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Published as doi: 10.1096/fj.08-108209.
 (The FASEB Journal. 2008;22:3370-3379.)
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T-cell-mediated and antigen-dependent differentiation of human monocyte into different dendritic cell subsets: a feedback control of Th1/Th2 responses

Sabrina Mariotti, Valeria Sargentini, Cinzia Marcantonio, Emiliano Todero, Raffaela Teloni, Maria Cristina Gagliardi, Anna Rita Ciccaglione and Roberto Nisini1

Dipartimento di Malattie Infettive, Parassitarie e Immunomediate, Istituto Superiore di Sanità, Rome, Italy

1Correspondence: Dipartimento Malattie Infettive, Parassitarie e Immunomediate, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. E-mail: roberto.nisini{at}iss.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It is well established that human monocytes differentiate into dendritic cells (DCs) when cultured with certain cytokine cocktails, such as granulocyte-macrophage colony-stimulating factor and interleukin-4. Conversely, it is not completely established which cell population synthesizes the cytokines required for monocyte differentiation and how their secretion is regulated. We show that on specific activation T cells induce the differentiation into DCs of antigen-presenting and bystander monocytes. Monocytes exposed to cytokines released by Th1 and Th0 lymphocytes differentiate into DCs with a reduced antigen uptake and antigen presentation capacity. Moreover, these DCs show a limited capacity to induce Th1 polarization of naive T cells but are capable of priming interleukin-10-secreting T cells. Conversely, DCs derived from monocytes sensing cytokines released by Th2 lymphocytes are antigen-presenting-cell (APC) endowed with a marked Th1 polarization capacity. Monocytes are corecruited with lymphocytes in chronic inflammation sites; thus our results suggest that functionally different DCs can be generated in environments characterized by the prevalent release of Th1-, Th0-, or Th2-associated cytokines. Because the APC capacities of these DCs have opposite functional consequences, a contribution in the regulation of the ongoing immune response by monocyte-derived inflammatory DCs is envisaged.—Mariotti, S., Sargentini, V., Marcantonio, C., Todero, E., Teloni, R., Gagliardi, M. C., Ciccaglione, A. R., Nisini, R. T-cell-mediated and antigen-dependent differentiation of human monocyte into different dendritic cell subsets: a feedback control of Th1/Th2 responses.


Key Words: antigen presentation/processing • cytokines • cell differentiation • chronic inflammation • T-cell clones


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DENDRITIC CELLS (DCS) ARE A heterogeneous population of cells with different myeloid or plasmacytoid origin and differential phenotype and function (1 , 2) .

In recent years DCs have been increasingly studied for their role in immune response against pathogens (3 , 4) and tumors (5) as well as in immune regulation and autoimmune responses (6) because DCs are crucial cells in the induction and regulation of adaptive immunity (7 , 8) . Moreover, DCs have been widely studied as promising "adjuvants" in vaccines for prevention of microbial infections, allograft rejection, and treatment of cancer and autoimmune diseases (9 10 11 12 13 14) . Finally, a large body of evidence on their importance in immunoregulation envisages the possibility for exploiting DCs for more general biomedical purposes (15) .

Several bone marrow and blood precursors of DCs have been identified, including monocytes (1 , 2) . Evidence for the capacity of monocytes to differentiate into DCs has been reported in the mouse models, where monocytes have been in vivo tracked using internalized fluorescent latex microspheres (16 17 18 19 20) or in the Listeria monocytogenes and Leishmania major infection system (21 , 22) . Interestingly, however, blood monocytes do not seem to contribute to the generation of splenic mouse DCs (23 24 25) . On the other hand, in humans it has not been defined which DC subset originates from monocytes, and experiments regarding transendothelial migration of DCs generated from monocytes (17) have not conclusively established which processes promote monocyte differentiation into DCs or into macrophages (26) .

In culture, human monocytes acquire a macrophage phenotype both in the presence and in the absence of added cytokines, such macrophage colony-stimulating factor (M-CSF) (27) ; thus, differentiation of monocytes into macrophages seems to represent a default differentiation program of monocytes on extravasation (28) . Conversely, well-defined cytokine cocktails are required for monocytes to differentiate into DCs (29 30 31 32) . Several cell types have been indicated as a possible source of cytokines capable of inducing monocyte differentiation into DCs in vitro (33 34 35 36) . However, the stimulus and context in which these cells would promote the monocyte differentiation into DCs instead of macrophages could not be unambiguously defined.

