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Special Susceptibility to Apoptosis of CD1a⁺ Dendritic Cell Precursors Differentiating from Cord Blood CD34⁺ Progenitors

Laboratoire de Biologie et Pathologie des Déficits Immunitaires and Laboratoire d'Immunologie Cellulaire de l'Ecole Pratique des Hautes Etudes, Faculté de Médecine et Hôpital Pitié-Salpêtrière, Paris, France

Key Words. Human • Dendritic cells • Differentiation • Apoptosis • Hematopoiesis

Dr. Bruno Canque, Laboratoire d'Immunologie, CERVI, Hôpital de la Pitié-Salpêtrière, 83 Blvd. de l'Hôpital, 75651 Paris CEDEX 13, France.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We analyzed the effect of tumor necrosis factor (TNF)- on the differentiation and viability of dendritic cells (DC) generated from cord blood CD34⁺ progenitors cultured for five days with GM-CSF, Flt-3 ligand (FL), and stem cell factor (SCF), and then with GM-CSF only [TNF(–) cultures]. Adding TNF- from the start [TNF(+) cultures] potentiated progenitor cell proliferation and promoted early differentiation of CD1a⁺ DC precursors without affecting differentiation of CD14⁺ cells, which comprise bipotent precursors of DC and macrophages, nor of CD15⁺ granulocytic cells. Use of TNF- was associated with increased cell mortality, which peaked on culture day 10 and mainly involved CD1a⁺ DC. Selective apoptosis of CD1a⁺ DC precursors was confirmed by showing that survival of day-7-sorted CD1a⁺CD14^– cells from TNF(+) cultures was lower than that of CD1a^–CD14⁺ cells. That similar findings were noted for sorted CD1a⁺CD14^– cells of TNF(–) cultures, further cultured with GM-CSF without or with TNF-, indicates that apoptosis of CD1a⁺ DC precursors was not induced by TNF-. Apoptosis of CD1a⁺ DC precursors occurred after the cells had lost the capacity to incorporate bromodeoxyuridin. Finally, using higher GM-CSF concentrations or adding interleukin 3 (IL-3) improved viability of CD1a⁺ cells. Other cytokines, such as IL-4 and transforming growth factor (TGF)-ß1, were ineffective in this respect, though they promoted differentiation of CD1a⁺ DC. These results indicate that TNF- promotes the differentiation of CD1a⁺ DC precursors, which display a high susceptibility to apoptosis that can be prevented by high concentrations of GM-CSF or use of IL-3, without affecting the differentiation of the CD14⁺ DC precursors.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Dendritic cells (DC) are professional antigen-presenting cells that prime naive T lymphocytes and sensitize cytotoxic T lymphocytes to antigens they present, which makes them promising tools for immunotherapy of cancer and severe viral diseases [1-4]. In vivo, DC are widely distributed in tissues where they display a heterogeneity that may reflect different maturation/activation stages but also different origins [5-9]. The latter possibility is supported by the finding that, in cultures of CD34⁺ hematopoietic progenitor cells (HPC) with GM-CSF and tumor necrosis factor (TNF)-, cells that resemble epidermal skin Langerhans cells (LC) differentiate from CD34⁺CLA⁺ progenitors via CD1a⁺ precursors, while blood/dermal-like DC derive from CD34⁺CLA^– progenitors via bipotent CD14⁺ precursors of either macrophages or DC [7, 10]. Another pathway of DC differentiation also appears to originate from a subset of CD34⁺ cells that are triggered to differentiate into CD1a^–CD40^– DC by CD40 ligation in the absence of exogenous cytokines [11]. The biological significance of this diversity is still unclear, though it has been reported that only CD14⁺-derived DC display a high capacity to take up soluble antigens and trigger IgM production by B lymphocytes [12].

In vitro, various cytokine cocktails elicit the preferential differentiation of DC in cultures of CD34⁺ HPC. Current methods are based on the association of early-acting factors (stem cell factor [SCF], Flt-3 ligand [FL], interleukin 3 [IL-3], and IL-6) with factors that promote DC differentiation (mainly GM-CSF, TNF-, IL-4, and IL-13) [7, 10, 13-19]. Most of these factors actually affect multiple steps of DC differentiation from the progenitor stage to that of fully mature DC. For example, TNF- [10, 20-24] and GM-CSF [10, 22, 25, 26] promote CD34⁺ HPC growth and commitment as well as DC maturation and survival; SCF and FL not only potentiate the growth of but also act as survival factors for HPC [27]. Similarly, IL-4 interferes with HPC proliferation, DC differentiation and function [14, 15, 18], and prevents apoptosis of myeloid cells [28].

