-3–dependent and granulocyte-macrophage colony-stimulating factor (GM-CSF)–independent .
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In vitro studies with CD34  progenitor cells have revealed two other subtypes of DCs belonging to myeloid lineage that emerge independently. One pathway involves CD1a CD14– cells that give rise to epidermal Langerhans cells (LCs). The other pathway includes bipotent CD1a– CD14  cells that can be induced to differentiate either into monocyte (Mo)-derived or interstitial/dermal DCs and macrophages .% W2 U3 ^) l: ?) a* Z7 z

# }6 r( l7 r* y6 Y' {: W& C  JGM-CSF and tumor necrosis factor alpha (TNF-) were found to be an efficient cytokine combination for in vitro generation of myeloid DCs from CD34  progenitor cells , but the addition of stem cell factor (SCF) to the culture system led to higher production of DCs by expanding progenitor cells  and also colony-forming unit DCs . More recently, FLT3-ligand (FL), another early-acting cytokine that induces the proliferation and survival of primitive hematopoietic progenitor and stem cells and shares similar receptors to SCF, has been demonstrated to enhance the expansion of DC precursors from CD34 DR– progenitor cells and to maintain their long-term production . When administered to mice and humans, it expands the number of both myeloid and lymphoid DCs . In most of these in vitro studies reported so far, the culture media used for inducing DC generation from CD34  cells was supplemented in addition to cytokines with serum or plasma. Strobl et al.  demonstrated that the basic cytokine combination of GM-CSF plus TNF- and SCF in the absence of serum or plasma induced only low percentages and low total yield of CD1a  cells from CD34  cord blood cells, but the addition of plasma or transforming growth factor (TGF)-? 1 to cytokine cocktail strongly induced cell proliferation and differentiation of CD1a  DC. TGF-?1 is one of the cytokines present in serum and plasma produced by many cell types that inhibits the expansion of immature CD34 CD38– progenitor cells  but in the presence of GM-CSF  or erythropoietin plus IL-3 and SCF  induces the proliferation of lineage-committed progenitor cells. Moreover, the effect of TGF-?1 on DC generation is dependent on the presence of GM-CSF and TNF- . It has also been reported that TGF-?1 is important for the formation of LCs . In addition, FL cooperates with TGF-?1, GM-CSF, TNF-, and SCF in the in vitro induction of DC/LC in serum-free culture conditions, being unable to generate CD1a  cells upon omission of TGF-?1 .# D' a/ t1 w% ]

$ S& S& H0 W' E. i- X) f* S- QThe growing interest in the use of DCs as cancer vaccines has obliged many groups of researchers to generate DCs ex vivo, but despite the progress achieved in this field, the results contrast with the extensive amplification of myeloid progenitors obtained from CD34  progenitor cells with early-acting cytokines with or without GM-CSF. We have previously described that in the presence of SCF, FL, thrombopoietin (TPO), and IL-6 or IL-3 in serum-free culture conditions, it is possible to expand CD34  cells and colony-forming units, maintaining or even increasing slightly long-term culture-initiating cells . However, the potential of generating DC precursors in this type of culture has not been extensively studied. To date, Arrighi et al. , using FL, TPO, and SCF in the presence of serum for some weeks, were able to induce a great expansion of DCs from CD34  cord blood cells, but taking into account the effects of serum addition to the cultures, it remains to be determined whether early-acting cytokines have substantial effects on DC precursor generation.
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The aims of this study were to expand CD34  cord blood cells in serum-free culture medium with SCF, FL, TPO, and IL-6 or IL-3 for a short period of time to generate hematopoietic progenitor cells, avoiding their maturation, and to study possible differences induced by IL-6 and IL-3 on the generation of myeloid DC precursors. Moreover, we asked whether the presence of IL-3 at the beginning of the culture was able to induce precursors of lymphoid DC. We report here that cytokines used were able to generate CD1a CD14– and CD1a– CD14  DC precursors, but their proportions were differently regulated by IL-6 and IL-3. These DC precursors became mature functional DCs. In addition, we have analyzed DC-specific intracellular adhesion molecule–grabbing nonintegrin (DC-SIGN) expression on generated cells and used it to discern the different myeloid DC subsets. Finally, this type of culture allowed us to identify the developmental pathway of functional plasmacytoid DCs.% T! N+ D1 J  Q5 s9 O
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MATERIALS AND METHODS1 k0 C1 g, L9 M7 K6 I( L6 t+ \