Starting from the hypothesis that monocytes could represent progenitor cells of tissue macrophages under physiological conditions and cells committed to the local replacement of migrating or dying DCs following an inflammatory process (1 , 2) , we analyzed the consequences of T-cell activation in the monocyte differentiation into DCs using a panel of Th1, Th2, and Th0 antigen-specific T-cell clones (TCCs). We obtained a model of monocyte differentiation not based on the use of synthetic cytokines or factors and that reasonably reproduces inflammatory microenvironments, allowing an easier extrapolation of data obtained in vitro.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Phytohemagglutinin was from Murex (Dartford, UK) and purified protein derivative (PPD) from Statens Serum Institute (Copenhagen, Denmark). Recombinant interleukin (IL) -2 was from EuroCetus (Milan, Italy). Recombinant IL-4 was from R&D Systems (Minneapolis, MN, USA), granulocyte macrophage colony-stimulating factor (GM-CSF) from Sandoz (Basel, Switzerland), and tritiated (3H)-thymidine from Amersham (Little Chalfont, UK). Lipopolysaccharide (LPS) of E. coli, phorbol 12-myristate 13-acetate (PMA), ionomycin, brefeldin-A, Staphylococcus aureus enterotoxin A (SEA), fluorescein isothiocyanate (FITC) -albumin, and Hepes were from Sigma Chemical Co. (St. Louis, MO, USA). Phosphatidylinositol dimannosides (PIM2) was kindly provided by Professor Gennaro De Libero (University Hospital Basel, Basel, Switzerland) (37) , green fluorescent protein recombinant Bacillus Calmette-Guérin (Gfp-rBCG) was provided by Dr. Marco R. Oggioni (Dipartimento Biologia Moleculare, Siena, Italy) (38) , and purified extract from Parietaria judaica (Parj1) was a kind gift of Dr. Gabriella Di Felice (Dipartimento MIPI, Isituto Superiore di Sanità, Rome, Italy) (38) . RPMI 1640 was used, supplemented with 100 U/ml kanamycin, 1 mM glutamine, 1 mM sodium pyruvate, 1% nonessential amino acids (complete medium, CM) (Euroclone Ltd., UK), and enriched with 10% fetal calf serum (FCS) (Hyclone, Logan, UT, USA).

Generation of antigen-specific TCCs
Human cells were obtained from healthy blood donor volunteers who gave their informed consent to the use of part of their blood donation for in vitro experiments not involving the screening for infectious diseases. The study is part of a European Community project, which was approved by Ethical Committee of Istituto Superiore di Sanità.

PPD and Parj1-specific TCCs were derived from peripheral blood mononuclear cells (PBMCs) of a normal donor and maintained in culture as described previously (39) with some variations. In particular, cultures were set up in the presence or absence of 100 U/ml IL-4 (40) to obtain Th2 TCCs. PPD or Parj1 specificity was assessed by proliferation assays using irradiated autologous PBMC prepulsed or not with PPD (10 µg/ml) or Parj1 (50 µg/ml). Cluster of differentiation (CD)1b-restricted TCCs were provided by G. De Libero and obtained as described previously (37) .

Monocyte differentiation into DCs
Monocytes were isolated from PBMCs of normal donors by direct magnetic sorting, and reference DCs were generated from monocytes using GM-CSF (25 ng/ml) and IL-4 (1000 U/ml), as described previously (41) .

TCCs were grown in medium without IL-2 for 24 h, irradiated (1500 RAD), and cocultured with autologous monocytes at 1:1 ratio in the absence or presence of antigen in CM plus 10% FCS without the addition of any known monocyte differentiation factor or cytokine. Experiments in transwell plates were set up using 24-well tissue culture plates (Falcon; Becton Dickinson, Franklin Lakes, NJ, USA) with cell culture inserts (pore size 0.4 µm). In the upper inserts, TCCs and autologous monocytes were placed at 1:1 ratio in the absence or presence of an antigen, and monocytes from the same or different donors were cultured in the lower chamber (at 4x105 cells/ml). After 6 days, monocytes from the lower chambers were analyzed. In some experiments monocytes were cultured for 6 days with autologous peripheral CD4+ lymphocytes, isolated by indirect magnetic sorting with the CD4+ T-Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) at 1:1 ratio in the presence or absence of SEA (0.1 µg/ml).

When indicated, on the 5th day of culture DCs were stimulated overnight with 0.2 µg/ml LPS of E. coli.

TCCs were also stimulated with plastic bound-anti-CD3 monoclonal antibody (mAb) (Immunotech, Fullerton, CA, USA; 2 µg/ml); supernatants were collected on day 3 and used diluted 1:2.

FACS analysis
Biotin conjugated anti-CD1c mAb was from Cymbus Biotechnology LTD (Hants, UK), and human-adsorbed FITC-conjugated streptavidin was from Sigma. All the others used mAb, and appropriate isotype controls were from Pharmingen (San Diego, CA, USA). For intracellular cytokines staining, T cells were stimulated with 10–7 M PMA and 1 µg/ml ionomycin for 5 h, with brefeldin-A added during the last 2 h, then PE-conjugated anti-IL-4 or IL-10 and FITC-conjugated anti-IFN-{gamma} or IL-2 were added after fixation and permeabilization using Cytofix/CytopermTM (Pharmingen).

