The FASEB Journal • Research Communication

T-cell-mediated and antigen-dependent differentiationof human monocyte into different dendritic cellsubsets: 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 Sanita,Rome, Italy

ABSTRACT It is well established that human mono-cytes differentiate into dendritic cells (DCs) whencultured with certain cytokine cocktails, such as granu-locyte-macrophage colony-stimulating factor and inter-leukin-4. Conversely, it is not completely establishedwhich cell population synthesizes the cytokines re-quired for monocyte differentiation and how theirsecretion is regulated. We show that on specific activa-tion T cells induce the differentiation into DCs ofantigen-presenting and bystander monocytes. Mono-cytes exposed to cytokines released by Th1 and Th0lymphocytes differentiate into DCs with a reducedantigen uptake and antigen presentation capacity.Moreover, these DCs show a limited capacity to induceTh1 polarization of naive T cells but are capable ofpriming interleukin-10-secreting T cells. Conversely,DCs derived from monocytes sensing cytokines re-leased by Th2 lymphocytes are antigen-presenting-cell(APC) endowed with a marked Th1 polarization capac-ity. Monocytes are corecruited with lymphocytes inchronic inflammation sites; thus our results suggest thatfunctionally different DCs can be generated in environ-ments characterized by the prevalent release of Th1-,Th0-, or Th2-associated cytokines. Because the APCcapacities of these DCs have opposite functional con-sequences, a contribution in the regulation of theongoing immune response by monocyte-derived inflam-matory DCs is envisaged.—Mariotti, S., Sargentini, V.,Marcantonio, C., Todero, E., Teloni, R., Gagliardi,M. C., Ciccaglione, A. R., Nisini, R. T-cell-mediated andantigen-dependent differentiation of human monocyteinto different dendritic cell subsets: a feedback controlof Th1/Th2 responses. FASEB J. 22, 3370–3379 (2008)

Key Words: antigen presentation/processing � cytokines � celldifferentiation � chronic inflammation � T-cell clones

Dendritic cells (DCs) are a heterogeneous popula-tion of cells with different myeloid or plasmacytoidorigin and differential phenotype and function (1, 2).

In recent years DCs have been increasingly studiedfor 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 crucialcells in the induction and regulation of adaptive immu-nity (7, 8). Moreover, DCs have been widely studied aspromising “adjuvants” in vaccines for prevention ofmicrobial infections, allograft rejection, and treatmentof cancer and autoimmune diseases (9–14). Finally, alarge body of evidence on their importance in immu-noregulation envisages the possibility for exploitingDCs for more general biomedical purposes (15).

Several bone marrow and blood precursors of DCshave been identified, including monocytes (1, 2). Evi-dence for the capacity of monocytes to differentiateinto DCs has been reported in the mouse models,where monocytes have been in vivo tracked usinginternalized fluorescent latex microspheres (16–20) orin the Listeria monocytogenes and Leishmania major infec-tion system (21, 22). Interestingly, however, bloodmonocytes do not seem to contribute to the generationof splenic mouse DCs (23–25). On the other hand, inhumans it has not been defined which DC subsetoriginates from monocytes, and experiments regardingtransendothelial migration of DCs generated frommonocytes (17) have not conclusively established whichprocesses promote monocyte differentiation into DCsor into macrophages (26).

In culture, human monocytes acquire a macrophagephenotype both in the presence and in the absence ofadded cytokines, such macrophage colony-stimulatingfactor (M-CSF) (27); thus, differentiation of monocytesinto macrophages seems to represent a default differ-entiation program of monocytes on extravasation (28).Conversely, well-defined cytokine cocktails are requiredfor monocytes to differentiate into DCs (29–32). Sev-eral cell types have been indicated as a possible sourceof cytokines capable of inducing monocyte differentia-tion into DCs in vitro (33–36). However, the stimulusand context in which these cells would promote the

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

doi: 10.1096/fj.08-108209

3370 0892-6638/08/0022-3370 © FASEB

monocyte differentiation into DCs instead of macro-phages could not be unambiguously defined.

Starting from the hypothesis that monocytes couldrepresent progenitor cells of tissue macrophages underphysiological conditions and cells committed to thelocal replacement of migrating or dying DCs followingan inflammatory process (1, 2), we analyzed the conse-quences of T-cell activation in the monocyte differen-tiation into DCs using a panel of Th1, Th2, and Th0antigen-specific T-cell clones (TCCs). We obtained amodel of monocyte differentiation not based on theuse of synthetic cytokines or factors and that reasonablyreproduces inflammatory microenvironments, allowingan easier extrapolation of data obtained in vitro.