Here, we re-examined the role of TNF- on the differentiation and viability of DC generated from CD1a⁺CD14^– and CD1a^–CD14⁺ precursors in cultures of human cord blood CD34⁺ HPC.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cord Blood CD34⁺ HPC Isolation and Culture
Normal cord blood (laboratoire Senders, hôpital Saint-Vincent de Paul; service de Gynécologie-Obstétrique, hôpital Saint-Antoine; Paris, France) was collected according to institutional guidelines. After Ficoll-Paque (Pharmacia; Uppsala, Sweden) centrifugation, mononuclear cells were centrifuged over Percoll (d = 1.070; Pharmacia) for enrichment in light-density cells. CD34⁺ HPC were purified with CD34 monoclonal antibody (mAb) 561-coated M-450 Dynabeads (Dynal; Oslo, Norway), as described [14], yielding 88% ± 9% pure viable CD34⁺ cells (n = 36). The cells (1-2 x 10⁴/ml) were cultured for five days in six-well plates (ATGC; Noisy le Grand, France) in humidified 5% CO₂ at 37°C, in RPMI 1640, 10% fetal calf serum ([FCS]; Dutscher; Brumath, France), 1% glutamine, 1% antibiotics (GIBCO BRL; Paisley, Scotland), with the following recombinant human growth factors, as described [15]: GM-CSF: 200 U/ml unless otherwise stated; SCF: 50 ng/ml; FL: 50 ng/ml; TNF-: 50 U/ml when used (all from Genzyme; Cambridge, MA). After five days, SCF and FL were discontinued, and cells were cultured with GM-CSF with or without TNF-. Other cytokines were: IL-3 (100 U/ml) (Sandoz; Rueil-Malmaison, France), IL-4 (50 ng/ml) and transforming growth factor-ß1 ([TGF-ß1]; 0.5 ng/ml) (both from Genzyme). Medium and cytokines were renewed every three days.

Determination of Cell Growth and Mortality
Viable nonadherent cell (NAC) counts at different time points were normalized relative to 1 x 10⁵ CD34⁺ cells seeded at culture initiation. Percentage of dead cells was determined by propidium iodide (PI) staining (2.5 µg/ml) and flow cytometry. To evaluate apoptosis, cells were washed in phosphate-buffered saline (PBS), cytospun onto glass slides, dried and fixed for five min in PBS, 1% formaldehyde, 0.2% glutaraldehyde; they were then incubated for 10 min with 0.5 µg/ml Hoechst 33342 (Calbiochem Nova-biochem; La Jolla, CA), dried at 37°C, washed three times in PBS, mounted in glycerol, and examined under UV fluorescence.

To assess proliferation, 2.5 x 10⁴ NAC were seeded on different days in 96-well plates and further cultured for 18 h under the same conditions, but with 1 µCi/well [³H]thymidine ([³H]TdR; Amersham; Amersham, UK). Results are shown as mean cpm of triplicates.

Flow Cytometry Cell Surface Marker Analysis and Secondary Sorting
Cells were incubated for 30 min at 4°C with 1:100 final fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)- and/ or quantum red (QR)-conjugated mAb in PBS, 2% FCS. After washing, cells were analyzed with a FACScan^® (Becton Dickinson; Mountain View, CA). The mAbs were: OKT6-FITC or T6RD1-PE (CD1a; Ortho; Raritan, NJ; Coulter Coultronics; Margency, France); LeuM7-PE (CD13), LeuM3-FITC (CD14), LeuM1-FITC (CD15), anti-HLA–DR (DR)-FITC or -PE and anti-HLA–DQ (DQ) (Becton Dickinson); CD40-FITC, CD86-PE (PharMingen; San Diego, CA); CD80-FITC (Valbiotech, Crawley Down; Sussex, UK); HB15a-PE (CD83) (Immunotech; Marseille, France); CD11b (Sigma; St Louis, MO). Isotype matched FITC-, PE-, and QR-conjugated irrelevant control mAbs were from Sigma. Cytokine receptors, and CD1a expression using mAb CD1a NA1/34 (DAKO; Glostrup, Denmark), were assessed by indirect labeling: anti-TNFR mAbs were biotinylated MR1-2/CD120a and MR2-1/CD120b (Monosan; Uden, the Netherlands) or non-conjugated HTR9/CD120a and UTR1/CD120b (gift from Dr. Brockaus, Hoffman-Laroche; Basel, Switzerland). Labeling was developed either by streptavidin Tri-color (Caltag; San Francisco, CA) or with a PE-conjugated goat anti-mouse IgG antibody (Southern Biotechnology; Birmingham, AL). Unless otherwise stated, only PI^- NAC were phenotyped.