Microscopic evaluation of DCs
Monocytes, differentiated on round-shaped coverslips seeded on the bottom of plastic 24-well culture plates, were analyzed in phase contrast mode. Images were acquired with Leica Image Manages 1000 software using a Leica DFC350FX camera mounted on a microscope Leica model DM 4000B using an x40 objective (Leica Microsystems, Wetzlar, Germany).

Phagocytosis and endocytosis assays
DCs were incubated 1 h at 37°C with Gfp-rBCG at a multiplicity of infection = 1:6 in CM plus 10% FCS, then washed by low-speed centrifugation (100 g) and analyzed by flow cytometry. Phagocytosis was measured as percentage of fluorescent cells within the electronic gate based on the DC side and forward scatter. Endocytosis was evaluated using FITC-albumin (1 mg/ml) in CM plus 25 mM Hepes and 10% FCS for 1 h at 37°C, as described (41) and measured as median channel of fluorescence. To test the responsiveness to LPS stimulation, results were expressed as mean values ± SD of the Gfp-rBCG phagocytosis (percent fluorescent cells) or FITC-albumin endocytosis (median channel of fluorescence) reduction in comparison to non-LPS-treated DCs.

Priming of naive T cells
Decreasing numbers of DCs were cultured with 3 x 104 cord blood CD4+ T cells purified by indirect magnetic sorting with the CD4+ T-Cell Isolation Kit. T-cell proliferative response was measured after 6 days of coculture by a 16 h pulse with 3H-thymidine (1 µCi/well). In parallel cultures, supernatants were harvested for cytokine determination by ELISA, and T cells were stimulated with PMA/ionomycin to cytometrically evaluate their intracellular cytokine accumulation.

Antigen presentation assays
Responder CD1b-restricted and major histocompatibility complex (MHC) class II-restricted TCCs (3x104 cells) were cocultured with non-LPS-stimulated DCs (4x103 cells) in 96-well flat-bottom plates. Antigens were added at 5-fold dilutions. T-cell proliferation was measured after 48 h of culture by a 16 h pulse with 3H-thymidine.

TCCs and autologous monocytes were cocultured in the presence of PPD (10 µg/ml). After 3 days, the supernatants were collected and the amounts of released cytokines measured by ELISA.

Cytokine determination
Cytokine content was determined using commercially available ELISA kits (R&D) according to the manufacturer’s instructions (detection limit of the assays: 15 pg/ml).

The IL-2 content was measured as U/ml in a biological assay measuring the CTLL2 cell line proliferation (42) and recombinant IL-2 as standard.

Real-time polymerase chain reaction (PCR)
Total RNA was extracted from 1.5 x 106 TCCs and DCs using RNeasy kits (Quiagen, Hilden, Germany) and was reverse transcribed using the high-capacity cDNA Archive Kit in a ABI Prism 7000 Sequence Detector System (Applied Biosystems, Foster City, CA, USA). PCRs were performed in triplicate using TaqMan chemistry with primer and probe sets from the Assay-on-Demand list (all from Applied Biosystems). Fold induction was calculated by the {Delta}{Delta}Ct method (43) using the 18S mRNA level to normalize values and the mRNA level of basal condition (nonactivated T cells or immature reference DCs) as a calibrator.

Statistical analysis
The statistical significance of the difference between groups of data with a normal distribution was determined by the ANOVA with Bonferroni-Dunn posttests using the Statview 4.1 program (Abacus Concepts, Inc., Berkeley, CA, USA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of antigen-specific TCCs with different functional polarization
We isolated a panel of PPD-specific TCCs from normal donors. The TCCs were tested for their capacity to secrete IFN-{gamma} and IL-4 on antigen-specific stimulation using autologous monocyte as APCs by intracellular cytokine staining and flow cytometric analysis (Fig. 1 A). For a more complete functional characterization, the most relevant cytokines produced by the selected pool of Th1, Th2, and Th0 TCCs was measured as mRNA transcript levels by real-time (RT)-PCR and as amount of proteins by ELISA. Figure 1B shows the cytokine release after TCC antigen-specific PPD stimulation using monocytes as APCs, and Table 1 reports data obtained stimulating TCCs with plastic bound anti-CD3, to avoid the possible detection of cytokines released by APCs. Th1 clones released IFN-{gamma}, GM-CSF, tumor necrosis factor (TNF)-{alpha}, and IL-2 and increased their IL-6, IL-1β, and IL-3 mRNA transcripts following activation. Conversely, the stimulation of Th2 clones was characterized by the release of IL-4 together with GM-CSF, IL-13, and IL-5. On activation Th0 clones, in addition to the IL-4 and IFN-{gamma} release, increased GM-CSF, TNF-{alpha}, IL-2, and IL-5 protein secretion and the mRNA induction of IL-1β, IL-3, with a remarkable expression of IL-6. All the tested cytokines were induced specifically on stimulation because cytokines in resting TCCs were not detectable by ELISA and were detectable at extremely low levels by RT-PCR (data not shown). Note that the amount of cytokines secreted by activated TCCs, and in particular GM-CSF and IL-4, was of the same order of magnitude of that required to obtain monocyte differentiation in vitro using the recombinant cytokines.