0 K/ K, l; i0 v0 xCD34  Cord Blood Cells Differentiate into CD1a  and CD14  Cells with Early-Acting Cytokines
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CD34  cord blood cells were grown in serum-free medium in the presence of SCF, FL, and TPO containing IL-6 (STF6) or IL-3 (STF3). After 7 days of culture, cells were expanded 12.7 ± 3.9-fold in the presence of IL-6 relative to 25.1 ± 5.8-fold, when IL-3 was added. Both culture conditions led to occurrence of nonadherent round cells (Figs. 1A and 1D). Both cultures were used as source of DCs and are referred in the text as primary cultures. Cell suspensions derived from both primary cultures were 80%–90% HLA-DR  and 70%–75% CD33 , but, surprisingly, whereas all cells derived from STF6 treatment were CD13 , approximately 20%–25% of cells from STF3 primary cultures did not express this Ag. CD34  cells remaining in the culture constituted 36.5 ± 9.8% and 15.5 ± 3.5% of the total cells for STF6 and STF3 primary cultures, respectively. Both treatments used in this study to expand hematopoietic progenitors induced the generation of the myeloid DC precursors CD34– CD1a  and CD34– CD14  (Fig. 2A). Because CD11c is a marker of myeloid DCs, we determined its expression in primary cultures. As can be seen in Table 1, 10% of the cells obtained from STF6 primary cultures expressed CD11c and approximately 20% when IL-3 was present, this difference being proportional to the different expansion capacity induced by these cytokines. A small percentage of expanded cells were CD1a , and again the presence of IL-3 induced a greater proportion of this cell subset (Table 1). However, despite the lesser increase in the total cell number produced by the cytokine combination, including IL-6, the percentage of CD14  cells was greater than that observed for IL-3–containing cultures (Table 1).
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) C2 A0 L( q! ]7 tFigure 1. Morphological changes of CD34  cells induced to differentiate into DCs. Light microscopy of CD34  cells cultured with stem cell factor, FLT3-ligand, thrombopoietin, and IL-6 (STF6) for 7 days (A) subsequently switched to GM-CSF plus IL-4 until day 14 and induced the DC maturation by adding TNF- during the last 2 days (B). Original magnification x 40. May-Grünwald Giemsa staining of freshly isolated CD34  cells (C), cultured with STF6 for 7 days (D), cultured with GM-CSF plus IL-4 the following 5 days (E), and cultured 2 days more with TNF- (F). Original magnification x 50. Abbreviations: DC, dendritic cell; IL, interleukin; TNF, tumor necrosis factor.7 \) g4 p- i( C+ Y( m
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Figure 2. Phenotypic development of CD34  cord blood cells expanded with early-acting cytokines. CD34  cells from cord blood were cultured with stem cell factor, FLT3-ligand, thrombopoietin, and IL-6 (STF6) or IL-3 (STF3) as described in Materials and Methods. On day 7 of culture, a representative sample of the cultures was stained with anti-CD14, anti-CD1a, anti-CD34, or anti-CD11c. One representative experiment showing forward scatter and side scatter profile of cultured cells as well as the lack of expression of CD11c, CD14, and CD1a within CD34  cells derived from (STF3) primary cultures (A) and myeloid dendritic cell precursors obtained from both primary cultures (B) is given. Abbreviations: APC, allophycocyanin; FITC, fluorescein isothiocyanate; IL, interleukin; PE, phycoerythrin.7 G% m) }- ~- M! P# j( ^

. U2 x, |' I# z( U6 u8 j2 R4 jTable 1. Frequency of cell-surface markers induced after ex vivo expansion of cord blood CD34  cells
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To assess whether the differential effect on the generation of DC precursors could be attributable to a specific effect of IL-6 or IL-3, we cultured CD34  cells with SCF, FL, and TPO and observed that the percentage of CD1a  and CD14  cells were 6.3 ± 2.1% and 4.9 ± 1.5%, respectively. Considering that total cell expansion induced by SCF, FL, and TPO was 1.6 ± 0.8-fold increased by addition of IL-6, it can be assumed that IL-6 induced a decrease in the proportion of CD1a  cells and an increase in that of CD14  cells similar in absolute numbers to fold expansion induced. However, when IL-3 was added, percentage of CD1a  cells increased in parallel with cell expansion, but the proportion of CD14  cells was not significantly affected; therefore, IL-3 prevented the generation of CD14  cells and IL-6 prevented the generation of CD1a  cells induced by SCF, FL, and TPO. Nevertheless, this cytokine combination was not additionally used in this study, because it had a low capacity to expand hematopoietic progenitors." d: c7 O, A. T/ u1 w