MATERIALS AND METHODS

Reagents

Phytohemagglutinin was from Murex (Dartford, UK) andpurified protein derivative (PPD) from Statens Serum Insti-tute (Copenhagen, Denmark). Recombinant interleukin (IL)-2 was from EuroCetus (Milan, Italy). Recombinant IL-4 wasfrom R&D Systems (Minneapolis, MN, USA), granulocytemacrophage colony-stimulating factor (GM-CSF) from San-doz (Basel, Switzerland), and tritiated (3H)-thymidine fromAmersham (Little Chalfont, UK). Lipopolysaccharide (LPS)of E. coli, phorbol 12-myristate 13-acetate (PMA), ionomycin,brefeldin-A, Staphylococcus aureus enterotoxin A (SEA), fluo-rescein isothiocyanate (FITC) -albumin, and Hepes werefrom Sigma Chemical Co. (St. Louis, MO, USA). Phosphati-dylinositol dimannosides (PIM2) was kindly provided by Pro-fessor Gennaro De Libero (University Hospital Basel, Basel,Switzerland) (37), green fluorescent protein recombinantBacillus Calmette-Guerin (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 Sanita, Rome, Italy) (38). RPMI 1640 wasused, supplemented with 100 U/ml kanamycin, 1 mM glu-tamine, 1 mM sodium pyruvate, 1% nonessential amino acids(complete medium, CM) (Euroclone Ltd., UK), and en-riched with 10% fetal calf serum (FCS) (Hyclone, Logan, UT,USA).

Generation of antigen-specific TCCs

Human cells were obtained from healthy blood donor volun-teers who gave their informed consent to the use of part oftheir blood donation for in vitro experiments not involvingthe screening for infectious diseases. The study is part of aEuropean Community project, which was approved by EthicalCommittee of Istituto Superiore di Sanita.

PPD and Parj1-specific TCCs were derived from peripheralblood mononuclear cells (PBMCs) of a normal donor andmaintained in culture as described previously (39) with somevariations. In particular, cultures were set up in the presenceor absence of 100 U/ml IL-4 (40) to obtain Th2 TCCs. PPDor Parj1 specificity was assessed by proliferation assays usingirradiated autologous PBMC prepulsed or not with PPD (10�g/ml) or Parj1 (50 �g/ml). Cluster of differentiation(CD)1b-restricted TCCs were provided by G. De Libero andobtained as described previously (37).

Monocyte differentiation into DCs

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

TCCs were grown in medium without IL-2 for 24 h,irradiated (1500 RAD), and cocultured with autologousmonocytes at 1:1 ratio in the absence or presence of antigenin CM plus 10% FCS without the addition of any knownmonocyte differentiation factor or cytokine. Experiments intranswell plates were set up using 24-well tissue culture plates(Falcon; Becton Dickinson, Franklin Lakes, NJ, USA) withcell culture inserts (pore size 0.4 �m). In the upper inserts,TCCs and autologous monocytes were placed at 1:1 ratio inthe absence or presence of an antigen, and monocytes fromthe same or different donors were cultured in the lowerchamber (at 4�105 cells/ml). After 6 days, monocytes fromthe lower chambers were analyzed. In some experimentsmonocytes were cultured for 6 days with autologous periph-eral CD4� lymphocytes, isolated by indirect magnetic sortingwith the CD4� T-Cell Isolation Kit (Miltenyi Biotec, BergischGladbach, Germany) at 1:1 ratio in the presence or absenceof SEA (0.1 �g/ml).

When indicated, on the 5th day of culture DCs werestimulated overnight with 0.2 �g/ml LPS of E. coli.

TCCs were also stimulated with plastic bound-anti-CD3monoclonal antibody (mAb) (Immunotech, Fullerton, CA,USA; 2 �g/ml); supernatants were collected on day 3 andused diluted 1:2.

FACS analysis

Biotin conjugated anti-CD1c mAb was from Cymbus Biotech-nology LTD (Hants, UK), and human-adsorbed FITC-conju-gated streptavidin was from Sigma. All the others used mAb,and appropriate isotype controls were from Pharmingen (SanDiego, CA, USA). For intracellular cytokines staining, T cellswere stimulated with 10�7 M PMA and 1 �g/ml ionomycinfor 5 h, with brefeldin-A added during the last 2 h, thenPE-conjugated anti-IL-4 or IL-10 and FITC-conjugated anti-IFN-� or IL-2 were added after fixation and permeabilizationusing Cytofix/Cytoperm™ (Pharmingen).

Microscopic evaluation of DCs

Monocytes, differentiated on round-shaped coverslips seededon the bottom of plastic 24-well culture plates, were analyzedin phase contrast mode. Images were acquired with LeicaImage Manages 1000 software using a Leica DFC350FXcamera mounted on a microscope Leica model DM 4000Busing an �40 objective (Leica Microsystems, Wetzlar, Ger-many).