Figure 1
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  		Figure 1. Antigen-specific T-cell activation induces monocyte differentiation. A) PPD-specific T clones were tested for their capacity to secrete IFN-{gamma} and IL-4 by intracellular cytokine staining and flow cytometry analysis. Similar cytokine pattern was obtained with several clones per each subset. B) Th1, Th0, and Th2 PPD-specific TCCs and autologous isolated monocytes were cocultured in the presence of PPD (10 µg/ml). After 3 days, the supernatants were collected and the amounts of released IFN-{gamma}, IL-4, and GM-CSF were measured by ELISA. Values indicate the mean of 4 different clones per each subset and are expressed as ng/ml ± SD. C) Reference DCs were generated in a 6-day culture of monocytes with GM-CSF and IL-4 (DCk). Autologous monocytes alone (Mo) or cocultured with irradiated PPD-specific Th2 or Th1 TCC were incubated for 6 days in the absence (Mo+TCC) or the presence of PPD (Mo+PPD+Th2 and Mo+PPD+Th1, respectively). Double staining analysis for CD1a and CD14 was performed on CD3-negative cells. Numbers indicate the percentage of cells in each quadrant. D) Reference DCs were generated in a 6-day culture of monocytes with GM-CSF and IL-4 (DCk). Monocytes were placed in the lower chamber of transwell plate, whereas in the upper insert PPD-pulsed monocytes were cocultured with autologous PPD-specific Th2 (Mo+PPD+Th2), Th1 (Mo+PPD+Th1), or Th0 (Mo+PPD+Th0) TCCs. As a control of antigen specificity, PPD-specific Th2 or Th1 TCCs were also cocultured with Parj1-pulsed autologous monocytes (Mo+PJ+TCC) in the upper insert of the transwell plate. After 6 days, CD1a/CD14 surface expression of monocytes of the lower insert was checked. Numbers indicate the percentage of cells in each quadrant. Results are from 1 experiment representative of 10. Similar results were obtained using at least 4 different clones per Th subset.


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  		Table 1. Th1, Th0, and Th2 TCCs synthesize different cytokines upon activation

Specific T-cell activation induces antigen-presenting monocyte to differentiate into DCs
First, we set up for 6 days cocultures of irradiated Th1 or Th2 TCCs and autologous monocytes in the presence or absence of antigen (PPD) without the addition of any known differentiation factor. Phenotypic analysis of CD3-negative cells revealed that after a 6 day culture monocytes alone or cocultured in the absence of PPD and irrespective of the Th1 or Th2 phenotype of the used TCCs showed a macrophage-like phenotype, as revealed by microscopy and by their forward and side scatters (not shown) and CD14 expression. On the other hand, in the presence of PPD, TCCs induced the differentiation of PPD-presenting monocytes into CD14–ve cells. These cells showed a diverse CD1a expression according to the Th1 or Th2 phenotype of the cocultured clone. In particular, CD1a surface level in monocytes cocultured with Th2 TCCs was comparable to that of reference DCs generated in the presence of GM-CSF and IL-4 (DCk), whereas monocytes cultured with activated Th1 TCCs exhibited a significantly reduced percentage of CD1a+ve cells (Fig. 1C ). These changes in monocyte phenotype were clearly dependent on the T-cell activation, because PPD added to the culture was unable to interfere with the fate of monocytes cultured with or without GM-CSF and IL-4 (data not shown). These data indicate that, in addition to T-cell activation, a consequence of antigen presentation is the differentiation of presenting monocytes.

T-cell-dependent monocyte differentiation into DCs does not require cell-to-cell contacts
To test whether soluble factors released by activated TCCs and/or their intimate interaction with monocytes were crucial to induce monocyte differentiation, we also performed experiments using a transwell culture system, in which soluble cytokines released from cells in the upper chamber could diffuse to the lower chamber and interact with monocytes in the absence of cell-to-cell contact with TCCs. In the upper inserts, PPD-specific Th1, Th2, or Th0 TCCs and autologous monocytes were placed with or without PPD or Parj1, while monocytes from the same or from a different donor were placed in the lower chamber. Analysis of cells in the lower inserts after 6 days of culture showed that monocytes differentiated into cells with a level of CD1a molecule expression mirroring that of cells derived from monocytes cocultured with Th1 and Th2 TCCs in the presence of PPD. In the absence of antigens (not shown) or in the presence of a nonrelated antigen, such as Parj1, monocytes did not differentiate into DCs and revealed a macrophage-like phenotype (Fig. 1D ). We also showed that more than 70% of cells derived from monocyte-sensing factors released by activated Th0 TCCs were CD1a+ve. Taken together, data indicate that monocytes that have not processed and presented a given antigen may undergo differentiation in a microenvironment in which other APCs are responsible for T-cell activation.

