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Immature Dendritic Cells (DCs) Use Chemokines and Intercellular Adhesion Molecule (ICAM)-1, But Not DC-Specific ICAM-3-Grabbing Nonintegrin, to Stimulate CD4⁺ T Cells in the Absence of Exogenous Antigen¹

Eliana Real^*, Andrew Kaiser, Graça Raposo, Ali Amara, Alessandra Nardin, Alain Trautmann^* and Emmanuel Donnadieu^2,*

^* Département de Biologie Cellulaire, Institut Cochin, Institut National de la Santé et de la Recherche Médicale, Unité 567, Centre National de la Recherche Scientifique (CNRS), Unité Mixte de Recherche (UMR)8104, IDM (Immuno-Designed Molecules), Institut Curie, CNRS UMR144, and Unité d’Immunologie Virale, Institut Pasteur, Paris, France

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Dendritic cells (DCs) possess a number of unique features that distinguish them from other APCs. One such feature is their ability to trigger Ag-independent responses in T cells. Previous studies have focused on mature DCs, but the prevalence of this phenomenon in the resting-state immature DCs has never been considered. In this study, we show that, in the absence of Ag, human immature DCs trigger multiple responses in autologous primary CD4⁺ T cells, namely, increased motility, small Ca²⁺ transients, and up-regulation of CD69. These responses are particularly marked in CD4⁺ memory T cells. By using several experimental approaches, we found that DC-specific ICAM-3-grabbing nonintegrin plays no role in the induction of T cell responses, whereas ICAM-1/LFA-1 interactions are required. In addition, DC-produced chemokines contribute to the Ag-independent T cell stimulatory ability of DCs, because pertussis toxin-treated T cells exhibit diminished responses to immature DCs. More particularly, CCL17 and CCL22, which are constitutively produced by immature DCs, mediate both T cell polarization and attraction. Thus, immature DCs owe part of their outstanding Ag-independent T cell stimulatory ability to chemokines and ICAM-1, but not DC-specific ICAM-3-grabbing nonintegrin.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Dendritic cells (DCs)³ have been implicated in the induction of both immunity and tolerance, depending on their maturation state (1). In the periphery, DCs continuously sample Ags found in their vicinity, until danger signals associated with inflammatory and infectious conditions trigger their migration to the closest draining lymph node (LN). By the time they reach secondary lymphoid organs, migrating DCs have matured and lost their ability to capture new Ags, but have become highly efficient at presenting the Ags previously captured and at stimulating T cells. In this setting, DC-induced T cell priming results in a robust and specific immune response.

In the absence of an inflammatory stimulus, Ag presentation has a different purpose, because the functional outcome of Ag recognition is tolerance instead of immunity. DCs in the steady state constitutively process and transport self-Ags from nonlymphoid tissues to draining LN (2). Indeed, secondary LN in resting mice were shown to contain a large proportion of immature DCs (3) that present self-Ags in a tolerogenic fashion (4). Thus, self-reactive T cells that have escaped central tolerance might still be eliminated upon encounter with immature DCs loaded with self-peptides. Several mechanisms have been reported that may contribute to peripheral tolerance, namely, T cell deletion, functional anergy, and induction of regulatory T cells that silence potentially harmful effector T cells (for review, see Ref. 5). In addition, immature DCs have been suggested to maintain a population of CD4⁺CD25⁺ regulatory T cells (Treg) that originate in the thymus and play an important role in the prevention of autoimmune disorders (6).

