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The Journal of Immunology, 1999, 162: 5821-5828.
Copyright © 1999 by The American Association of Immunologists

Functional and Phenotypic Analysis of Thymic CD34+CD1a- Progenitor-Derived Dendritic Cells: Predominance of CD1a+ Differentiation Pathway1

Ali H. Dalloul2,*, Claire Patry{dagger}, Jean Salamero{dagger}, Bruno Canque{ddagger}, Fernanda Grassi* and Christian Schmitt*

* Laboratoire d’Immunologie Cellulaire, Unité Mixte de Recherche 7627, Centre National de la Recherche Scientifique, Hôpital Pitié-Salpêtrière, Paris, France; {dagger} Unité Mixte de Recherche, Centre National de la Recherche Scientifique 144, Institut Curie, Paris, France; and {ddagger} Laboratoire d’Immunologie Cellulaire de l’École Pratique des Hautes Études, Hôpital Pitié-Salpêtrière, Paris, France


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


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Dendritic cells (DC)3 are the most efficient APC able to stimulate primary and secondary response in T and B lymphocytes (1, 2). Several types of DC with subtle differences in phenotypes and functions have been described in the blood, skin, and lymphoid organs (2, 3). Whether these differences are related to purification/culture conditions or to the differentiation from distinct progenitors or whether it reflects the action of various microenvironmental factors on the same lineage (2, 3, 4, 5, 6) is a matter of debate, inasmuch as activation events may dramatically change the morphology, phenotype, and function of DC.

DC in humans can arise both from CD34+ bone marrow progenitors under GM-CSF + TNF-{alpha} (7, 8) and from peripheral blood monocytes under GM-CSF + IL-4 (2). DC were generally considered to belong to the myeloid lineage because they arise from monocytes (2). The fact that CD34+ cord blood progenitors cultured in GM-CSF + TNF-{alpha} lead independently to cells expressing CD14+, a marker of monocytes, and to CD14-CD1a+ precursor cells indicated the existence of two independent pathways for the generation of DC (8).

Thymic DC do not behave only as classical APC and appear to have distinct biological properties compared with DC in other tissues. Although they can be as good as peripheral DC in stimulating allogeneic T cells (9), their role in vivo is to determine clonal deletion (negative selection) of autoreactive thymocytes, thereby inducing central tolerance as demonstrated in murine models (10, 11). Human thymic DC have generally been regarded as lymphoid because they arise from CD34+ progenitors with little if any myeloid differentiation potential and because they express lymphoid surface markers such as CD2, CD5, and CD7 (6, 12). The existence of oligopotent progenitors in the fetal liver and the bone marrow with lymphoid but no myeloid maturation capacity and a phenotype matching that of thymic precursors suggests that they migrate to seed the thymus (13, 14). The existence of a common T lymphocyte/DC progenitor (5) or of a common NK/dendritic precursor in the human or mouse thymus was demonstrated (15) or indirectly suggested (16), further supporting the general view that thymic DC arise from an independent lymphoid-committed lineage. Although data concerning human thymic DC are rather sparse, cells isolated in vivo or cultured from precursors have not been reported to express typical surface lymphoid markers (6, 17) contrary to murine DC which express CD8{alpha}{alpha} (2, 5, 11, 18). The recently reported NK/DC precursor in the human thymus is CD33low (15), whereas most CD34+ thymic precursors are CD33 negative (19). This subset was reported to mature into DC with a CD13+CD33high phenotype closer to the myeloid than to the lymphoid lineage (15). Altogether, thymic DC may express either lymphoid or myeloid markers or both. Therefore, the reasons to classify thymic DC as "lymphoid" rely on their ontogeny rather than on their phenotype. The growth/maturation factors for thymic DC have also been questioned. Although GM-CSF seems central in most studies of DC generation from monocytes and bone marrow progenitors, one study in mice (18) and another in humans (20) shows that it is dispensable. Indeed, human CD34+CD44intermediate thymic precursors cultured in IL-7 generated DC and CD14+ cells. In our previous work aimed at determining the myeloid capacity of thymic progenitors, several conditions were tested, but we did not add macrophage CSF (M-CSF) to the cultures (19). Since M-CSF is known to support the survival of monocytes and the proliferation of their precursors (21), we studied its effects on thymic precursors.