Different TCC subpopulations induce monocytes to differentiate into DCs with distinct phenotypes
Expression of group I CD1 molecules is considered a distinct but not unique characteristic of monocyte-derived DCs. Moreover, CD1 expression is not a DC marker, because CD1–ve DCs have been described (44 , 45) . To establish clearly the nature of cells derived from monocytes cultured with activated TCCs, we performed a larger phenotypic characterization of monocytes cultured with activated Th2, Th1, or Th0 PPD-specific TCCs in comparison to DCk cells. Analysis revealed that cells derived from monocytes cultured with activated TCCs were indeed DCs. Monocytes cultured with Th2 TCCs differentiated into DCs (DCh2) not distinguishable from DCk (Fig. 2 A–C). In fact, both these DC populations homogeneously expressed high levels of presenting molecules, that is, group I CD1 molecules (CD1a, CD1b, and CD1c; Fig. 2A ) and MHC class I and class II (DR) molecules (Fig. 2B ) and were equally able to undergo maturation following LPS treatment as assessed by the upregulation of activation markers (CD83, CD80, CD86, CD40, MHC class I and II) (Fig. 2B ). Monocytes cultured with activated Th1 and Th0 TCCs differentiated into DCs (DCh1 and DCh0, respectively) with phenotypic characteristics different from DCk. In fact, they showed a reduced CD1 group I molecule expression (Fig. 2A ), and the level of maturation markers (CD83, CD86, DR) was higher than that of reference immature DCs (Fig. 2B ). It is interesting to note that DCh1 and DCh0 cells did not markedly modify their phenotype after LPS treatment (Fig. 2B ), with the exception of CD86, which was upregulated in DCh0 cells. All the DC subsets were CXCR4–ve and did not upregulate this receptor after LPS (data not shown). On the other hand, DCk and DCh2 cells turned CCR7+ve after LPS stimulation, whereas DCh1 and DCh0 cells were CCR7+ve even if the expression was lower than in the mature DCk cells and did not change after LPS treatment. All the DC subset were CCR5+ve, but although DCh2 and DCk cells turned CCR5–ve, DCh0 and DCh1 cells reduced the expression of this receptor only after LPS treatment (Fig. 2B ).


Figure 2
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  		Figure 2. Monocytes differentiate into DCs with distinct phenotypes following activation of different T helper subpopulations. Monocytes were cultured for 6 days with GM-CSF and IL-4 (DCk) or in the lower chamber of a transwell device in which Th2, Th1, or Th0 TCCs were cocultured with autologous antigen-pulsed monocytes in the upper chamber. DCs derived from monocytes sensing cytokine released from Th2, Th1, or Th0 TCCs were defined as DCh2, DCh1, and DCh0 cells, respectively. A) Flow cytometric analysis of CD1 molecules. The percentage of positive cells is reported in the relative histogram. B) Flow cytometric analysis of indicated surface molecules with or without LPS stimulation. The open histograms show staining of non-LPS-stimulated cells; filled histograms show staining of the corresponding LPS-treated cells. C) A CD1a/DC-SIGN surface expression. Numbers indicate the percentage of cells in the correspondent quadrant. Data are from 1 experiment representative of 8 independent experiments. D) Typical morphological appearance of the DC cultures on day 6 without LPS treatment. Cells were allowed to differentiate on round-shaped coverslips seeded onto the bottom of 24-well culture plastic plates and analyzed in phase contrast mode (x400).

In comparison to reference DCk cells, a reduced percentage of DCh1 cells were DC-SIGN+ve, and the majority of DC-SIGN+ve were CD1a–ve (Fig. 2C ). The presence of T-cell-released IL-2 did not affect the surface expression of IL-2 receptor {alpha} chain (CD25) in T-cell-induced DCs. All the mature DCs showed levels of CD25 comparable to that of mature DCk cells (data not shown). Moreover all the DCs were CD11c+ve, as expected according to their myeloid origin (data not shown).

DCh2 cells as well as reference DCk cells were nonadherent cells, whereas DCh1, and partially DCh0, cells were adherent, with long dendrites (Fig. 2D ) and had a morphology recalling that of DCs recently activated by maturation stimuli such as LPS.

The same differentiation of monocytes was observed using antigen-activated Parj1-specific Th1, Th2, and Th0 TCCs (data not shown).

Freshly isolated T lymphocytes ex vivo induce monocyte to differentiate into DCs
To test whether the capacity to induce monocyte differentiation into DCs was an in vitro acquired function of cultured and IL-2 expanded TCCs or a general function of activated T lymphocytes, we set up an ex vivo test analyzing the phenotype of monocyte after a 6 day culture in the presence of superantigen-activated freshly isolated autologous CD4+ lymphocytes. After 6 days of coculture with SEA activated, but not with resting T lymphocytes, monocytes differentiated into CD14–ve and CD1a+/–ve DCs (Fig. 3 ) with phenotypic characteristic similar to DCh1 cells (data not shown).