9 G/ v6 D: _9 H, R* sAmong CD14  cells, those expressing CD1a represented 14.0 ± 5.3% for STF6 primary cultures but 50.1 ± 15.6% for STF3 cultures; therefore, the proportion of the intermediate DC precursor, CD14  CD1a , obtained in these two primary cultures was also differentially regulated by cytokines (Table 1, Fig. 2B). We assessed the presence of CD11c Ag on the different DC subsets obtained from both type of cultures. Approximately 40% of CD1a  cells were also CD11c  in STF6-derived cells and 50% in STF3 cultures; however, most CD14  cells expressed CD11c, reaching values of 71% and 81% for STF6 or STF3 primary cultures, respectively (Fig. 2B). Moreover, CD14  CD1a  cells were CD11c , thus implying a different time acquisition of CD11c Ag by DC precursors.1 L- J1 Z8 u' x; o0 d# u
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It has been described that DC-SIGN is a molecule present in some immature DC subsets; therefore, we wondered whether DC precursors obtained could express it. In fact, as shown in Table 1, a very low percentage of cells were DC-SIGN , and cytometric analysis demonstrated that 58 ± 9% and 86 ± 9% of the DC-SIGN  cells expressed CD11c in STF6 and STF3 primary cultures, respectively. In addition, 20%–27% of CD1a  cells expressed DC-SIGN , irrespective of the primary culture.
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* _$ c9 J6 B1 j7 sMyeloid DC Precursors Become DCs with GM-CSF Plus IL-4: l0 r8 a4 R" k* K
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Cells derived from primary cultures were harvested, washed, and resuspended in serum-free medium containing GM-CSF, IL-4, SCF, and FL to induce generation of immature DCs. After 5 days of culture, twofold cell expansion was obtained with 75% viability, determined by trypan blue exclusion, and only 2% of total cells expressed CD34 in both primary cultures. Most cells became elongated, and veiled cells were also observed. When TNF- was added to maturate DCs, cell viability was similar, no CD34  cells were detected, and the morphological changes induced on cultured cells were more noticeable (Fig. 1B). Giemsa staining revealed that after exposure to GM-CSF plus IL-4, approximately 20% of cells increased their size with a large cytoplasm, some of them containing granules and dendritic prolongations (Fig. 1E). The nucleus was eccentric, indented, and sometimes double. After incubation with TNF-, these effects were more remarkable (Fig. 1F). Based on cytometric analysis of cell size and granularity, two cell populations were observed: R1, containing cells with a low side scatter and resembling primary cultures; and R2, including cells with a high scatter profile and a high expression of HLA-DR, similar to DCs under cytometric criteria (Fig. 3). Percentages of total viable cells gated on R2 after incubation with GM-CSF plus IL-4 were 22 ± 8% and 15 ± 10% for STF6- or STF3-derived cells, respectively. After exposure to TNF-, this gate included 27 ± 10% and 15 ± 6% of STF6- or STF3-derived cells, respectively. Therefore, phenotypic analysis was performed separately on gates R1 and R2. As shown in Table 2, most cells belonging to R2 expressed CD11c Ag, and approximately 40% of cells were CD1a , but approximately 15% of CD1a  cells did not display CD11c yet. Although cell cultures contained IL-4 during this period of time, down modulation of CD14 was not complete, and in fact 12% of cells gated on R2 were CD14 . Of that 12%, 52% of cells derived from STF6 expressed CD1a and 63% of cells derived from STF3 expressed CD1a.: J  k/ Z9 M+ s  c
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Figure 3. Phenotypic evolution of cells derived from primary cultures incubated with GM-CSF plus IL-4 and with TNF-. Cells derived from STF3 or STF6 primary cultures were induced to differentiate into DCs by incubation for 5 days with GM-CSF and IL-4 and 2 days more with TNF-. Forward scatter versus side scatter of cultured cells at both stages of the culture showing regions R1 and R2 is given. Phenotypic analysis from a representative experiment showing different cell-surface molecules at both stages of DC maturation from a sample of primary cultures is shown. Abbreviations:APC, allophycocyanin; DC, dendritic cell; FITC, fluorescein isothiocyanate; IL, interleukin; PE, phycoerythrin; TNF-, tumor necrosis factor alpha.5 k2 U, n1 r) B0 i2 S( ?7 b