The present study was undertaken to evaluate the growth factors needed for the generation of DC from thymic progenitors with a particular focus on GM-CSF, M-CSF, and TNF-{alpha}. We also investigated whether thymic DC differentiated along the same CD1a+- or CD14+-derived pathways already evidenced in cord blood (8). The effect of TNF-{alpha} on thymic DC was investigated and was shown to be acceleration of the differentiation of DC from progenitors as well as the activation of DC. Activation was evidenced by the improved capacity of DC to induce allogeneic MLR and to express high MHC class II levels at the plasma membrane as shown by confocal microscopy. The capacity of cells to present Ags is conditioned by their level of class II molecule synthesis and by intracellular trafficking of class II and invariant chains (22, 23). This led us to investigate whether thymic DC were different from monocyte-derived DC in this respect. Metabolic labeling and immunoprecipitation of class II/Ii chain complexes were thus performed in cultured cells from both lineages.

Altogether we present evidence that thymic DC-differentiating capacity, albeit much lower than that of cord blood, took place along the same pathways. In addition, thymic DC were very similar to monocyte-derived DC regarding their expression and trafficking of MHC class II and their ability to induce allogeneic MLR.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Abs

Flow cytometric studies were performed with control mouse-Ig-FITC or MSIg-PE, CD1a (T6-PE/), CD2 (T11-FITC), CD3 (T3-FITC), CD4 (T4-FITC), CD5 (T1-PE), CD7 (3A1-PE), CD8 (T8-PE), CD10 (J5-FITC), CD14 (My4-FITC), CD33 (My9-PE), CD45RA (2H4-FITC), CD56 (NKH1-PE), CD116 (SCO6-PE), CD40 (MAB89-PE), CD11-b (Bear1-FITC), and CD83 (HB15a-PE), all from Coulter-Immunotech, Marseille, France; anti-HLA-DR-FITC (1243/class II), CD4-PE (Leu-3a), CD34 (HPCA-2-FITC), and CD38 (Leu-17-PE), all from Becton Dickinson, Mountain View, CA; CD115 (c-fms, Santa Cruz Biotech, Santa Cruz, CA); CD80 (mAb 104-FITC), CD86 (FUN/B7.2, PharMingen, San Diego, CA), and E-cadherin (HECD-1, Takara Shuzo, Japan); and CD1a-FITC (Dako, Copenhagen, Denmark).

Purification of thymic cell subpopulations

Populations were obtained as previously described (19). Normal thymuses were obtained from children undergoing corrective cardiac surgery. Samples were minced in cold HBSS with 2% FCS and 1% penicillin/streptomycin (HFA medium). Thymocytes were isolated by Ficoll gradient density centrifugation. Cells at the interface were washed with HFA, and sequential depletion in CD3-, CD4-, CD8-, and CD19-expressing cells was performed in HFA using mAb-coated beads (Dynal Biosys, Compiègne, France). To this end, cells (20 x 106/ml) were incubated with UCHT1 CD3 mAb for 30 min at 4°C, washed, and incubated with sheep anti-mouse IgG-coated magnetic beads for 30 min at 4°C, at 2 beads/cell. After magnetic depletion, uncoated cells were resuspended with beads coated with CD4, CD8, and CD19 mAb at the above bead/cell ratio. When needed, an anti-glycophorin A mAb (JC159, Dako) was added to deplete residual RBC. Cells were thereafter stained with 20 µl/107 cells of CD34-FITC (HPCA-2 mAb) and 5 µl/107 cells of CD1a (T6) mAb for 45 min at 4°C. After washing, cells resuspended in HFA were sorted with the FACStarPlus equipped with an argon laser emitting at 488 nm. Cells were kept at 4°C during the sorting procedure.

Immunofluorescence and confocal microscopy

DC were allowed to adhere for 30 min at 37°C on glass coverslips precoated with a 0.1% poly-L-lysine in water. Cells were fixed in 3% paraformaldehyde for 10 min at room temperature and permeabilized with 0.05% saponin in PBS supplemented with 0.2% BSA. Cells were stained with anti-MHC class II (HLA-DRab dimer) and either CD1a or CD63 mAbs. Cells were first incubated with BL6 Ab (IgG1 CD1a, Immunotech, Marseille, France) for 30 min, washed three times, and incubated with Texas red-conjugated secondary Abs (Jackson Immunoresearch Laboratories, West Grove, PA). Alternatively, cells were incubated with cyanine 3-conjugated H5C6 Abs (IgG1 anti-CD63, provided by Dr. F. Lanza, Establissement Transfusion Sanguine, Strasbourg, France). Conjugation was performed using the Cy3 monoreactive dye pack (Amersham, Les Ulis, France). For double staining, cells were incubated with FITC-conjugated L243 (IgG2a, Becton Dickinson) conjugated in our laboratory using FLUOS (Boehringer Mannheim, Mannheim, Germany). Coverslips were then mounted in Mowiol (Sigma Aldrich, L’isle d’Abeau, France).