For secondary FACS sorting, 5-10 x 10⁶/ml washed day-7 cells were incubated at 4°C for 30 min with PE-CD14 and FITC-CD1a mAbs diluted 1:20. Cells were then resuspended in PBS, 2% FCS, and CD1a⁺CD14^– versus CD1a^–CD14⁺ cells were sorted as reported [29]. Cell populations obtained in this manner were 95% ± 3% pure (n = 42).

MLR Assay
Responder allogeneic T lymphocytes from adult blood were enriched to 85%-90% as described [30]. These cells (5 x 10⁴ cells/well in 96-well U-bottomed culture microplates; Costar; Cambridge, MA) were cultured for six days in RPMI 1640, 10% heat-inactivated human AB serum, 1% glutamine, 1% antibiotics, with 0.1 to 1 x 10³ culture day-7 sorted CD1a⁺CD14^– cells as stimulator cells. [³H]TdR incorporation was assessed by 18-h pulse with 1 µCi/well. Results are shown as mean cpm of triplicates.

Cell Cycle Analysis
Bromodeoxyuridin (BrdU) incorporation was assessed as described [31]. BrdU (30 µg/ml) was added to cultures for 60 min; cells were washed, stained as usual for surface markers, and samples were fixed at 4°C in 0.5% paraformaldehyde (100 µl/sample). After four h, 25 µl PBS, 5% Tween 20 (Sigma) were added and cells were allowed to permeabilize overnight at 4°C. They were then washed in PBS and resuspended in 20 µl PBS, 10 mg/ml DNAse I (Boehringer Mannheim; Mannheim, Germany), 4 µg/ml anti-BrdU-FITC mAb (Becton Dickinson). After two h, 250 µl PBS were added, and samples were analyzed by FACS.

Statistics
Results are presented as means ± SD of data from individual cultures. Statistical analysis was performed with the paired Student's t test (Excel 5, Microsoft; Redmond, WA).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
TNF- Potentiates HPC Growth and Promotes Differentiation of CD1a⁺ DC Precursors
CD34⁺ HPC were cultured with GM-CSF, SCF and FL for five days, and then with GM-CSF only. As reported [10], addition of TNF- from the start [TNF(+)] enhanced cell growth during the first week of culture relative to cultures without TNF- [TNF(–)] ( Fig. 1A): starting from 1 x 10⁵ CD34⁺ seeded cells, viable NAC recovery averaged 4.2 x 10⁶ on day 7 in TNF(+) cultures versus 1.9 x 10⁶ in TNF(–) cultures; NAC numbers dramatically decreased later on in TNF(+) cultures, while they reached a plateau in TNF(–) cultures, to average 2 x 10⁶ on day 12 in both cases. Cell proliferation assays confirmed these findings by showing limited but significantly higher [³H]TdR uptake by NAC in TNF(+) cultures than in TNF(–) on days 3 and 5, and lower uptakes thereafter ( Fig. 1B). Thus, TNF- appeared to potentiate the early effect of GM-CSF, SCF, and FL on HPC proliferation in culture. We then examined how TNF- affected early DC differentiation.

View larger version (22K): [in this window] [in a new window]   Figure 1. Effect of TNF- on CD34+ HPC-derived cell growth. (A) Viable NAC numbers from TNF(+) and TNF(–) cultures were normalized relative to 1 x 105 seeded CD34+ HPC; mean ± SD of 10 experiments (with 9 and 7 points examined on days 10 and 12, respectively); differences were statistically significant on days 5 (p = 0.025) and 7 (p <0.001). (B) [3H]TdR incorporation into 2.5 x 104 cells from TNF(+) or TNF(–) cultures, further cultured for 18 h under the same conditions but with [3H]TdR; mean ± SD of six experiments; differences were statistically significant on days 3 (n = 5, p = 0.009), 5 (n = 6, p = 0.049), 7 (n = 6, p = 0.014) and 10 (n = 6, p = 0.007), but not on day 12 (n = 3).