Phagocytosis and endocytosis assays

DCs were incubated 1 h at 37°C with Gfp-rBCG at a multi-plicity of infection � 1:6 in CM plus 10% FCS, then washed bylow-speed centrifugation (100 g) and analyzed by flow cytom-etry. Phagocytosis was measured as percentage of fluorescentcells within the electronic gate based on the DC side andforward scatter. Endocytosis was evaluated using FITC-albu-min (1 mg/ml) in CM plus 25 mM Hepes and 10% FCS for1 h at 37°C, as described (41) and measured as medianchannel of fluorescence. To test the responsiveness to LPSstimulation, results were expressed as mean values � sd of theGfp-rBCG phagocytosis (percent fluorescent cells) or FITC-

3371HUMAN T CELLS DRIVE DC DIFFERENTIATION OF MONOCYTE

albumin endocytosis (median channel of fluorescence) re-duction in comparison to non-LPS-treated DCs.

Priming of naive T cells

Decreasing numbers of DCs were cultured with 3 � 104 cordblood CD4� T cells purified by indirect magnetic sorting withthe CD4� T-Cell Isolation Kit. T-cell proliferative responsewas measured after 6 days of coculture by a 16 h pulse with3H-thymidine (1 �Ci/well). In parallel cultures, supernatantswere harvested for cytokine determination by ELISA, and Tcells were stimulated with PMA/ionomycin to cytometricallyevaluate their intracellular cytokine accumulation.

Antigen presentation assays

Responder CD1b-restricted and major histocompatibilitycomplex (MHC) class II-restricted TCCs (3�104 cells) werecocultured with non-LPS-stimulated DCs (4�103 cells) in96-well flat-bottom plates. Antigens were added at 5-folddilutions. T-cell proliferation was measured after 48 h ofculture by a 16 h pulse with 3H-thymidine.

TCCs and autologous monocytes were cocultured in thepresence of PPD (10 �g/ml). After 3 days, the supernatantswere collected and the amounts of released cytokines mea-sured by ELISA.

Cytokine determination

Cytokine content was determined using commercially avail-able ELISA kits (R&D) according to the manufacturer’sinstructions (detection limit of the assays: 15 pg/ml).

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

Real-time polymerase chain reaction (PCR)

Total RNA was extracted from 1.5 � 106 TCCs and DCs usingRNeasy kits (Quiagen, Hilden, Germany) and was reversetranscribed using the high-capacity cDNA Archive Kit in a ABIPrism 7000 Sequence Detector System (Applied Biosystems,Foster City, CA, USA). PCRs were performed in triplicateusing TaqMan chemistry with primer and probe sets from theAssay-on-Demand list (all from Applied Biosystems). Foldinduction was calculated by the ��Ct method (43) using the18S mRNA level to normalize values and the mRNA level ofbasal condition (nonactivated T cells or immature referenceDCs) as a calibrator.

Statistical analysis

The statistical significance of the difference between groupsof data with a normal distribution was determined by theANOVA with Bonferroni-Dunn posttests using the Statview4.1 program (Abacus Concepts, Inc., Berkeley, CA, USA).

RESULTS

Characterization of antigen-specific TCCs withdifferent functional polarization

We isolated a panel of PPD-specific TCCs from normaldonors. The TCCs were tested for their capacity tosecrete IFN-� and IL-4 on antigen-specific stimulation

Figure 1. Antigen-specific T-cell activation induces monocytedifferentiation. A) PPD-specific T clones were tested for theircapacity to secrete IFN-� and IL-4 by intracellular cytokinestaining and flow cytometry analysis. Similar cytokine patternwas obtained with several clones per each subset. B) Th1,Th0, and Th2 PPD-specific TCCs and autologous isolatedmonocytes were cocultured in the presence of PPD (10�g/ml). After 3 days, the supernatants were collected and theamounts of released IFN-�, IL-4, and GM-CSF were measuredby ELISA. Values indicate the mean of 4 different clones pereach subset and are expressed as ng/ml � sd. C) ReferenceDCs were generated in a 6-day culture of monocytes withGM-CSF and IL-4 (DCk). Autologous monocytes alone (Mo)or cocultured with irradiated PPD-specific Th2 or Th1 TCCwere incubated for 6 days in the absence (Mo�TCC) or thepresence of PPD (Mo�PPD�Th2 and Mo�PPD�Th1, re-spectively). Double staining analysis for CD1a and CD14 wasperformed on CD3-negative cells. Numbers indicate thepercentage of cells in each quadrant. D) Reference DCs weregenerated in a 6-day culture of monocytes with GM-CSF andIL-4 (DCk). Monocytes were placed in the lower chamber oftranswell plate, whereas in the upper insert PPD-pulsedmonocytes were cocultured with autologous PPD-specificTh2 (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 withParj1-pulsed autologous monocytes (Mo�PJ�TCC) in theupper insert of the transwell plate. After 6 days, CD1a/CD14surface expression of monocytes of the lower insert waschecked. Numbers indicate the percentage of cells in eachquadrant. Results are from 1 experiment representative of 10.Similar results were obtained using at least 4 different clonesper Th subset.