- P. J6 Z# R$ H9 x: [- j& wTable 2. Phenotypic analysis of R1 and R2 cell populations induced by GM-CSF plus interleukin-4 (IL-4) and matured with tumor necrosis factor alpha (TNF-)+ }2 s: D. [+ v/ ]! f
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The percentage of DC-SIGN  cells was similar to that of CD1a  cells. Based on DC-SIGN expression, two different CD1a  cell subsets were revealed, CD1a DC-SIGN– and CD1a DC-SIGN , that represented 55% of CD1a  cells, irrespective of the type of primary culture. In addition, 17%–28% of cells gated on R2 displayed the molecules of costimulation, CD86, CD80, and CD40, and 8%–12% of cells also were CD83 .
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Maturation of DCs induced by TNF- was accompanied by a significant decrease in the percentage of CD14  cells, which only comprised 4% of cells gated on R2. The analysis of coexpression of DC-SIGN and CD1a confirmed again the presence of two distinct myeloid DC subsets. Thus, only 60% of CD1a  cells were also DC-SIGN . Therefore, TNF- did not alter this selective Ag distribution. As expected, after 2 days of incubation with TNF-, an overexpression of costimulatory molecules as well as an increase in the content of cells positive for these molecules was observed (Table 2, Fig. 3). However, it is noteworthy that whereas percentages of CD40  and CD83  cells were similar in both derived cell cultures, a significant increase in CD80  and CD86  cells was observed in STF6 with respect to STF3-derived cells. Additionally, FACS analysis indicated that CD83 was induced on DC-SIGN  and CD1a  myeloid DCs. Thus, averaged numbers demonstrated that, irrespective of primary cultures, the proportions of DC-SIGN  and CD1a  cells expressing CD83 increased after addition of TNF- from 13% and 15% to 45% and 75%, respectively.
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In contrast to R2, only 20%–30% of the cells gated on R1 exhibited a high expression of HLA-DR, and 12.5% or 16.6% of cells derived from STF6 or STF3 primary cultures expressed CD11c, respectively, a very similar percentage to that observed after 7 days of CD34  cell expansion. After incubation with GM-CSF and IL-4, the proportions of CD14  and CD1a  cells were considerably lower than those present in R2 (Table 2). To assess whether the presence of factors generating immature DCs induced the expression of CD1a on CD14  cells, as happened with cells gated on R2, we analyzed the coexpression of these two Ags. Only 23% and 37% of CD14  cells derived from STF6 or STF3 primary cultures, respectively, expressed CD1a. Percentage of DC-SIGN  cells gated on R1 was slightly increased, but not statistically significant, compared with primary cultures (Table 2). Within the CD1a  cell subset, 44% and 71% of cells derived from STF6 primary cultures were DC-SIGN  and CD11c , respectively, whereas 69% and 59% of cells derived from STF3 primary cultures expressed DC-SIGN and CD11c, respectively. Therefore, this coexpression was similar in both gates R1 and R2. The addition of TNF- also induced a significant increase in percentage of cells expressing costimulatory molecules, which was more remarkable in STF6-derived primary cultures, and a significant decrease in the number of CD14  cells (Table 2). Taken together, these results indicate that a continuous generation of DCs is taking place; thus we observed DCs at different stages of maturation.5 e" h! H/ J' s0 P7 c8 [- u+ A8 U
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Functional Assays of Myeloid DCs