Thus, in LN, both immature and mature DCs are expected to exert important functions. Immature DCs presumably present a large panel of self-Ag. This is equivalent to an in vitro situation with no exogenous Ag added. What will be termed hereafter "Ag-independent T-DC interactions," consists therefore in interactions occurring in the presence of self or environmental Ags. In a seminal work, Nussenzweig and Steinman (7) have shown that, under these conditions, mature murine DCs were able not only to interact with syngeneic T cells but also to induce a modest T cell proliferation that they called autologous MLR. We have previously analyzed at the single-cell level the consequences of the interaction between mature murine DCs and syngeneic T cells in the absence of exogenous Ag. Multiple T cell responses were described, including the relocalization of various cell surface and signaling molecules at the T-DC interface as well as the induction of a small Ca²⁺ signal. These phenomena led to enhanced T cell survival (8, 9). In addition, Ag-independent responses induced by mature DCs in human T cell clones have also been reported (10, 11). The question of whether these Ag-independent signaling events are specific to fully mature immunogenic DCs or may also be triggered by immature DCs had not yet been addressed.

Compared with other APCs, DCs have an exceptional ability to trigger Ag-independent T cell responses. However, the identity of the DC molecules that initiate the T-DC interaction remains elusive. DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN) is a DC-specific C-type lectin that binds and internalizes several pathogens such as HIV-1 (12). In addition, it has been proposed to initiate the contact between human naive T cells and DCs. Indeed, DC-SIGN binds efficiently ICAM-3, which is highly expressed on resting T cells, and blocking the ICAM-3/DC-SIGN interaction with mAbs results in a marked decrease in allogeneic T cell proliferation (13). Hence, DC-SIGN was considered a good candidate for the Ag-independent clustering of resting T cells with DCs.

In addition, soluble molecules secreted by DCs such as cytokines and chemokines are expected to modulate the T-DC interaction. Even if the role of chemokines in promoting the adhesion between leukocytes and endothelial cells has been proven, relatively little is known concerning their participation in the formation of a T-DC synapse (however, see Refs. 14 and 15).

In this study, we have identified multiple cellular responses induced by autologous immature DCs in CD4⁺ primary human T cells in the absence of exogenous Ag, and tested the involvement of DC-SIGN. Surprisingly, it appears that DC-SIGN plays no role in the induction of these responses. On the contrary, they require that the interaction between ICAM-1 and its ligand is intact, and they show a marked sensitivity to DC-produced chemokines, in particular to CCL17 and CCL22.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
T lymphocytes and in vitro generation of DCs

Human CD4⁺ T cells were purified from PBMCs by negative selection using a CD4⁺ T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Naive and memory CD4⁺ T cells were further purified with CD45RO and CD45RA Microbeads, respectively (Miltenyi Biotec). DC differentiation was performed with VacCell processor (IDM, Paris, France) as previously described (16). Briefly, PBMCs were cultured for 7 days in serum-free VacCell medium (Invitrogen Life Technologies, Carlsbad, CA), supplemented with 500 U/ml GM-CSF (Novartis Pharmaceuticals, East Hanover, NJ) and 50 ng/ml IL-13 (Sanofi-Synthélabo, Paris, France). DCs were then isolated by elutriation. Purity ranged from 80 to 99%; viability was >95%. The following Abs were used to phenotype DCs: anti-HLA-A, -B, -C (G46-2.6; BD Biosciences, Mountain View, CA), anti-DR, -DP, -DQ (TÜ39; BD Biosciences), anti-B7.1 (L307.4; BD Biosciences), anti-B7.2 (IT2.2; BD Biosciences), anti-ICAM-1 (HA58; BD Biosciences), and anti-DC-SIGN (1B10; Ref. 17). For experiments with supernatants, immature DCs cultured in 24-well plates were washed and incubated in serum-free VacCell medium without any cytokines for 24 h. Culture supernatants were then collected and frozen at –20°C.

Generation of DC-SIGN transfectants

A DC-SIGN cDNA was generated by PCR using cDNA prepared from immature DCs (see below) as template, a forward primer (5'-AGAGTGGGGTGACATGAGTG), and a reverse primer (5'-GAAGTTCTGCTACGCAGGAG). The PCR product was subcloned into pCR2.1 (Invitrogen Life Technologies) and subsequently ligated into the HindIII, XhoI restriction sites of pREP7 (Invitrogen Life Technologies). This eukaryotic expression plasmid is maintained episomally and confers resistance to hygromycin in transfected cells.