3372 Vol. 22 September 2008 MARIOTTI ET AL.The FASEB Journal

using autologous monocyte as APCs by intracellularcytokine staining and flow cytometric analysis (Fig. 1A).For a more complete functional characterization, themost relevant cytokines produced by the selected poolof Th1, Th2, and Th0 TCCs was measured as mRNAtranscript levels by real-time (RT)-PCR and as amountof proteins by ELISA. Figure 1B shows the cytokinerelease after TCC antigen-specific PPD stimulation us-ing monocytes as APCs, and Table 1 reports dataobtained stimulating TCCs with plastic bound anti-CD3, to avoid the possible detection of cytokines re-leased by APCs. Th1 clones released IFN-�, GM-CSF,tumor necrosis factor (TNF)-, and IL-2 and increasedtheir IL-6, IL-1, and IL-3 mRNA transcripts followingactivation. Conversely, the stimulation of Th2 cloneswas characterized by the release of IL-4 together withGM-CSF, IL-13, and IL-5. On activation Th0 clones, inaddition to the IL-4 and IFN-� release, increased GM-CSF, TNF-, IL-2, and IL-5 protein secretion and themRNA induction of IL-1, IL-3, with a remarkableexpression of IL-6. All the tested cytokines were in-duced specifically on stimulation because cytokines inresting TCCs were not detectable by ELISA and weredetectable at extremely low levels by RT-PCR (data notshown). Note that the amount of cytokines secreted byactivated TCCs, and in particular GM-CSF and IL-4, wasof the same order of magnitude of that required toobtain monocyte differentiation in vitro using the re-combinant cytokines.

Specific T-cell activation induces antigen-presentingmonocyte to differentiate into DCs

First, we set up for 6 days cocultures of irradiated Th1or Th2 TCCs and autologous monocytes in thepresence or absence of antigen (PPD) without theaddition of any known differentiation factor. Pheno-typic analysis of CD3-negative cells revealed that aftera 6 day culture monocytes alone or cocultured in theabsence of PPD and irrespective of the Th1 or Th2phenotype of the used TCCs showed a macrophage-like phenotype, as revealed by microscopy and bytheir forward and side scatters (not shown) andCD14 expression. On the other hand, in the pres-ence of PPD, TCCs induced the differentiation ofPPD-presenting monocytes into CD14�ve cells. Thesecells showed a diverse CD1a expression according to

the Th1 or Th2 phenotype of the cocultured clone.In particular, CD1a surface level in monocytes cocul-tured with Th2 TCCs was comparable to that ofreference DCs generated in the presence of GM-CSFand IL-4 (DCk), whereas monocytes cultured withactivated Th1 TCCs exhibited a significantly reducedpercentage of CD1a�ve cells (Fig. 1C). These changesin monocyte phenotype were clearly dependent onthe T-cell activation, because PPD added to theculture was unable to interfere with the fate ofmonocytes cultured with or without GM-CSF and IL-4(data not shown). These data indicate that, in addi-tion to T-cell activation, a consequence of antigenpresentation is the differentiation of presentingmonocytes.

T-cell-dependent monocyte differentiation into DCsdoes not require cell-to-cell contacts

To test whether soluble factors released by activatedTCCs and/or their intimate interaction with monocyteswere crucial to induce monocyte differentiation, wealso performed experiments using a transwell culturesystem, in which soluble cytokines released from cells inthe upper chamber could diffuse to the lower chamberand 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 mono-cytes were placed with or without PPD or Parj1, whilemonocytes from the same or from a different donorwere placed in the lower chamber. Analysis of cells inthe lower inserts after 6 days of culture showed thatmonocytes differentiated into cells with a level of CD1amolecule expression mirroring that of cells derivedfrom monocytes cocultured with Th1 and Th2 TCCs inthe presence of PPD. In the absence of antigens (notshown) or in the presence of a nonrelated antigen,such as Parj1, monocytes did not differentiate into DCsand revealed a macrophage-like phenotype (Fig. 1D).We also showed that more than 70% of cells derivedfrom monocyte-sensing factors released by activatedTh0 TCCs were CD1a�ve. Taken together, data indicatethat monocytes that have not processed and presenteda given antigen may undergo differentiation in a mi-croenvironment in which other APCs are responsiblefor T-cell activation.