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; X9 R3 s' G/ pDCs derived from CD34  cells were compared with Mo-derived DCs for their capacity for endocytosis examined by uptake of FITC-dextran. Expanded cells generated in the presence of early-acting cytokines were incapable of internalizing FITC-dextran. A slight uptake in GM-CSF plus IL-4–derived cultures was observed, with most of the cells responsible for this moderate uptake belonging to R1 (Fig. 4). Averaged numbers indicated that within R1, 21.7 ± 6.6% and 21.3 ± 1.6% of cells derived from STF3 or STF6 cultures, respectively, internalized FITC-dextran, compared with only 4%–8% of cells corresponding to R2, and these percentages were not modified by incubation with TNF- (Fig. 4). The low number of cells gated on R2 capable of taking up dextran probably indicated a great proportion of DC in a high maturation stage. In contrast, freshly Mo- and immature Mo-derived DCs showed a high level of FITC-dextran uptake, and, as expected, after exposure to TNF- for 2 days, mature Mo-derived DCs diminished their ability to internalize FITC-dextran (Fig. 4).
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Figure 4. Differential uptake of FITC-dextran by CD34 - and monocyte-derived cells induced to differentiate into myeloid dendritic cells. Endocytosis assays were performed as described in Materials and Methods. CD34  cord blood cells were incubated with early-acting cytokines for 7 days and additionally cultured with GM-CSF and IL-4 for 7 days, the last 2 days with TNF-. R1 and R2 correspond to the gates in Figure 3. Monocytes were from cord blood, freshly isolated and cultured for 5 days with GM-CSF plus IL-4 and 2 days more with TNF-. Thin lines in histograms are negative controls incubated at 4oC for 1 hour with FITC-dextran. A representative experiment from each type of culture is shown. Abbreviations: FITC, fluorescein isothiocyanate; IL, interleukin; TNF-, tumor necrosis factor alpha.. r& Y% O' @) n4 o7 g
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A comparative assessment of the MLR-stimulating potency of both CD34 -derived cultures is shown in Figure 5A. Neither STF3- nor STF6-treated cells were capable of stimulating allogenic lymphocytes from adult peripheral blood (data not shown). When cells derived from primary cultures were incubated with GM-CSF plus IL-4, a slight allostimulatory capacity was observed, increasing after exposure to TNF-, thus indicating that mature DCs were obtained. As can be seen in Figure 5A, cells derived from STF6 treatment were more potent stimulators, but not statistically significant, than those derived from primary cultures incubated with STF3 at any cell dose tested, probably because of the higher percentage of cells expressing costimulatory molecules. Because mature DCs are responsible for lymphocyte proliferation and are characterized by the presence of CD83 molecule, we correlated cell proliferation with the cell number expressing CD83 present in the cultures incubated with TNF-. Independently of early-acting cytokines used to expand CD34  cells, a very good correlation (R2 = 0.92) between these two parameters was obtained, as observed in Figure 5B, demonstrating that the allostimulatory capacity is closely related to CD83  cell content.
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  }! M) U- O/ E/ k: ~1 @( E+ y, n, sFigure 5. Stimulatory capacity of myeloid DCs derived from CD34  cells. (A): CD34  cells from cord blood were expanded in the presence of FLT3-ligand, thrombopoietin, stem cell factor plus IL-3 (STF3), or IL-6 (STF6) and induced to generate immature or mature DCs by addition of GM-CSF plus IL-4 and TNF- , as described in Materials and Methods. A total of 1 x 105 allogenic CD3  T cells from adult peripheral blood were incubated with graded doses of CD34 -derived cells generated in culture. Results are presented as mean ± standard error of duplicate samples corresponding to three independent experiments. Cells derived from STF3 cultures () or STF6 () were incubated with GM-CSF plus IL-4 for 5 days; cells derived from STF3 cultures () or STF6 () were incubated with GM-CSF plus IL-4 for 7 days, the last 2 days with TNF-. (B): 5'-bromo-2'deoxy-uridine incorporation is represented versus CD83  cells present in primary cultures cultured with GM-CSF, IL-4, and TNF-, independently of the combination of early-acting cytokines used to expand CD34  cells. Absorbance values correspond to those of (A). Abbreviations: DC, dendritic cell; IL, interleukin; TNF-, tumor necrosis factor alpha.7 ]4 G2 ~% p. H# J( E7 L+ R