B cells from the EBV-positive Burkitt’s lymphoma Raji (1 x 10⁷ in RPMI 1640 with 10% FCS) were electroporated in a Gene Pulse cuvette (Bio-Rad, Hercules, CA) with 10 µg of plasmid at 950 µF and 240 V. After 48 h, cells were cultured in normal medium supplemented with 0.8 mg/ml hygromycin. Cells were analyzed for expression of DC-SIGN 2 wk after transfection by flow cytometry.

Single-cell video imaging

Measurement of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) was performed as previously described (18). In brief, 1 x 10⁵ DCs were left to adhere to polylysine-pretreated glass coverslips (2 µg/ml) for 20 min and washed with mammalian saline buffer (18) supplemented with 2% of autologous human serum. T cells (5 x 10⁵) were incubated for 20 min at 37°C with 1 µM fura 2-AM (Molecular Probes, Eugene, OR) or CFSE (Molecular Probes), washed, and added to the DC layer. When indicated, T cells were pretreated overnight with 50 ng/ml pertussis toxin (PTX) or the B subunit of the toxin (Calbiochem, San Diego, CA). T cells were sometimes incubated for 20 min before the beginning of the experiment with 1 µg/ml TAK-779 (kindly provided by Dr. F. Arenzana-Seisdedos, Institut Pasteur, Paris, France). In some experiments, DCs were incubated with 10 µg/ml blocking Abs at 37°C for 20 min before the experiment. The blocking Abs anti-CCL17 (54026.11; R&D Systems, Minneapolis, MN), anti-CCL22 (57226.11; R&D Systems), anti-DC-SIGN (AZN-D1; provided by Drs. Y. van Kooyk and C. Figdor, University Medical Center, Nijmegen, The Netherlands), and anti-ICAM-1 (HA58; BD Biosciences) were used. For reconstitution experiments, glass coverslips were coated overnight at 4°C with 3 µg/ml human ICAM-1 Fc (provided by N. Hogg, Cancer Research, London, U.K.), washed with PBS, and blocked with PBS containing 1% BSA for 30 min at 37°C. T cells were loaded with either fura 2-AM or CFSE, placed on the ICAM-1 layer, and stimulated or not with DC culture supernatant. For experiments with Raji cells reconstituted or not with DC-SIGN, B cells were left to adhere to polylysine-pretreated glass coverslips (2 µg/ml) for 20 min. During that time, B cells were incubated with 0.5 µg/ml bacterial staphylococcal enterotoxin E (SEE) (Toxin Technology, Sarasota, FL). Raji cells were then washed with mammalian saline buffer and used in imaging experiments.

CD69 induction assay

DCs (2 x 10⁵) and T cells (1 x 10⁵) were washed in RPMI 1640 with 10% human autologous serum and incubated together in 96-round-well plates for 12–24 h. Cells were then dual stained with FITC-labeled anti-CD3 and PE-labeled anti-CD69 Abs (BD Biosciences) to determine CD69 expression on CD3-positive cells. In some experiments, T cells were first incubated overnight with either PTX or the B subunit of the toxin at 10 ng/ml, and then washed once before adding them to DCs. When stated, blocking anti-DC-SIGN and anti-ICAM-1 Abs were used at 10 µg/ml and present during the interaction time.

Electron microscopy

For cell surface immunogold labeling, DCs and T cells were fixed with 4% paraformaldehyde in 0.2 M phosphate buffer (pH 7.4), and then stained with anti-DC-SIGN (AZN-D1) and anti-ICAM-3 (TU41; BD Biosciences) Abs, respectively, for 1 h at room temperature. After several washes with PBS containing 0.2% FCS, Abs were visualized with protein A coupled to 10-nm gold particles (PAG 10; Department of Cell Biology, University of Utrecht, Utrecht, The Netherlands). Samples were then processed as described (8). Electron micrographs were scanned using an Arcus scanner (Agfa, Ridgefield Park, NJ). The length of the cell body and villi was measured using Metamorph software (Universal Imaging, Downington, PA), and the number of gold particles in these two regions was manually counted. Microvilli were clearly distinguished from the cell body by visual inspection.