Figure 3
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  		Figure 3. Ex vivo T lymphocytes stimulation by a superantigen induces monocytes to differentiate into DCs. Freshly isolated CD4+ T lymphocytes and autologous monocytes were cocultured in the presence (Mo+SEA+CD4) or absence (Mo+CD4) of SEA at 0.1 µg/ml. After a 6 day culture, CD1a/CD14 expression was analyzed in the CD3–ve population. Reference DCs were generated in a 6 day culture of monocytes with GM-CSF and IL-4 (DCk). Numbers indicate the percentage of cells in the relative quadrant. In the absence of SEA, monocytes acquired a macrophage-like phenotype (CD14low CD1a–ve), whereas in the presence of SEA, monocytes lose CD14 expression and acquired CD1a molecules. SEA at the same concentration did not induce DC differentiation (Mo+SEA). Results are from 1 experiment representative of 3 independent experiments performed with different healthy donors.

DCs differentiated following T-cell activation show a differential cytokine secretion pattern
To measure the cytokines released by DCs, cultures were washed after 5 days, cells counted and adjusted to 3 x 105 cell/ml and cultured in the presence or absence of 0.2 µg/ml LPS for an additional 18 h before supernatants collection. DCh1 and DCh0 cells produced low amounts of IL-12p70 even after LPS stimulation, and their secretion was statistically different from that of DCk cells (P<0.001). On the other hand, LPS-matured DCh2 cells release amount of IL-12p70 not statistically different from that released by mature DCk cells (Fig. 4 ). Notably, DCh1 cells were characterized by their capacity to release spontaneously amounts of IL-10 significantly (P<0.05) higher than LPS-stimulated DCk cells, and the maturation stimulus did not significantly increase its release. DCh0 cells secreted IL-10 after LPS treatment at a level significantly (P<0.05) higher than mature DCk cells. RT-PCR analysis also showed that IL-1β and IL-6 mRNA transcripts were detected following activation in both DCh1 and DCh0 cells, which showed the highest expression of TNF-{alpha} transcripts among the different DC populations (data not shown).


Figure 4
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  		Figure 4. Cytokine secretion pattern of DC populations differentiated following T-cell activation. At day 5 of culture, the DCk, DCh2, DCh1, and DCh0 cells were incubated in the presence or absence of 0.2 µg/ml LPS for an additional 18 h. Supernatants were collected at the end of the culture and examined for IL-12p70 and IL-10 contents by ELISA. Values indicate the mean of 3 independent experiments and are expressed as pg/ml ± SD. *P < 0.05 vs. IL-10 released by DCk + LPS; °P < 0.001 vs. IL-12 released by DCk + LPS. n.s., nonsignificant differences vs. DCk + LPS.

DCs induced by activated T lymphocytes show different degree of bacterial phagocytosis and soluble antigen uptake
The capacity of DCs derived from monocyte following TCC activation to phagocytose Gfp-rBCG is reported in Fig. 5 . The percentage of fluorescent-bacteria-associated cells was comparable between DCh2 and reference DCk populations (DCh2=20% and DCk=23%). As expected, these percentages were reduced on LPS stimulation. On the contrary, a lower percentage of DCh1 (10%) and DCh0 (13%) bound Gfp-rBCG after 1 h of incubation, as compared to reference cells, consistent with the partial maturation state of these DCs (Fig. 5A ). Interestingly, however, the low internalization capacity of these cells was further reduced after LPS stimulation, which suggests a sensitivity to this maturation stimulus that was not observed in terms of surface molecule expression (Fig. 5A, B ). We obtained similar results also evaluating the capacity of FITC-albumin endocytosis (Fig. 5C, D ) as a measure of soluble antigen uptake.


Figure 5
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  		Figure 5. Phagocytosis and endocytosis of DC populations differentiated following T-cell activation. At day 5 of culture, the DCk, DCh2, DCh1, and DCh0 cells were incubated in the presence or absence of 0.2 µg/ml LPS for an additional 18 h. A) At the end of cell culture, DCs were incubated 1 h at 37°C with Gfp-rBCG at a multiplicity of infection cell:BCG = 1:6 in CM supplemented with 10% FCS, then washed by low-speed centrifugation (100 g). Percentage of cells that bound Gfp-rBCG was evaluated by flow cytometry; number is indicated in the quadrant. Results are from 1 experiment representative of 4 independent experiments. B) Reduction of phagocytic activity after LPS-induced maturation. Results are expressed as reduction ± SD in percentage of Gfp-rBCG phagocytosis on LPS stimulation of 4 independent experiments. C) Endocytic capacity of DC subsets. Analyses by flow cytometry of FITC-albumin uptake after 1 h at 0°C (negative control; dotted histograms) or 37°C of immature (empty black histograms) and LPS-stimulated (filled gray histograms) DCs. D) Reduction of endocytosis activity after LPS-induced maturation. Results are expressed as reduction ± SD in mean fluorescent intensity on LPS-stimulation of 4 independent experiments.