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

TCC

Protein levela mRNA transcript levelb

TNF- (ng/ml) IL-5 (ng/ml) IL-13 (ng/ml) IL-10 (ng/ml) IL-2 (U/ml)c IL-6 (FI) IL-1 (FI) IL-15 (FI) IL-3 (FI)

Th1 19.1 4.5 1.1 0.5 361 593 48 1 1150Th0 12.7 19.8 n.d. 0 146 1893 47 1 790Th2 4.2 17.6 6.5 0 55 212 7 1 25

aAmount of secreted protein after anti-CD3 stimulation of 5 � 105/ml TCCs as measured by ELISA, unless noted otherwise. bCalculatedby ��Ct method using 18S mRNA level to normalize values and the mRNA level in nonactivated TCC as a calibrator. cMeasured using the CTLL2proliferation assay. FI, fold induction; n.d., not determined.

3373HUMAN T CELLS DRIVE DC DIFFERENTIATION OF MONOCYTE

Different TCC subpopulations induce monocytes todifferentiate into DCs with distinct phenotypes

Expression of group I CD1 molecules is considered adistinct but not unique characteristic of monocyte-derived DCs. Moreover, CD1 expression is not a DCmarker, because CD1�ve DCs have been described (44,45). To establish clearly the nature of cells derived frommonocytes cultured with activated TCCs, we performeda larger phenotypic characterization of monocytes cul-tured with activated Th2, Th1, or Th0 PPD-specificTCCs in comparison to DCk cells. Analysis revealed thatcells derived from monocytes cultured with activatedTCCs were indeed DCs. Monocytes cultured with Th2TCCs differentiated into DCs (DCh2) not distinguish-able from DCk (Fig. 2A–C). In fact, both these DCpopulations homogeneously expressed high levels ofpresenting molecules, that is, group I CD1 molecules(CD1a, CD1b, and CD1c; Fig. 2A) and MHC class I andclass II (DR) molecules (Fig. 2B) and were equally ableto undergo maturation following LPS treatment asassessed by the upregulation of activation markers(CD83, CD80, CD86, CD40, MHC class I and II) (Fig.2B). Monocytes cultured with activated Th1 and Th0TCCs differentiated into DCs (DCh1 and DCh0, respec-

tively) with phenotypic characteristics different fromDCk. In fact, they showed a reduced CD1 group Imolecule expression (Fig. 2A), and the level of matu-ration markers (CD83, CD86, DR) was higher than thatof reference immature DCs (Fig. 2B). It is interesting tonote that DCh1 and DCh0 cells did not markedlymodify their phenotype after LPS treatment (Fig. 2B),with the exception of CD86, which was upregulated inDCh0 cells. All the DC subsets were CXCR4�ve and didnot upregulate this receptor after LPS (data notshown). On the other hand, DCk and DCh2 cellsturned CCR7�ve after LPS stimulation, whereas DCh1and DCh0 cells were CCR7�ve even if the expressionwas lower than in the mature DCk cells and did notchange after LPS treatment. All the DC subset wereCCR5�ve, but although DCh2 and DCk cells turnedCCR5�ve, DCh0 and DCh1 cells reduced the expressionof this receptor only after LPS treatment (Fig. 2B).

In comparison to reference DCk cells, a reducedpercentage of DCh1 cells were DC-SIGN�ve, and themajority of DC-SIGN�ve were CD1a�ve (Fig. 2C). Thepresence of T-cell-released IL-2 did not affect thesurface expression of IL-2 receptor chain (CD25) inT-cell-induced DCs. All the mature DCs showed levelsof CD25 comparable to that of mature DCk cells (data

Figure 2. Monocytes differentiate into DCs with distinct phenotypes following activation ofdifferent 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 werecocultured with autologous antigen-pulsed monocytes in the upper chamber. DCs derived frommonocytes 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 ofpositive cells is reported in the relative histogram. B) Flow cytometric analysis of indicated surfacemolecules 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) ACD1a/DC-SIGN surface expression. Numbers indicate the percentage of cells in the correspon-

dent quadrant. Data are from 1 experiment representative of 8 independent experiments. D) Typical morphological appearanceof the DC cultures on day 6 without LPS treatment. Cells were allowed to differentiate on round-shaped coverslips seeded ontothe bottom of 24-well culture plastic plates and analyzed in phase contrast mode (�400).

3374 Vol. 22 September 2008 MARIOTTI ET AL.The FASEB Journal

not shown). Moreover all the DCs were CD11c�ve, asexpected according to their myeloid origin (data notshown).

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

The same differentiation of monocytes was observedusing antigen-activated Parj1-specific Th1, Th2, andTh0 TCCs (data not shown).

Freshly isolated T lymphocytes ex vivo inducemonocyte to differentiate into DCs

To test whether the capacity to induce monocyte dif-ferentiation into DCs was an in vitro acquired functionof cultured and IL-2 expanded TCCs or a generalfunction of activated T lymphocytes, we set up an ex vivotest analyzing the phenotype of monocyte after a 6 dayculture in the presence of superantigen-activatedfreshly isolated autologous CD4� lymphocytes. After 6days of coculture with SEA activated, but not withresting T lymphocytes, monocytes differentiated intoCD14�ve and CD1a�/�ve DCs (Fig. 3) with phenotypiccharacteristic similar to DCh1 cells (data not shown).