Confocal laser scanning microscopy and immunofluorescence analysis were performed with a TCS4D confocal microscope based on a dichroic mirror microscope interfaced with an argon/krypton laser. Simultaneous double-fluorescence acquisitions were performed using the 488 nm and the 568 nm laser lines to excite FITC and Texas red dyes using a 100x oil immersion plan Apo objective (numerical aperture, 1.4). The fluorescence was selected with the appropriate double-fluorescence dichroic mirrors and band pass filters and measured with blue-green sensitive and red side-sensitive-one photomultipliers. The images shown correspond to the merging of four confocal microscopy slices.

Liquid cultures and cytokines for thymic and monocyte-derived DC

The following final concentrations of factors were used: stem cell factor (SCF) (50 ng/ml) and IL-7 (20 ng/ml), both from Valbiotech (Paris, France); flt3L (50 ng/ml), IL-3 (10 ng/ml), GM-CSF (10 ng/ml), TNF-{alpha} (10 U/ml), and IL-4 (200 U/ml), all from Genzyme (Cambridge, MA); and M-CSF (20 ng/ml) from R&D Systems (Minneapolis, MN). Sorted CD34+CD1a- or CD34+CD1a+ thymocytes were cultured at 106/ml in RPMI supplemented with 2 mM glutamine, penicillin/streptomycin (all from Life Technologies, Gaithersburg, MD) and 10% FCS (Valbiotech) from day 1 with various cytokine combinations as indicated in the text. CD1a+HLA-DR+, CD1a+CD14-, and CD1a-CD14+ cells were sorted with the FACStarPlus at days 7–14. Monocytes were isolated from the blood by continuous flow centrifugation leukapheresis, and counterflow centrifugation elutriation as described (24). They were cultured in complete RPMI 1640 as above with 50 ng/ml recombinant human GM-CSF and 200 U/ml IL-4 for 7 days.

Mixed leukocyte reaction

Variable numbers of stimulating cells from culture on day 7–8 or day 14 were seeded in 96-well round-bottom plates with 105 PBMC in 200 µl final volume. Random PBMC samples were obtained from our HLA-typing laboratory and used as responder cells. After 3 days of culture, 1 µCi of [3H]thymidine (sp. act., 25 Ci/mmol, Amersham) was added per well for 15 h and counted. Tests were carried in triplicates, and results are expressed as mean cpm ± SD.

Radiolabeling, biotinylation, immunoprecipitation, and electrophoresis

Cells (5 x 106) were pulse labeled with 35S Promix (Amersham) for 10 min at 37°C and chased for various periods of time as described (25). At the indicated times, cells were chilled in cold PBS and cell surface biotinylated with a solution containing 25 mg of sulfosuccinimidyl-(2 biotinamide)-ethyl-1,3-dithio propionate (Pierce, Rockford, IL) in 1 ml of cold PBS for 5 min at 4°C. The reaction was quenched with 50 mM ice cold glycine in PBS. Cells were solubilized in 1% Triton X-100, 150 mM NaCl, 20 mM Tris-HCl, 5 mM EDTA, 0.2% BSA, and protease inhibitors. Postnuclear lysates were precleared for 2 h with protein A-Sepharose at 4°C. Lysates were thereafter immunoprecipitated sequentially with Abs L243 and DA6.147 previously bound on protein A-Sepharose CL-4B (Pharmacia, Uppsala, Sweden). This allowed distinction between the two forms of MHC-II molecules (L243 recognizes {alpha}ß dimers while mAb DA6.147 recovers {alpha}ßIi complexes). Immunoprecipitates were washed and eluted with 10 µl of 10% SDS at 95°C for 5 min. Eluted material was resuspended in 100 µl of lysis buffer without BSA. The efficiency of biotinylation being 10–15%, biotinylated proteins were recovered with streptavidin-agarose on 90% of the elution volume, and the remaining 10% was left untreated. Samples were boiled in Laemmli’s buffer on a 10–15% SDS-PAGE and run under reducing conditions (100 mM DTT). Autoradiographies were scanned with a video camera (Bio Print System, Vilbert Lourmat, Marne la Vallée, France).