View larger version (16K): [in this window] [in a new window]   Figure 2. FACS analysis of viable culture day 5 NAC from TNF(+) and TNF(–) cultures. NAC were labeled with CD13-PE and with CD40-FITC or CD95-FITC mAbs; open histograms: control labeling with an irrelevant mAb; solid histograms: staining by the relevant FITC- or PE-labeled mAb. Data are from one representative experiment out of 10.

View larger version (27K): [in this window] [in a new window]   Figure 3. Kinetics of CD1a+, CD14+ and CD15+ cell differentiation in TNF(+) and TNF(–) cultures. Viable NAC percentages (upper panels) or numbers (x 10–6/105 seeded CD34+ HPC; lower panels); mean ± SD from nine experiments; statistical significance of differences was as follows:

View larger version (81K): [in this window] [in a new window]   Figure 4. Phenotype and function of CD1a+ cells on culture day 7. (A) FACS analysis of cells from TNF(+) and TNF(–) cultures; cells were labeled with CD1a-FITC or -PE mAb (OKT6), and with (CD11a, CD11c, CD80, CD86, CD83)-PE, CD11b-QR or (CD40, CD95, DR, DQ)-FITC mAbs; data are from one representative experiment out of three; open and solid histograms are as in Figure 2. (B) MLR-stimulating capacity for allogenic T lymphocytes of day 7-sorted CD1a+CD14– DC precursors from TNF(+) and TNF(–) cultures; mean ± SD cpm of 3 experiments; differences were not statistically significant.

Selective Death of CD1a⁺ DC Precursors in the Presence of TNF-
Cell mortality was then assessed. The proportion of PI⁺ cells was greater in TNF(+) than in TNF(–) cultures, in a limited manner on day 7 and more prominently on day 10 (57% ± 14% versus 23% ± 11%) ( Fig. 5A), and more apoptotic cells were present in TNF(+) cultures at that time ( Figs. 5B , 5C). This may account for the leveling of cell number recovery noted from day 7 to 10. Thus, TNF- apparently promoted cell death after the first week of culture.

View larger version (180K): [in this window] [in a new window]   Figure 5. Effect of TNF- on cell mortality. (A) Cell death was assessed by PI staining of total NAC; mean ± SD %PI+ cells from six experiments; differences were significant on days 7 (n = 6, p = 0.037) and 10 (n = 4, p = 0.001). Apoptotic cells were examined by Hoechst 33342 staining on day 8 in (B) TNF(+) and (C) TNF(–) cultures. (D) Day 10 labeling of NAC from a TNF(+) culture with both PI and CD1a-, CD14- and CD15-FITC mAbs. Data are from one representative experiment out of 13.

To confirm these data, day 7 viable CD1a⁺CD14^– and CD1a^–CD14⁺ cells from TNF(+) cultures were sorted and further cultured with GM-CSF and TNF-. After three days, 66% ± 7% of initially CD1a⁺CD14^– cells were PI⁺ versus 24% ± 12% for initially CD1a^–CD14⁺ cells (p = 0.001, n = 4). This was associated with a drop in viable NAC recovery five days post-sort: 0.1 ± 0.05 x 10⁶ cells per 1 x 10⁶ sorted CD1a⁺CD14^– cells versus 0.4 ± 2 x 10⁶ for CD1a^–CD14⁺ cells (p = 0.02). Most viable DC were then CD1a^hiCD83⁺ ( Fig. 6), suggesting a correlation between DC maturation and survival.

View larger version (17K): [in this window] [in a new window]   Figure 6. Phenotype of cells recovered five days after sorting of day 7 viable CD1a+CD14– and CD1a–CD14+ cells from TNF(+) cultures, and further cultured with GM-CSF and TNF-. FACS analysis of viable cells labeled with CD1a-FITC or CD83-PE mAbs; open and solid histograms are as in Figure 2. Data are from one representative experiment out of five.

Death of CD1a⁺ DC Precursors is Not Induced by TNF-

These data indicate that CD1a⁺ DC found in the culture at that time (i.e., mostly CD1a⁺ DC precursors) display a high susceptibility to apoptosis independently of the use of TNF-.