DCs differentiated following T-cell activation show adifferential cytokine secretion pattern

To measure the cytokines released by DCs, cultureswere washed after 5 days, cells counted and adjusted to3 � 105 cell/ml and cultured in the presence orabsence of 0.2 �g/ml LPS for an additional 18 h beforesupernatants collection. DCh1 and DCh0 cells pro-duced low amounts of IL-12p70 even after LPS stimu-lation, 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-12p70not statistically different from that released by matureDCk cells (Fig. 4). Notably, DCh1 cells were character-ized by their capacity to release spontaneously amountsof IL-10 significantly (P�0.05) higher than LPS-stimu-lated DCk cells, and the maturation stimulus did notsignificantly increase its release. DCh0 cells secretedIL-10 after LPS treatment at a level significantly(P�0.05) higher than mature DCk cells. RT-PCR anal-ysis also showed that IL-1 and IL-6 mRNA transcriptswere detected following activation in both DCh1 andDCh0 cells, which showed the highest expression ofTNF- transcripts among the different DC populations(data not shown).

DCs induced by activated T lymphocytes showdifferent degree of bacterial phagocytosis and solubleantigen uptake

The capacity of DCs derived from monocyte followingTCC activation to phagocytose Gfp-rBCG is reported inFig. 5. The percentage of fluorescent-bacteria-associ-ated cells was comparable between DCh2 and referenceDCk populations (DCh2�20% and DCk�23%). Asexpected, these percentages were reduced on LPSstimulation. On the contrary, a lower percentage ofDCh1 (10%) and DCh0 (13%) bound Gfp-rBCG after1 h of incubation, as compared to reference cells,consistent with the partial maturation state of these DCs(Fig. 5A). Interestingly, however, the low internaliza-tion capacity of these cells was further reduced afterLPS stimulation, which suggests a sensitivity to thismaturation stimulus that was not observed in terms ofsurface molecule expression (Fig. 5A, B). We obtainedsimilar results also evaluating the capacity of FITC-

Figure 3. Ex vivo T lymphocytes stimulation by a superantigeninduces monocytes to differentiate into DCs. Freshly isolatedCD4� T lymphocytes and autologous monocytes werecocultured in the presence (Mo�SEA�CD4) or absence(Mo�CD4) of SEA at 0.1 �g/ml. After a 6 day culture,CD1a/CD14 expression was analyzed in the CD3�ve popula-tion. Reference DCs were generated in a 6 day culture ofmonocytes with GM-CSF and IL-4 (DCk). Numbers indicatethe percentage of cells in the relative quadrant. In theabsence of SEA, monocytes acquired a macrophage-like phe-notype (CD14low CD1a�ve), whereas in the presence of SEA,monocytes lose CD14 expression and acquired CD1a mole-cules. SEA at the same concentration did not induce DCdifferentiation (Mo�SEA). Results are from 1 experimentrepresentative of 3 independent experiments performed withdifferent healthy donors.

Figure 4. Cytokine secretion pattern of DC populationsdifferentiated following T-cell activation. At day 5 of culture,the DCk, DCh2, DCh1, and DCh0 cells were incubated in thepresence or absence of 0.2 �g/ml LPS for an additional 18 h.Supernatants were collected at the end of the culture andexamined for IL-12p70 and IL-10 contents by ELISA. Valuesindicate the mean of 3 independent experiments and areexpressed 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.

3375HUMAN T CELLS DRIVE DC DIFFERENTIATION OF MONOCYTE

albumin endocytosis (Fig. 5C, D) as a measure ofsoluble antigen uptake.

DCh1 and DCh0 cells have a reduced capacity toprime naive CD4� T lymphocytes

The antigen-presenting capability of the diversemonocyte-derived DC populations was evaluated in amixed lymphocyte reaction (MLR) using a sortedpopulation of allogeneic cord blood CD4� T lympho-

cytes as responder cells. DCh1, DCh2, and DCh0 cellsgenerated from monocytes in the lower chamber of atranswell device were used to obtain DCs noncon-taminated by irradiated T cells. All the non-LPS-treated DCs were unable to stimulate an efficientproliferation of naive T cells, which, on the otherhand, proliferated extensively when stimulated byLPS-matured DCh2 and reference DCk cells (Fig.6A). Interestingly, LPS-matured DCh1 and DCh0cells induced a T-cell proliferation that was con-