* v! G& z9 `$ ^) |% b8 D. cPrimary Cultures Derived from STF3 Also Contain Plasmacytoid DC Precursors: ?0 N6 W# P( M' i% i& @( A

- [; _5 M4 J: a- ~# {/ gThe presence of CD13– cells within STF3 primary cultures led us to consider the possibility that these cultures could also generate precursors of plasmacytoid DCs (pro-DC2). To study this, we carried out the isolation of CD34  cells from two cord blood units and first characterized phenotypically CD34  cells. We found that 91 ± 5% of CD34  cells expressed CD13, 92 ± 3% were HLA-DR , 33 ± 11% were CD45RA , 59 ± 7% were CD123 , and 9 ± 5% were CD7 , whereas less than 0.2% were CD1a  and expression of CD4 was not detected. According to CD45RA and CD7 expression, CD45RA CD7 , CD45RA– CD7 , and CD45RA CD7– cells represented 4.5 ± 2.6%, 1.5 ± 0.4%, and 27.7 ± 8.2% of CD34  cells, respectively. After 7 days of culture with STF3 combination, we examined the presence of pro-DC2 by staining cells with a lineage cocktail of mAb against CD3, CD19, CD20, CD16, CD56, and CD14 and analyzed the expression of selected Ag on Lin– HLA-DR  cell fraction. In the primary cultures, 20 ± 6% of cells were CD13– , from which 92 ± 2% lacked CD11c expression; 13 ± 1% of cells were CD45RA ; 5 ± 2% were CD4 ; 29 ± 5% were CD34 ; and 34 ± 5% were CD123 , from which 63 ± 1% were CD11c–. CD7  cells were not detected. Taking into account that pro-DC2 develops from a CD34 CD45RA  late progenitor, we performed extensive phenotypic evaluation on CD45RA  cells. Therefore, 87 ± 1% lacked CD11c, 91 ± 8% were CD33 , 73 ± 2% expressed CD34, and 7 ± 4% were CD4 . Regarding CD34  and CD4  cells, 21 ± 3% and 17 ± 2% were CD45RA , respectively.; V( r: x9 T% T6 C

7 w6 I9 y- t9 [  E. A; NIL-3 with TNF- Promotes the Differentiation into Plasmacytoid DCs- `2 C1 Q/ S! y( F
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It has been reported that plasmacytoid cells (DC2) die rapidly by apoptosis unless IL-3 has been added; therefore, cells derived from primary cultures were induced to differentiate into DC2 by incubation with IL-3 plus TNF-, maintaining SCF and FL. Three and 6 days after addition of these cytokines, viability of cells, determined by trypan blue exclusion, was 94% and 91%, respectively, and cells were expanded three and four more times. This cytokine combination induced morphological changes in cultured cells, with the appearance of clusters of cells with dendritic prolongations as well as veiled cells that were more abundant after 6 days (Figs. 6A and 6B). Giemsa staining results showed that DC2 derived from STF3 primary cultures exhibited typical plasmacytoid morphology (Fig. 6C), displaying most cell pseudopods and veiled morphology after 6 days with IL-3 plus TNF- (Fig. 6D).% ~/ W$ s2 _0 E: `7 K) i5 T