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Low numbers of DC are generated from CD34+CD1a- thymocytes under various conditions with or without GM-CSF

In previous studies, we have defined the most immature thymocyte population as being CD34+CD1a- (19, 26). This subset expressed CD45RA, CD38, CD7, CD2, CD5, and CD10 surface molecules on almost all the cells and was negative for CD14 and CD33 markers (not shown). This immature subset proliferated strongly in response to SCF + IL-7 (19) and generated CD1a+HLA-DR+ DC when cultured in SCF + IL-7 + flt3L+ GM-CSF + TNF-{alpha} in keeping with other results (7, 8) (Fig. 1Go). As shown, only a minority of cells matured in DC, whereas most of the cells (HLA-DR/CD1a negative (lower left quadrant)) remained CD7+, which were IL-7-driven prothymocytes as previously reported (19). The CD34+1a- subset also generated a pure NK cell population when cultured in SCF + IL-7 + flt3L + IL-2. In contrast, the more mature CD34+1+ population was unable to make DC and generated only low numbers of NK cells (Fig. 1Go). Thus, in liquid culture and in absence of thymic stroma, CD34+1- could generate DC or NK cells but not T cells even if IL-1 and IL-6 were added to the mixture (data not shown). DC derived from thymic progenitors under SCF + IL-7 + flT3L + GM-CSF + TNF-{alpha} localized within large cells (R1) and expressed the following membrane Ags (Fig. 2Go): HLA-DR, CD1a, CD2, CD4, CD5, CD7, CD11b, CD11c, CD33, CD40, CD83, CD86, and E-cadherin. In our hands, CD83 expression was low but tended to increase at the late stage of cultures, day 14 (not shown). Importantly, and in contrast to what has been reported for cord blood, CD34+-derived DC (8), no CD14+ intermediates were observed in our cultures from day 1 to day 10. Thus, thymic DC generated in vitro shared common markers with monocyte-derived DC, such as CD33 (28), and common markers with CD1a+-derived cord blood DC, such as E-cadherin (8, 21).



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  		FIGURE 1. The capacity to generate DC from thymic precursors is limited to the CD34+CD1a- subset. Triple-negative thymocytes were prepared by magnetic bead depletion. CD34+CD1a- and CD34+CD1a+ subpopulations were then separated by FACS according to the gates represented and cultured in SCF + flt3L + IL-7 supplemented with GM-CSF + TNF-{alpha}. (DC) or with IL-2 (NK). Cultures were analyzed by flow cytometry for their content on DC (day 7) or NK (day 14). No DC could be recovered from the CD34+CD1a- population (not shown).

 


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  		FIGURE 2. Expression of surface markers on thymic DC following 10 days of culture of CD34+CD1a- cells. Sorted CD34+CD1a- thymocytes were cultured for 10 days in medium containing SCF + IL-7 + flt3L + GM-CSF + TNF-{alpha} and analyzed by flow cytometry gated on large cells (R1) as shown. Data are representative of 8–12 samples.

 
Given that GM-CSF and TNF-{alpha} did not appear to be indispensable for the generation of murine (18) as well as human (20) thymic DC, we evaluated the role of different cytokine mixtures on the growth of DC. Table IGo shows the absolute number of DC obtained at day 14 from 2 x 105 CD34+1- thymocytes. DC could be generated under a mixture of SCF + flt3L + IL-7. IL-7 was necessary since almost no DC were generated in its absence. We could, however, dispense IL-7 for the first 3 days of culture instead of 14 days (IL-7 was added at day 1 and day 3) without affecting the final number of DC. This indicates that the role of IL-7 is to induce the early proliferation of thymocyte precursors, some of which would differentiate into DC without further proliferation. Interestingly, DC were also generated under M-CSF which enhanced the generation of DC when added to the mixture. By contrast, adding IL-3 to the mixture did not appear to increase significantly the yield of DC. Thus, exogenous GM-CSF was dispensable for the differentiation of thymic progenitors into DC, although it was more potent than M-CSF and particularly of IL-3. Finally, even under the best conditions (SCF + flt3L + IL-7 + GM-CSF with or without TNF-{alpha}), the number of DC recovered was very low. Indeed, we could generate ~1.5 DC from two seeded CD34+1- thymocytes. We found that adding TNF-{alpha} to a mixture comprising SCF + flt3L + IL-7 + GM-CSF did not significantly enhance the number of DC generated at day 14. This is also true when TNF-{alpha} was added to mixtures comprising IL-3 or M-CSF.