Figure 5. Phagocytosis and endocytosis of DCpopulations differentiated following T-cell acti-vation. At day 5 of culture, the DCk, DCh2,DCh1, and DCh0 cells were incubated in thepresence or absence of 0.2 �g/ml LPS for anadditional 18 h. A) At the end of cell culture,DCs were incubated 1 h at 37°C with Gfp-rBCGat a multiplicity of infection cell:BCG � 1:6 inCM supplemented with 10% FCS, then washedby low-speed centrifugation (100 g). Percentageof cells that bound Gfp-rBCG was evaluated byflow cytometry; number is indicated in thequadrant. Results are from 1 experiment repre-sentative of 4 independent experiments. B)Reduction of phagocytic activity after LPS-in-duced maturation. Results are expressed asreduction � sd in percentage of Gfp-rBCGphagocytosis on LPS stimulation of 4 indepen-dent experiments. C) Endocytic capacity of DCsubsets. Analyses by flow cytometry of FITC-albumin uptake after 1 h at 0°C (negativecontrol; 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 fluorescentintensity on LPS-stimulation of 4 independent experiments.

Figure 6. DC populations differentiated follow-ing T-cell activation have different capacity toprime naive T cells and to stimulate memory Tcells. A) DCk (squares), DCh2 (circles), DCh1(triangles), and DCh0 (crosses) cells at day 5 ofculture were incubated in the absence (opensymbols) or presence of 0.2 �g/ml LPS (filledsymbols) for an additional 18 h and used asAPCs at different cell numbers to stimulate 3 �104 cord blood-purified CD4� T cells. TheT-cell proliferative response was measured after6 days by 3H-thymidine incorporation; resultsare expressed as mean counts per minute(cpm) of triplicate wells. One experiment rep-resentative of 6 is shown. B) Cytokine produc-tion by naive CD4� T cells after coculture withallogeneic LPS-treated DCk, DCh2, DCh1, andDCh0 cells. Dot plots represent the flow cyto-metric analysis of intracellular cytokine accu-mulation; numbers indicate the percentage ofcells in the corresponding quadrant. Histo-grams represent cytokines released in superna-tants as measured by ELISA (ng/ml�sd) withthe exception of IL-2, which was measured by abiological assay using the cell line CTLL-2 andexpressed as U/ml � sd. C, D) To test thecapacity of the different DC population tostimulate memory T cells, DCk, DCh2, DCh1, or DCh0 cells were used to stimulate PPD-specific MHC class II-restricted (C) orPIM2-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.

3376 Vol. 22 September 2008 MARIOTTI ET AL.The FASEB Journal

stantly of lower magnitude than the proliferationinduced by mature DCk cells (Fig. 6A).

Analysis of intracellular cytokine production by naiveCD4� T cells expanded in an MLR showed that matureDCh2 cells induced the expansion of a number ofIFN-� and IL-2 producing T cells comparable to DCkcells (Fig. 6B). Note that DCh2 cells primed a reducednumber of cells secreting IL-4, which indicates that theyare capable of inducing a more marked Th1 responsethan reference DCk cells. Inversely, among the T cellspolarized by mature DCh1 and DCh0 cells, a statisticallysignificant reduced percentage of IFN-�- and IL-2-secreting cells and a higher proportion of cells produc-ing IL-10 in T expanded by DCh0 cells was observed.ELISA measurement also confirmed that IL-10 wasreleased by T cells stimulated by DCh0 cells in amountsstatistically higher (P�0.01) than those released by Tcells primed by other APCs (Fig. 6B).

DCh1 and DCh0 cells have a reduced capacity toactivate antigen-specific TCCs

The ability of the different DCs to present PPD andPIM2 to specific MHC class II and CD1-restricted TCCs,respectively, was analyzed. As shown in Fig. 6C, theefficiency of presentation, measured as the PPD con-centration required to give 50% of maximum TCCproliferative response, varies with APCs. The antigenpresentation capacity of DCh2 cells was comparable tothat of DCk cells, whereas DCh1 and DCh0 cells showeda reduced efficiency. Interestingly, DCh2 cells wereeven more efficient than DCk cells in presenting alipidic antigen to a specific CD1b-restricted TCCs (Fig.6D). On the other hand, DCh1 and DCh0 cells showeda reduced capacity to activate the specific TCCs, interms of both maximum response and amount ofantigen required, that can be only partially attributedto the reduced expression of surface CD1b molecules.

DISCUSSION

In the present study we demonstrate that human CD4�

T lymphocytes can induce monocytes to differentiateinto DCs following antigen-specific activation. DC dif-ferentiation is induced in both antigen-presenting andin bystander monocytes, which sense the cytokinesreleased by activated T cells. The in vitro model de-scribed here is of particular relevance because it doesnot include the addition of any known DC differentia-tion factor, but reproduces the functional conse-quences of specific T-cell activation, which followsantigen presentation, on monocyte differentiation.