Semiquantitative RT-PCR

Total RNA from immature DCs was extracted using TRIzol reagent (Invitrogen Life Technologies). cDNA was prepared using Moloney murine leukemia virus reverse transcriptase and oligo(dT) primers from Advantage RT-for-PCR kit (BD Biosciences). PCR amplification of cDNA samples was conducted using the primers whose sequences and expected size of amplified products are shown in the supplemental table.⁴ All reactions were performed in 50-µl final volume with 10 µl of reverse transcriptase reaction (equivalent to 0.1 µg of RNA), 1 U of Taq polymerase (Finnzymes, Espoo, Finland), 200 µM dNTPs, 50 pmol of forward and reverse primers, and 1.5 mM MgCl₂. As a positive control, human G3PDH cDNA (983 bp of the expected size) was amplified by PCR using specific primers (BD Biosciences). PCR cycling conditions were as follows: denaturation at 94°C for 45 s, annealing at 60°C for 45 s, and extension at 72°C for 60 s (25 and 30 cycles). PCR products were resolved on a 1% agarose gel containing ethidium bromide.

ELISA

The chemokines CCL2, CCL17, and CCL22 contained in supernatants derived from 24-h DC culture were quantified by ELISA using Abs from R&D Systems.

Polarization, chemotaxis, and motility assays

T cell polarization was quantified by visual inspection of cell morphology following exposure to 100 ng/ml recombinant chemokines (PeproTech, London, U.K.) or DC culture supernatant. For chemotaxis assays, 5 x 10⁵ T cells were placed on the upper chamber of a Transwell plate, 6.5 mm in diameter, with 5-µm polycarbonate filters (Corning, Corning, NY). The lower chamber contained either DC supernatant diluted in RPMI 1640 supplemented with 0.5% BSA or control medium. After 3 h of assay at 37°C, cells were collected in the lower compartment, and either counted by FACS using Flow-check beads (Beckman Coulter, Fullerton, CA) or labeled with the following Abs: anti-CD45RA (HI100; BD Biosciences), anti-CD45RO (UCHL1; BD Biosciences), anti-CCR7 (3D12; BD Biosciences), and anti-CD62L (DREG56; Beckman Coulter). In some experiments, T cells were incubated overnight with 50 ng/ml PTX. Anti-CCL17 and anti-CCL22 (R&D Systems) were used at a concentration of 10 µg/ml. For motility assays, the percentage of cells showing a displacement of >20 µm from their starting positions during a 15-min recording was measured in several experiments, with always >30 cells scored per experiment.

Adhesion assays

Ag-independent adhesion between immature DCs and autologous CD4⁺ T cells was quantified as follows. A total of 4 x 10⁵ DCs was plated for 15 min on microscopic glass slides pretreated with 2 µg/ml polylysine. During that time, DCs were incubated or not with 10 µg/ml blocking Abs. Cells were then washed, and the glass slide was assembled in a parallel plate laminar flow chamber (Immunetics, Cambridge, MA). The flow chamber was mounted on the stage of an Eclipse TE300 inverted microscope equipped with a x20 objective. A total of 5 x 10⁵ T cells was loaded for 5 min with 2.5 µM CFSE, washed, and infused in the chamber. After 7 min of T-DC adhesion under static conditions at 37°C, increasing flow rates (from 0.5 to 4 dyne/cm²) were applied, and the percentage of T cells remaining adherent to DCs was scored from processed fluorescent images acquired during the experiment.