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  		Table I. Effect of various cytokine combinations on the production of DC from CD34+CD1a- thymic precursors

 
CD14+CD1a- and CD14-CD1a+ DC precursors are generated under M-CSF

Having found that DC could be generated in cultures with M-CSF we investigated whether they passed through CD14+ intermediate cells generated from thymic precursors. While as shown above, several cytokines such as IL-3, M-CSF, and GM-CSF generated HLA-DR+CD1a+ DC, only progenitors cultured with M-CSF were able to generate CD14+ cells (Table IGo). FACS profiles (Fig. 3GoA) show that two distinct populations arose from thymic progenitors after 7 days of culture with SCF + IL-7 + flt3L + M-CSF: a CD1a+CD116+CD14- and a CD1alowCD116intermediateCD14+ population. By contrast, progenitors cultured with GM-CSF led only to the CD116+ CD14- population. Strikingly, GM-CSF down-regulated CD116 (GM-CSF receptor) expression on CD14- and on CD14+ cells. This cannot be attributed to a competition between GM-CSF and the Ab for binding to the receptor given that the CD116 mAb (see Materials and Methods) is not blocking and recognizes the CD116-GM-CSF complex. We observed that when progenitors were cultured for 7 days with M-CSF and recultured for 48 h with GM-CSF, the CD14+ population disappeared and the expression of CD116 diminished, giving the same picture as culture in GM-CSF from the start (not shown). To determine whether CD14+ cells could generate DC, CD14+ cells were sorted and recultured in GM-CSF or in M-CSF alone. Within 48–72 h, we observed a down-regulation of CD14 and an up-regulation of CD1a expression with GM-CSF, whereas cells kept in M-CSF remained CD14highCD1alow (Fig. 3GoB). Cell shape was investigated by immunostaining and confocal microscopy to determine whether our cultures generated cells with monocytic and dendritic morphology. M-CSF-cultured cells appeared heterogeneous in shape, some round with lobulated nuclei and others expressing typical dendritic morphology. Cells stained weakly with CD1a (BL6) and had surface and intracellular MHC class II molecules. The latter colocalized in the endosomal compartment as demonstrated by double staining with anti-MHC class II L243 and anti-LAMP (lysosomal-associated membrane protein, CD63) H5C6 Abs (Fig. 4Go). As shown by confocal microscopy, the same cells recultured with GM-CSF (GM-CSF + M-CSF) had homogeneous DC features with endosomal and membrane MHC class II and a stronger CD1a expression. Thus, thymic DC differentiated directly from CD1a+CD14- precursors under GM-CSF and indirectly from CD1a-CD14+ cells if sequentially cultured with M-CSF and GM-CSF.



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  		FIGURE 3. CD34+CD1a- thymocyte progenitors differentiate into CD14+CD1a- and CD14-CD1a+ DC precursors in culture with M-CSF. Sorted CD34+1a- thymocytes were cultured for 7 days in medium containing SCF + IL-7 + flt3L in combination with M-CSF, GM-CSF, or M-CSF + GM-CSF as indicated. A, at day 7 cells were washed and analyzed by flow cytometry for the expression of CD14/CD1a or CD14/CD116 (GM-CSFR). B, at day 7, CD14-expressing cells were FACS sorted from a M-CSF-supplemented culture and reincubated for 48 h in M-CSF or GM-CSF before flow cytometric analysis. Numbers in the plots refer to the percentage of cells in each quadrant. Data are representative of 6–12 experiments.

 


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  		FIGURE 4. Aspect on confocal microscopy of cells generated from CD34+CD1a- thymocyte progenitors cultured for 10 d with IL-7 + SCF (+ M-CSF) or (+ M-CSF + GM-CSF). A, M-CSF-cultured cells have mixed monocytic and DC morphology, B, M-CSF + GM-CSF-cultured cells have a more typical DC morphology. Localization of MHC class II molecules (L243 Ab) in the endosomal compartment (H5C6 anti-CD63). DC stained with anti-CD1a (BL6). Data are representative of five samples.

 
TNF-{alpha} accelerates the differentiation of progenitors into DC and increases MHC class II membrane expression and MLR-inducing capacity of DC

As shown in Table IGo, TNF-{alpha} did not significantly increase the number of GM-CSF-induced DC at day 14. Kinetic analysis of DC generated from progenitors cultured in GM-CSF or GM-CSF + TNF-{alpha} showed, however, that TNF-{alpha} accelerated the differentiation into DC (Fig. 5Go), which peaked at day 8–10, while culture without TNF-{alpha} peaked at day 14. TNF-{alpha} also accelerated the differentiation into DC whether it was added to GM-CSF or to M-CSF. Confocal microscopy studies (Fig. 6Go) showed that DC cultured with GM-CSF + TNF-{alpha} were larger and had more class II molecules than DC cultured in GM-CSF alone. In TNF-{alpha}-stimulated cells, MHC class II molecules localized predominantly to the membrane, whereas they were mostly intracellular in unstimulated cells. This is in keeping with our observations that TNF-{alpha}-cultured DC were more active in inducing allogeneic MLR (Fig. 7Go) whether TNF-{alpha} was added to M-CSF or to GM-CSF-cultured cells. Thus, TNF-{alpha} had a dual activity. It accelerated the differentiation of DC from thymic or cord blood progenitors. On the other hand, it increased the capacity of DC to induce allogeneic MLR, consistent with an increased membrane expression of MHC class II molecules. Fig. 5Go shows also that TNF-{alpha} had similar activity on CD34+ cells from the cord blood or from the thymus, cultured in the same conditions. However, much higher absolute numbers (80-fold) of DC were generated from cord blood than from thymic CD34+ precursors. This further confirmed that DC progenitors from the cord blood or from the thymus responded in a way similar to that of cytokines, although the latter had a very low proliferation capacity.