Monocytes, cocultured with autologous T cells in theabsence of antigens, differentiated into CD14lowCD1�ve

cells similar to macrophages derived from monocytescultured with or without M-CSF, indicating that thedefault differentiation pathway in vitro of monocytesleads to macrophages and that nonactivated T cells donot interfere with this process. Conversely, antigen-

pulsed monocytes cocultured with specific TCCs differ-entiated into DCs, and because the cell-to-cell contactof monocytes with activated T cells was not required forDC generation, we conclude that T-cell-released solu-ble factors are involved in monocyte differentiation. Itis interesting to note that the phenotype of differenti-ated DCs varied according to the functional polariza-tion of T cells. DCh2 cells showed a phenotype indis-tinguishable from reference DCk cells, whereas DCh1and DCh0 cells showed a reduced expression of groupI CD1 molecules and a more mature phenotype, whichwas not significantly modified by LPS stimulation.These data are in line with the fact that Th1-secretedcytokines such as GM-CSF, IL-3, TNF-, or IFN-� havebeen previously described to induce generation of DCs(46, 47) with characteristics different from those differ-entiated with Th2 cytokines, namely, GM-CSF, IL-4, orIL-5 (41, 48). Our findings expand preliminary dataindicating 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 witha recent paper proving the inability of MHC class IIrestricted T cells, unlike NK T cells, to promote mono-cyte differentiation into DCs (35). We obtained mono-cyte-derived DCs using 2 diverse pools of TCCs withdifferent antigen specificity and notably also in ex vivoexperiments using freshly isolated CD4� T cells stimu-lated with a superantigen. Thus, a diverse in vitro settingcould be responsible for the different result obtained inthe cited paper (35). A previous paper suggested theability of activated T cells to differentiate monocytesinto DCs through the CD40 ligation (36), but the use ofadherent instead of purified monocytes and the lack ofa formal demonstration of CD40 ligand involvementmade the results of that paper nonconclusive. However,even if our findings prove that soluble factors areresponsible for monocyte differentiation, we cannotexclude that a monocyte-T-cell interaction throughCD40 ligation may positively concur to the DC genera-tion.

DCs derived from monocytes sensing a Th1 or Th0inflammatory microenvironment had a reduced capac-ity to allow the expansion of naive T cells and, inagreement with their reduced IL-12 and increasedIL-10 synthesis, showed a hampered ability to inducetheir functional polarization into Th1 cells. It is inter-esting to note that DCh1 and particularly DCh0 cellswere capable to prime IL-10-secreting T cells with apossible regulatory role. Expansion of IL-10-secreting Tcells was shown to be dependent on the presence ofIL-10 itself (49). However, since both DCh1 and DCh0cells secrete IL-10, but the expansion of IL-10-secretingT cells is mediated mainly by DCh0 cells, other charac-teristics of DCh0 cells, including their high TNF-synthesis (47, 50) and CD86 expression (51), must beinvolved and are now under investigation.

DCh1 and DCh0 cells were shown to be less efficientthan DCk and DCh2 cells in stimulating antigen-spe-cific TCCs, which reproduce in vitro experienced/

3377HUMAN T CELLS DRIVE DC DIFFERENTIATION OF MONOCYTE

memory T cells (52). Because CD1 molecule expressionin DCh1 and DCh0 cells was reduced, it was notsurprising to observe a decreased capacity of theseAPCs to present a lipid antigen to CD1-restricted TCCs(53). Unexpectedly, DCh1 and DCh0 cells had a re-duced ability to present antigen to MHC class II re-stricted TCCs, even if their DR expression was higherthan reference DCk cells, which may be attributed totheir decreased antigen uptake and abundant secretionof IL-10 (54, 55), together with other not yet knowncharacteristics.

Although it is not always possible to translate in vivowhat observed in vitro, the phenomenon that we de-scribe herein is likely to occur in chronic inflammationsites where monocytes and lymphocytes are corecruited(56), and our data suggest that the generation ofmonocyte-derived DCs described in infected rodents(22) has the possibility to occur also in humans. To-gether with T-cell-derived cytokines, it is likely thatother microenvironmental factors related to thechronic inflammation and to its causes could contrib-ute to the differentiation of monocytes into “inflamma-tory” DCs with diverse phenotype and functional ability(26). However, the demonstration that DCh2, DCh1,and DCh0 cells have different capacity to induce thefunctional polarization of naive T cells and to stimulatememory T lymphocytes is suggestive for a feedbackcontrol of Th1/Th2 immune responses orchestrated bymonocyte-derived DCs. Responses characterized by astrong Th2 polarization could be counterbalanced bythe differentiation of DCh2 cells that, in turn, wouldprime new generation of antigen/allergen-specific Th1cells. On the other hand, the differentiation of DCh1and DCh0 cells could be instrumental in limiting theTh1-associated tissue damage in chronic inflammationsites.

We thank Prof. Antonio Cassone and Prof. VincenzoBarnaba for suggestions and critical reading of the manu-script. This paper was partially supported by the EC projectMILD-TB, contract no. 037326. The authors declare nofinancial conflict of interest.

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

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