' r. T: s6 d5 k- ?Figure 6. Morphology of STF3 primary cultures induced to differentiate into plasmacytoid dendritic cells. CD34  cells were cultured for 7 days with SCF, FL, thrombopoietin, and IL-3 (STF3) and incubated with SCF, FL, IL-3, and TNF- for 3 days (A) or 6 days (B). Original magnification x20. May-Grünwald Giemsa staining of STF3-derived cells exposed to TNF- for 3 (C) and 6 (D) days. Original magnification x50. Abbreviations: FL, FLT3-ligand; IL, interleukin; SCF, stem cell factor; TNF-, tumor necrosis factor alpha.0 x4 W% j- y& {: Q
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After 3 days with IL-3 plus TNF-, 14 ± 4% of cells expressed CD4 , 17 ± 2% were CD45RA , and the remaining CD34  cells constituted 15 ± 5% of the total cells. Three days later, CD34  cells represented less than 3% of cells, and the percentages of CD4  and CD45RA  reached 30 ± 2% and 22 ± 2% of cells, but the proportion of cells coexpressing these two Ags constituted 10% of bulk cells. It is assumed that within Lin–HLA-DR  cells, those with a high expression of CD123 and lacking CD11c are DC2. In our culture conditions, CD123  cells constituted 30%–35% of cells, from which approximately 40% and 21% did not express CD11c at the third and sixth day after addition of TNF-, respectively, but the remaining cells exhibited a low expression of CD11c. FACS analysis revealed that only a small proportion of cells expressed CD86 (10 ± 2%), CD40 (3 ± 1%), and CD83 (3 ± 1%), unlike the proportions observed when cells derived from primary cultures were incubated with GM-CSF, IL-4, and TNF-.
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To provide support for the notion that hematopoietic late progenitor cells could give rise to pro-DC2, precursors of DC2 (pre-DC2), and finally DC2, we analyzed by FACS the presence of CD34 CD45RA , CD34 CD4 , and CD34 CD123  within Lin– HLA-DR  cell subset. After 3 days with TNF-, 31 ± 10%, 67 ± 5%, and 63 ± 11% of CD34  cells remaining in the culture expressed CD4, CD45RA, and CD123, respectively (Fig. 7), thus demonstrating the presence of cells at different stages of DC2 development.
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Figure 7. IL-3 plus TNF- induced the generation of precursors of plasmacytoid dendritic cells. CD34  cord blood cells were expanded in the presence of SCF, FL, thrombopoietin, and IL-3 during 7 days and thereafter switched to culture medium containing SCF, FL, and IL-3 plus TNF. The figure depicts a representative experiment showing the presence of preDC2 as CD34  cells that exhibit a high expression of HLA-DR coexpressing CD45RA, CD123, and CD4 after 3 days of incubation with IL-3 plus TNF-. Abbreviations: FL, FLT3-ligand; IL, interleukin; SCF, stem cell factor; TNF-, tumor necrosis factor alpha.4 t+ s7 D! |3 I& C" {+ v7 _

7 R) y$ G# G0 J8 l4 I& k& qFunctional Properties of Plasmacytoid DCs  G' f2 O1 j' x- z0 o

- A5 K! Z, G1 m  ]4 UTo investigate the function of DC2 as a stimulator of allogenic CD3  T cells, its ability to stimulate an allogenic MLR was assessed. As it is shown in Figure 8A, after 6 days in the presence of TNF-, allostimulatory capacity was slightly increased. Nevertheless, DC2 generated in culture was found to be a less-potent stimulator of allogenic MLR than myeloid DCs. Plasmacytoid DCs are a major source of type I IFN upon exposure to virus and bacteria. It has been demonstrated that oligodeoxynucleotides containing CpG motifs (CpG ODN) mimic bacterial DNA, and are able to induce IFN-I secretion by acting on toll-like-receptor 9 (TLR9) . Plasmacytoid DCs, unlike myeloid DCs, express high levels of TLR9  and have been identified as a primary target for CpG ODN ; thus, we determined IFN- production after stimulation with ODN. As can be observed in Figure 8B, STF3-derived primary cultures maturated with TNF- were able to secrete IFN- when stimulated with ODN, demonstrating the generation of true plasmacytoid DCs.! k/ Z+ V9 N- H# l7 ?3 I3 K

; L5 e+ n' }2 R) ^. @Figure 8. Plasmacytoid dendritic cells derived from STF3 primary cultures are functional dendritic cells. Cells derived from STF3 primary cultures were incubated during 3 or 6 days with interleukin-3 plus TNF- as described in Materials and Methods. These cells were used as stimulators in graded doses to 1 x105 allogenic CD3  T cells from adult peripheral blood in an MLR assay (A) or were cultured in the presence of CpG ODN 2006 at 2 μg/ml for 24 or 48 hours for IFN- production (B). Results are presented as mean ± standard error of duplicate samples corresponding to three independent experiments. MLR activity after 3 days () or 6 days () of exposure to TNF-. Open bars and filled bars correspond to IFN- secretion after 24 or 48 hours of ODN stimulation, respectively. Abbreviations: IFN, interferon; MLR, mixed leukocyte reaction; ODN, oligodeoxynucleotide; TNF-, tumor necrosis factor alpha.8 w6 |/ ~+ r9 O/ |# `

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We thank members of the laboratory of Cord Blood Bank of Valencian Community for providing umbilical cord blood specimens. This research was supported by grant FIS 01/0066-03.- m; c9 ?, v6 }  a
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