DCh1 and DCh0 cells have a reduced capacity to prime naive CD4+ T lymphocytes
The antigen-presenting capability of the diverse monocyte-derived DC populations was evaluated in a mixed lymphocyte reaction (MLR) using a sorted population of allogeneic cord blood CD4+ T lymphocytes as responder cells. DCh1, DCh2, and DCh0 cells generated from monocytes in the lower chamber of a transwell device were used to obtain DCs noncontaminated by irradiated T cells. All the non-LPS-treated DCs were unable to stimulate an efficient proliferation of naive T cells, which, on the other hand, proliferated extensively when stimulated by LPS-matured DCh2 and reference DCk cells (Fig. 6 A). Interestingly, LPS-matured DCh1 and DCh0 cells induced a T-cell proliferation that was constantly of lower magnitude than the proliferation induced by mature DCk cells (Fig. 6A ).


Figure 6
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  		Figure 6. DC populations differentiated following T-cell activation have different capacity to prime naive T cells and to stimulate memory T cells. A) DCk (squares), DCh2 (circles), DCh1 (triangles), and DCh0 (crosses) cells at day 5 of culture were incubated in the absence (open symbols) or presence of 0.2 µg/ml LPS (filled symbols) for an additional 18 h and used as APCs at different cell numbers to stimulate 3 x 104 cord blood-purified CD4+ T cells. The T-cell proliferative response was measured after 6 days by 3H-thymidine incorporation; results are expressed as mean counts per minute (cpm) of triplicate wells. One experiment representative of 6 is shown. B) Cytokine production by naive CD4+ T cells after coculture with allogeneic LPS-treated DCk, DCh2, DCh1, and DCh0 cells. Dot plots represent the flow cytometric analysis of intracellular cytokine accumulation; numbers indicate the percentage of cells in the corresponding quadrant. Histograms represent cytokines released in supernatants as measured by ELISA (ng/ml±SD) with the exception of IL-2, which was measured by a biological assay using the cell line CTLL-2 and expressed as U/ml ± SD. C, D) To test the capacity of the different DC population to stimulate memory T cells, DCk, DCh2, DCh1, or DCh0 cells were used to stimulate PPD-specific MHC class II-restricted (C) or PIM2-specific CD1b-restricted TCCs (D). After 48 h of coculturing, 3H-thymidine was added, and cells were harvested 18 h later. Results are expressed as mean ± SD cpm in triplicate wells. One experiment representative of 3 is shown.

Analysis of intracellular cytokine production by naive CD4+ T cells expanded in an MLR showed that mature DCh2 cells induced the expansion of a number of IFN-{gamma} and IL-2 producing T cells comparable to DCk cells (Fig. 6B ). Note that DCh2 cells primed a reduced number of cells secreting IL-4, which indicates that they are capable of inducing a more marked Th1 response than reference DCk cells. Inversely, among the T cells polarized by mature DCh1 and DCh0 cells, a statistically significant reduced percentage of IFN-{gamma}- and IL-2-secreting cells and a higher proportion of cells producing IL-10 in T expanded by DCh0 cells was observed. ELISA measurement also confirmed that IL-10 was released by T cells stimulated by DCh0 cells in amounts statistically higher (P<0.01) than those released by T cells primed by other APCs (Fig. 6B ).

DCh1 and DCh0 cells have a reduced capacity to activate antigen-specific TCCs
The ability of the different DCs to present PPD and PIM2 to specific MHC class II and CD1-restricted TCCs, respectively, was analyzed. As shown in Fig. 6C , the efficiency of presentation, measured as the PPD concentration required to give 50% of maximum TCC proliferative response, varies with APCs. The antigen presentation capacity of DCh2 cells was comparable to that of DCk cells, whereas DCh1 and DCh0 cells showed a reduced efficiency. Interestingly, DCh2 cells were even more efficient than DCk cells in presenting a lipidic antigen to a specific CD1b-restricted TCCs (Fig. 6D ). On the other hand, DCh1 and DCh0 cells showed a reduced capacity to activate the specific TCCs, in terms of both maximum response and amount of antigen required, that can be only partially attributed to the reduced expression of surface CD1b molecules.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study we demonstrate that human CD4+ T lymphocytes can induce monocytes to differentiate into DCs following antigen-specific activation. DC differentiation is induced in both antigen-presenting and in bystander monocytes, which sense the cytokines released by activated T cells. The in vitro model described here is of particular relevance because it does not include the addition of any known DC differentiation factor, but reproduces the functional consequences of specific T-cell activation, which follows antigen presentation, on monocyte differentiation.