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  		FIGURE 5. TNF-{alpha} accelerates DC maturation from CD34+CD1a- progenitors from the thymus or cord blood but does not increase the final number of DC. Absolute numbers of DC, estimated as in Table IGo, recovered from 2 x 105 progenitors cultured with various cytokine combinations. Standard (std): SCF + Flt3L + IL-7. Note that 50–100-fold DC were generated from cord blood than from thymic progenitors. HPC, hemopoietic precursor cells. Mean of six experiments.

 


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  		FIGURE 6. TNF-{alpha} induces MHC class II migration to the membrane of DC: aspect on confocal microscopy. A, GM-CSF-cultured cells. MHC class II (L243 Ab) is localized in endosomes stained with anti-CD63 (H5C6 Ab). B, GM-CSF + TNF-{alpha}-cultured cells. MHC class II is localized predominantly at the membrane. Both cells express CD1a (BL6 Ab).

 


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  		FIGURE 7. TNF-{alpha} enhances the capacity of thymic DC to induce allogeneic MLR. PBMC were cultured with graded amounts of DC (0 to 1000/well) obtained from cultures with SCF + flt3L + IL-7 (cytokine mixture) + M-CSF or GM-CSF with or without of TNF-{alpha}. [3H]Thymidine was incorporated into PBMC (105/well in 200 µl final volume) at day 4. Mean cpm (SD was <20%) of six cultures in triplicates.

 
CD1a+ thymic DC and monocyte-derived DC but not monocytes express class II {alpha}ßIi complexes at the membrane

Thymic DC could be activated by TNF-{alpha}; this was in keeping with microscopy findings that MHC class II was essentially expressed at the plasma membrane in activated DC. Given the importance of intracellular trafficking of {alpha}ß and invariant chains in the function of monocyte-derived DC (22, 23, 27), we investigated whether thymic DC could be differentiated from monocyte-derived DC, based on their respective patterns of MHC class II and Ii expression and the early steps of their intracellular trafficking (25). We have recently studied intracellular vs membrane expression of MHC class II complexes in monocyte-derived DC, monocytes, and EBV-transformed B cells (25). Monocyte-derived DC synthesized a particularly high level of the p41 isoform. Most of the newly synthesized {alpha}ßIi complexes were shown to be transported first to the plasma membrane in these cells, before internalization of the whole complex and the generation of new {alpha}ß dimers devoid of intact Ii chains. The same technique was applied to the study of thymic DC, in particular to CD1a+-derived DC using IL-7 + SCF + GM-CSF + TNF-{alpha}. However, given the paucity of CD14+-derived DC in the thymus, especially after sorting, we used DC derived from CD14+ blood monocytes as control.

Monocytes, blood monocyte-derived DC, and CD1a+-derived thymic DC were pulse chase labeled and surface biotinylated at different times. They were first immunoprecipitated with L243 mAb to retrieve mature {alpha}ß dimers and precipitated thereafter with DA6.147 mAb to recover precursor {alpha}ßIi complexes. This method allows the study of the kinetics of synthesis and transport and the relative expression of both types of class II complexes. Here are shown the autoradiography of the second immunoprecipitation step. We found that MHC class II in thymic DC are synthesized within 30 min and that oligomerization of {alpha}ßIi was complete within 2 h after synthesis which was comparable in all three cell types. The main finding was that {alpha}ßIi complexes reached the membrane of thymic DC within 30 min–1 h after their synthesis (Fig. 8Go). As previously reported (25), this was also found on myeloid DC but not on monocytes in which most of the class II molecules remained intracellular. All Ii isoforms including p47, the sialylated form of p41, were detected on both DC lineages, and mature forms of Ii were transiently detected at the membrane. We could also detect surface Ii by FACS and confocal microscopy using supernatant from the BU 45 hybridoma as described on monocyte-derived DC (25) (data not shown). Thus, transient membrane transit of {alpha}ßIi complexes is a general feature of DC but not of monocytes or B-EBV cells (25). CD1a-derived thymic DC could be differentiated from monocytes but had no special features regarding MHC class II trafficking as compared with CD14-derived myeloid DC.