Cell Cycle Analysis of CD1a⁺ DC Precursors
Sequential labeling with BrdU and FITC-anti-BrdU mAb showed that the proportion of NAC in S-phase decreased over time ( Fig. 7) from 38% ± 6% on days 5 and/or 6 to 8% ± 5% on days 9 and/or 10 (p = 0.002, n = 5), confirming the data of the [³H]TdR uptake experiments. The kinetics of BrdU uptake by CD1a⁺ DC and CD14⁺ cells were parallel: there were 36% ± 9% (n = 5) and 6% ± 3% (n = 3) CD1a⁺BrdU⁺ DC on culture days 5 and/or 6 and 9 and/or 10, respectively, relative to 27% ± 6% and 13% ± 4% CD14⁺BrdU⁺ cells on the same days ( Fig. 7).

View larger version (33K): [in this window] [in a new window]   Figure 7. BrdU incorporation into CD1a+ and CD14+ cells on culture days 6 and 9. Cells were double-labeled with BrdU and CD1a- or CD14-PE mAbs as indicated in Materials and Methods. Data are representative of one experiment out of five on days 5-6 or out of three on days 9-10.

Survival of CD1a⁺ DC Precursors is Improved by Increasing GM-CSF Concentration in or Adding IL-3 to Cultures
Because our data indicated that a significant proportion of CD1a⁺ DC were programmed to die from culture days 7 to 10, we examined whether additional signals provided by increasing GM-CSF concentration, continuously using early-acting factors SCF and FL, or adding other cytokines improved their viability ( Fig. 8). Increasing GM-CSF concentration to 1,000 U/ml (3.5 ng/ml) or using SCF and FL until day 12 did not interfere with cell differentiation during the first culture week, while CD1a⁺ cell percentages were lower when 100 U/ml IL-3 were used (26% ± 5% versus 35% ± 8%, p = 0.04, n = 6). Both high GM-CSF concentration and IL-3 also significantly decreased day 10 PI⁺ cell percentages, resulting in a six- and threefold increase in viable culture day 12 CD1a⁺ cell numbers, respectively; in contrast, the continuous use of SCF and FL failed to prevent day 10 cell mortality; the twofold increase in CD1a⁺ cell numbers noted two days later under this condition was not statistically significant ( Fig. 8).

View larger version (31K): [in this window] [in a new window]   Figure 8. Effect of a high GM-CSF concentration, of IL-3, or of continuously using SCF and FL on CD1a+ cell numbers and cell mortality. TNF(+) cultures were conducted with either 1,000 U/ml GM-CSF, adding 100 U/ml IL-3, or using SCF and FL for the whole culture period. Upper panels: viable CD1a+ cell numbers (x 10–6/105 seeded CD34+ HPC); lower panels: percentages of PI+ cells; mean ± SD. Statistical significance of differences was as follows:

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We re-examined here the role of TNF- on the differentiation of DC from cord blood CD34⁺ HPC in light of recent advances in this field [7, 12, 17, 18, 32]. To this end, CD34⁺ HPC were cultured with GM-CSF, with or without TNF-, SCF and FL being used for the first five days. TNF- potentiated GM-CSF-, SCF-, and FL-induced HPC proliferation during the first days of culture, as reported [20, 24]. TNF- also affected the phenotype of day 5 immature cells, which then expressed more CD40, and it promoted the differentiation of CD1a⁺ DC precursors as early as culture day 5, and more prominently on days 7 and 10, but it did not affect differentiation of CD14⁺ cells, among which are bipotent precursors of macrophages and DC [32], nor of CD15⁺ granulocytes. These data indicate that TNF- does not promote differentiation of CD1a⁺ DC precursors at the expense of CD14⁺ cells, but rather through its positive effect on cell expansion; it appears mainly to act at the HPC level, either by recruiting DC progenitors or by committing recruited progenitors to the DC rather than to the macrophage/DC differentiation pathway, arguing thus for early divergence of CD1a⁺-derived and CD14⁺-derived DC [7]. The kinetics of CD15⁺ cell differentiation differed from that of the two other cell types in that they already represented about 25% of cells as early as culture day 3 and reached a plateau from day 5 onward, confirming the early branching-off of granulocytes, a hypothesis supported by other findings in both human [33] and mouse [34] models.