Monocytes, cocultured with autologous T cells in the absence of antigens, differentiated into CD14lowCD1–ve cells similar to macrophages derived from monocytes cultured with or without M-CSF, indicating that the default differentiation pathway in vitro of monocytes leads to macrophages and that nonactivated T cells do not interfere with this process. Conversely, antigen-pulsed monocytes cocultured with specific TCCs differentiated into DCs, and because the cell-to-cell contact of monocytes with activated T cells was not required for DC generation, we conclude that T-cell-released soluble factors are involved in monocyte differentiation. It is interesting to note that the phenotype of differentiated DCs varied according to the functional polarization of T cells. DCh2 cells showed a phenotype indistinguishable from reference DCk cells, whereas DCh1 and DCh0 cells showed a reduced expression of group I CD1 molecules and a more mature phenotype, which was not significantly modified by LPS stimulation. These data are in line with the fact that Th1-secreted cytokines such as GM-CSF, IL-3, TNF-{alpha}, or IFN-{gamma} have been previously described to induce generation of DCs (46 , 47) with characteristics different from those differentiated with Th2 cytokines, namely, GM-CSF, IL-4, or IL-5 (41 , 48) . Our findings expand preliminary data indicating a role of nonantigen-specific CD8+ T cells (34) and of natural killer (NK) and NK T cells (33 , 35) in the induction of monocyte differentiation into DCs. On the other hand, our data are in partial contrast with a recent paper proving the inability of MHC class II restricted T cells, unlike NK T cells, to promote monocyte differentiation into DCs (35) . We obtained monocyte-derived DCs using 2 diverse pools of TCCs with different antigen specificity and notably also in ex vivo experiments using freshly isolated CD4+ T cells stimulated with a superantigen. Thus, a diverse in vitro setting could be responsible for the different result obtained in the cited paper (35) . A previous paper suggested the ability of activated T cells to differentiate monocytes into DCs through the CD40 ligation (36) , but the use of adherent instead of purified monocytes and the lack of a formal demonstration of CD40 ligand involvement made the results of that paper nonconclusive. However, even if our findings prove that soluble factors are responsible for monocyte differentiation, we cannot exclude that a monocyte-T-cell interaction through CD40 ligation may positively concur to the DC generation.

DCs derived from monocytes sensing a Th1 or Th0 inflammatory microenvironment had a reduced capacity to allow the expansion of naive T cells and, in agreement with their reduced IL-12 and increased IL-10 synthesis, showed a hampered ability to induce their functional polarization into Th1 cells. It is interesting to note that DCh1 and particularly DCh0 cells were capable to prime IL-10-secreting T cells with a possible regulatory role. Expansion of IL-10-secreting T cells was shown to be dependent on the presence of IL-10 itself (49) . However, since both DCh1 and DCh0 cells secrete IL-10, but the expansion of IL-10-secreting T cells is mediated mainly by DCh0 cells, other characteristics of DCh0 cells, including their high TNF-{alpha} synthesis (47 , 50) and CD86 expression (51) , must be involved and are now under investigation.

DCh1 and DCh0 cells were shown to be less efficient than DCk and DCh2 cells in stimulating antigen-specific TCCs, which reproduce in vitro experienced/memory T cells (52) . Because CD1 molecule expression in DCh1 and DCh0 cells was reduced, it was not surprising to observe a decreased capacity of these APCs to present a lipid antigen to CD1-restricted TCCs (53) . Unexpectedly, DCh1 and DCh0 cells had a reduced ability to present antigen to MHC class II restricted TCCs, even if their DR expression was higher than reference DCk cells, which may be attributed to their decreased antigen uptake and abundant secretion of IL-10 (54 , 55) , together with other not yet known characteristics.

Although it is not always possible to translate in vivo what observed in vitro, the phenomenon that we describe herein is likely to occur in chronic inflammation sites where monocytes and lymphocytes are corecruited (56) , and our data suggest that the generation of monocyte-derived DCs described in infected rodents (22) has the possibility to occur also in humans. Together with T-cell-derived cytokines, it is likely that other microenvironmental factors related to the chronic inflammation and to its causes could contribute to the differentiation of monocytes into "inflammatory" DCs with diverse phenotype and functional ability (26) . However, the demonstration that DCh2, DCh1, and DCh0 cells have different capacity to induce the functional polarization of naive T cells and to stimulate memory T lymphocytes is suggestive for a feedback control of Th1/Th2 immune responses orchestrated by monocyte-derived DCs. Responses characterized by a strong Th2 polarization could be counterbalanced by the differentiation of DCh2 cells that, in turn, would prime new generation of antigen/allergen-specific Th1 cells. On the other hand, the differentiation of DCh1 and DCh0 cells could be instrumental in limiting the Th1-associated tissue damage in chronic inflammation sites.


   ACKNOWLEDGMENTS
 
We thank Prof. Antonio Cassone and Prof. Vincenzo Barnaba for suggestions and critical reading of the manuscript. This paper was partially supported by the EC project MILD-TB, contract no. 037326. The authors declare no financial conflict of interest.

Received for publication February 15, 2008. Accepted for publication May 2, 2008.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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