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  		FIGURE 8. Synthesis, intracellular transport, and membrane appearance of MHC class II precursor forms in thymic DC (middle), monocyte-derived DC (down), and monocytes (up) as determined by autoradiography. Cells were pulse labeled with 35S for 10 min, chased after the indicated time, and surface biotinylated at 4°C for each time point. Lysates were sequentially precipitated with Abs that recognize {alpha}ß dimers (not shown) and {alpha}ßIi complexes (DA6.147). Biotinylated proteins were separated in streptavidin-agarose before analysis by SDS-PAGE (surface) and compared with aliquots of the total immunoprecipitated material. {alpha}ßIi complexes are transported to the membrane as early as 30 min (lane 7) postsynthesis in thymic-derived and in monocyte-derived DC but not in monocytes. Total amounts are comparable in the three cell types, but much higher membrane expression is detected at the surface of DC than monocytes. Ii mature isoforms (p35 and p47) are detected at the surface of thymic and monocyte-derived DC (arrows) at early time points. Data are representative of three samples.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have shown that, similar to what was found in cultures of CD34+ cord blood cells, human thymic progenitors are able to differentiate into DC directly from CD1a+ or indirectly from CD14+ cell stages. However, the production of the latter cells from the thymus, but not from cord blood, required M-CSF. DC arising from the CD14+ subset may be considered as "myeloid DC" similar to the ones arising from blood CD14+ monocytes (28, 30). Consistent with this is the cellular distribution of various markers and the cell morphology observed by microscopy, and also (data not shown) that our CD14+ cells became adherent when sorted and kept in M-CSF. As opposed to myeloid DC, the ones directly differentiating from CD34+ progenitors through CD14-CD1a+ cells may be considered as "lymphoid" similar to their bone marrow/cord blood counterparts (8, 29). Indeed, the CD1a+-derived DC subset is the only population generated from thymic precursors provided that M-CSF is not added to the culture. Thus, the response of thymic progenitors to M-CSF helps by defining a predominant CD1a+-derived pathway as "lymphoid" and a minor CD14+-derived pathway as "myeloid." The description of a lymphoid DC lineage is based on the finding in the bone marrow (13), and in the neonatal or fetal thymus from humans (14, 31) or mice (5), of pluripotent progenitors able to differentiate into T, B, NK, and DC but not into myeloid cells. This definition is much more reliable than that based on surface phenotype since DC express several lymphocytic and myelocytic markers. In addition, phenotypes may vary according to purification conditions. In one study, for instance (6), freshly separated thymic DC did not express CD1a but reexpressed it in culture. Nevertheless, our thymic lymphoid DC expressed E-cadherin a feature shared by their cord blood counterpart, further linking them to a lymphoid-Langerhans lineage (8, 29). Although the affiliation of at least some thymic DC to lymphoid lineage was strongly suggested, some experiments showed that thymic progenitors still conserved a monocytic maturation capacity (20). Nevertheless, thymic CD34+ cells probably represent downstream progenitors more committed to lymphoid lineage than to myeloid lineage. The significance of CD33 on our thymic DC is unclear; although it is a myeloid marker, it was claimed to be present on a common NK/DC progenitor (15) and on the CD44bright DC precursors arising from IL-7-cultured CD34+ thymocytes (20). Could thymic DC be the precursors of CD33-positive plasmacytoid T cells (32)? This is unlikely because thymic DC are CD11c positive and did not apoptose in the absence of IL-3. Instead, IL-3 was not very effective in our hands in generating DC from CD34 thymic progenitors (data not shown). The low number of CD14+ cells observed, even in the presence of M-CSF, is further evidence of the lymphoid commitment of thymic progenitors. The requirement for M-CSF in thymic but not in cord blood precursors is also suggestive of a more advanced commitment toward lymphocytes.4 By contrast, progenitors from the bone marrow or fetal liver are more primitive multipotent cells and show a greater capacity to generate myeloid and lymphoid DC (33, 34). Whether "myeloid/DC" progenitors in these experiments were different from "lymphoid/DC" progenitors was not ruled out.