Use of TNF- was also associated with high cell mortality peaking on culture day 10, which preferentially involved CD1a⁺ DC. Given the known pro-apoptotic activity of TNF- [20, 35], this suggested first the special susceptibility of CD1a⁺ DC precursors to TNF--induced apoptosis. Such hypothesis was ruled out by showing that cell death did not depend on expression of TNFR1/CD120a and TNFR2/CD120b, expressed as well by CD1a⁺, CD14⁺, and CD15⁺ cells, and that viability of CD1a⁺CD14^– DC precursors sorted from TNF(-) cultures was comparable to that of their TNF(+) counterparts and independent of TNF- use after sorting. Survival of sorted CD1a^–CD14⁺ cells was greater than that of CD1a⁺CD14^– cells from the same cultures and cultured under the same conditions, an indication that only CD1a⁺ DC precursors are susceptible to apoptosis. These findings are in line with a previous report showing that TNF- is mainly a survival factor for more mature human skin LC [21], and they indicate that high mortality in TNF(+) cultures is primarily due to occurrence of greater percentages of CD1a⁺ DC precursors.

BrdU incorporation experiments showed, in addition, that apoptosis of CD1a⁺ DC precursors coincided with the decrease of their proliferative capacity, since only a minority of them still incorporated BrdU on culture day 9/10. Also, the fact that the kinetics of BrdU incorporation into both CD1a⁺ and CD14⁺ cells were closely parallel indicates that the commitment along these two pathways does not depend on cell proliferation. These results suggest that the high susceptibility to apoptosis of CD1a⁺ DC precursors is stage-dependent, an hypothesis supported by the observation that five days after sorting of CD1a⁺CD14^– cells, mostly viable mature CD1a^hiCD83⁺ cells remained in culture.

Apoptosis of DC differentiated in vitro from cord blood CD34⁺ HPC has previously been reported [36, 37]. However, several lines of evidence indicate that the phenomenon described here differs from those reports, which involved either early CD34⁺ HPC [36] or mature DC populations [37]. At variance with these studies, TGF-ß1 did not prevent apoptosis of CD1a⁺ DC precursors, and culture of CD1a⁺CD95⁺ DC precursors in the presence of a CD95 mAb or with CD40L-expressing murine fibroblasts did not affect their viability (data not shown).

We finally tested whether a significant fraction of CD1a⁺ precursors was irreversibily committed to apoptosis as early as culture day 7, or if increasing GM-CSF concentration or adding other factors known for their anti-apoptotic activity in different systems [27, 28, 36, 37, 40,] could prevent cell death. Using a high concentration of GM-CSF or adding IL-3 improved CD1a⁺ cell viability in bulk cultures, an indication that these cells were not irreversibly committed to apoptosis. Such redundant effect of GM-CSF and IL-3 on cell survival was not unexpected because their receptors utilize a common ß-chain that, upon dimerization, converts from low affinity to high affinity receptors [38]. Our results are in line with previous ex vivo studies showing that GM-CSF preserves the viability and allows maturation of murine skin LC [25, 39], and that IL-3 both rescues human plasmacytoid T cells from apoptosis and cooperates with CD40L to allow their differentiation into functional DC [9]. Thus, maturation of in vitro differentiated CD1a⁺ precursors into functional DC not only requires activation/maturation signals provided by TNF-, IL-4, CD40 ligation or LPS [14, 15, 17, 18, 26, 40], but also the presence of survival factors such as IL-3 or GM-CSF.

In conclusion, the present study demonstrates that while TNF- promotes the differentiation of CD1a⁺ DC precursors, it does not interfere with that of the bipotent CD14⁺ precursors of DC and macrophages, and that the formers display a high susceptibility to apoptosis that can be prevented by increasing the concentration of GM-CSF or adding IL-3.

	Acknowledgments

 
This work was supported by the Agence Nationale de Recherche sur le SIDA, Université Paris 6, the Centre National de la Recherche Scientifique (ERS 107), and the Association pour la Recherche sur les Déficits Immunitaires Viro-Induits (Paris, France).

We are grateful to Prof. J. Milliez and his staff of the Service de Gynécologie-Obstétrique, Hôpital Saint-Antoine (Paris, France) for the gift of cord blood samples, to Dr. M. Brockaus (Hoffman-Laroche, Basel, Switzerland) for the gift of HTR9/CD120a and UTR1/CD120b mAbs, and to R&D Systems for the gift of the Annexin V apoptosis detection kit.

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