DC were generated from human (22) or murine (18) precursors without addition of GM-CSF. These results together with ours are likely to be due to the outgrowth of a "lymphoid" committed DC precursor under the influence of IL-7 + SCF and to a lesser extent of flt3L. Thymic precursors would mature in vitro into DC when cytokines necessary for their survival are present in the culture and provided that IL-2 (and possibly IL-15) are not added. This would avoid the outgrowth of NK cells at the expense of DC (Ref. 15 and our unpublished data). If this hypothesis is correct, the role of M-CSF in our experiments was to differentiate thymic precursors into CD14+ cells (35, 36) while sparing DC-committed precursors of lymphoid origin, which differentiated under IL-7. It could be argued that M-CSF-stimulated monocytes produced various factors including GM-CSF (37), which synergizes with M-CSF in enhancing monocyte and multipotential colony formation (38). However, it seems unlikely that "lymphoid" DC were generated through an indirect effect of M-CSF, since they were produced under IL-7 + SCF with or without GM-CSF. Finally, the addition of M-CSF to our cultures demonstrated that thymic precursors retain some monocytic differentiation capacity.

The low number of DC generated from thymic precursors is striking as compared with the high capacity of cord blood progenitors. We were unable to generate DC colonies in semisolid medium from CD34+CD1a- thymocytes (not shown). This difference may be related either to the very low number of committed DC progenitors in the thymus or more likely to their more mature state as compared with cord blood progenitors. Indeed the CD34+CD1a- fraction in the thymus is CD7high (19) and preferentially proliferates to IL-7 + SCF (26). Their counterparts represent <10% of the cord blood CD34+ cells with similarly low DC differentiation capacity (our unpublished data) by contrast to the bulk CD34+ population. Thus, it is possible that thymic DC arise from bone marrow DC directly homing into the thymus. Indeed, irradiated SCID mice injected with bone marrow cells had their thymuses colonized with DC before thymocyte expansion occurred (39). Alternatively, DC might be produced in situ from CD34 precursors. This is indirectly suggested from experiments in which ~70% of CD4low thymic precursors showed a DC differentiation capacity following in vivo transfer to mice (5). In humans, the recently described CD33low fraction in the thymus (15) represented 20% of most immature CD34+1- cells used in our study. Therefore, ~0.1% of thymocytes differentiated into NK or DC, the latter showing a 15-fold amplification from the initial progenitors seeded. This should result in a 3-fold amplification of the CD34+CD1a- fraction as compared with 1.5-fold in our own hands.

We have shown the dual role of TNF-{alpha} on thymic progenitors and on DC. Although it was not needed to generate DC, it accelerated their maturation from CD34+ cells and activated them as evidenced by MLR and microscopy studies. TNF-{alpha} may work through inhibition of growth of the monocyte lineage in synergy with GM-CSF as described (40), thereby shifting the differentiation of CD34+ cells toward CD1a+-derived "lymphoid" pathway. On the other hand, differentiation of DC by TNF-{alpha} and other inflammatory stimuli such as IL-1, LPS, and bacterial products has been demonstrated (41, 42). Following culture with TNF-{alpha}, DC up-regulate adhesion and costimulatory molecules and no longer endocytose. As shown here by confocal microscopy, MHC class II molecules are mostly intracellular in immature cells while mainly located at the membrane following stimulation with TNF-{alpha}. Our results also show that thymic DC behave like myeloid DC, although differently from fresh monocytes, both for the early steps of intracellular transport of {alpha}ßIi chains and for the composition of MHC-II complexes. We have studied the predominant thymic lymphoid DC subset (generated without M-CSF), and compared it with blood monocyte-derived DC. We assume that the latter is similar to the CD14+-derived thymic subset, which showed a monocytic morphology. It has long been suggested that thymic DC had no specific features that would explain their role in clonal deletion of self-reactive thymocytes. Indeed they could be replaced for this function by splenic DC (43), indirectly demonstrating that deletion is due to the reactivity of immature thymocytes themselves, with self MHC. Moreover, thymic DC are as good as peripheral ones in presenting Ags (9). Altogether, we present additional arguments that although DC can arise from different progenitors, they can be functionally very similar provided that they are cultured in the same conditions.


   Acknowledgments
 
We thank Professor Leca for providing thymuses; Professor J.-C. Gluckman for critical review of the manuscript; Mrs. C. Laurent for technical help in cell sorting; and Dr. A. Hosmalin, Dr. M. Rozenzwajg, and Mr. M. Yagello for helpful advice.


   Footnotes
 
1 This work was supported by grants from the Association de Recherche Contre le Cancer. Back

2 Address correspondence and reprint requests to Dr. Ali H. Dalloul, Laboratoire d’Immunologie Cellulaire, CERVI, Hôpital Pitié-Salpêtrière, 83 Blvd de l’Hôpital, 75651 Paris, France. E-mail address: Back

3 Abbreviations used in this paper: DC, dendritic cells; M-CSF, macrophage CSF; SCF, stem cell factor. Back

4 B. Canque et al. Submitted for publication. Back

Received for publication November 18, 1998. Accepted for publication February 26, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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