Statistics

Data are expressed as mean ± SD, and the significance of differences between two series of results was assessed using the Student’s unpaired t test. Values of p < 0.05 were considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Immature DCs induce multiple signals in human CD4⁺ resting T cells in the absence of exogenous Ag

The early responses of primary nonstimulated CD4⁺ human T cells induced by autologous immature DCs derived from monocytes were analyzed by video imaging. The DCs used in this study express MHC class I and class II molecules as well as ICAM-1 and the DC-specific marker DC-SIGN (Fig. 1). These cells possess an immature phenotype, as judged by the low expression of the costimulation molecules B7.1 and B7.2. Fig. 2A and supplemental movie 1 show a sequence of images in which two CFSE-loaded CD4⁺ T cells can be seen interacting with immature DCs in the absence of exogenous Ag. The first detectable event was the acquisition by the T cells of an active motile phenotype enabling them to scan multiple DCs. T cell paths were not continuous but rather chaotic with sudden and frequent turns. Marked shape changes, characterized by the formation of a leading edge and a trailing uropod, were observed during periods of accelerated motion. This polarization contrasted with the rounding-up of the T cells that often preceded the direction shifts. The velocity of the T cells shown in Fig. 2A were not constant, but presented quiet periods alternating with sudden accelerations (B). On average, the mean T cell velocity was 6.8 ± 3.9 µm/min (n = 18 cells), with peaks exceeding 30 µm/min. Trajectory analysis of several T cells indicated that their migration was nondirectional, with abrupt changes in their paths and frequent U-turns (Fig. 2C). When other APCs were used, like the human B cell line Raji or autologous monocytes, the percentage of motile T cells was lower than with DCs (11 ± 3.6%, n = 3 experiments with Raji, in which >30 cells were analyzed per experiment, 1.7 ± 1.4%, n = 2 with monocytes, and 18.1 ± 4.6%, n = 4 with immature DCs).

View larger version (20K): [in this window] [in a new window]   FIGURE 1. Surface phenotype of immature DCs. DCs were labeled with Abs against HLA-A, -B, -C; HLA-DR, -DP, -DQ; DC-SIGN; ICAM-1; B7.1; and B7.2; and expression was measured by flow cytometry. Solid line, Specifically labeled DCs. Thin line, Staining with an isotype control Ab.

View larger version (57K): [in this window] [in a new window]   FIGURE 2. Immature DCs induce multiple responses in CD4+ T cells in the absence of exogenous Ag. A, Two CFSE-loaded CD4+ T cells (green) scanning the surface of several DCs during 30 min. Red and blue dots mark the position of the T cells at successive 30-s intervals. A time-lapse animation of these cells is shown in supplemental movie 1. B, Velocity of the T cells represented in A. C, Paths of nine individual T cells during their interaction with DCs. Tracks have been overlaid and normalized to their starting coordinates. D, Two single-cell Ca2+ responses in red overlaid with velocity in black. The arrows indicate the time points of initial interactions with successive DCs. Supplemental movies 2 and 3 represent time-lapse animations of these cells. E, CD69 expression measured by flow cytometry on CD3-positive CD4+ T cells after 24 h of culture with immature DCs. One representative result of at least three experiments is shown.

Thus, in the absence of exogenous Ag, immature DCs transmit multiple signals to CD4⁺ T cells, which respond with a migratory phenotype followed by small and transient Ca²⁺ responses and the up-regulation of CD69.

ICAM-1 but not DC-SIGN is involved in the Ag-independent T cell stimulatory ability of DCs

Next, we investigated the molecular basis of DC-induced T cell responses in the absence of exogenous Ag. Several mechanisms could explain the observed DC efficiency, including the expression of a DC-restricted molecule such as DC-SIGN (13). To examine the importance of this molecule, we first studied by immunogold electron microscopy the ultrastructural localization of DC-SIGN and of its ligand ICAM-3 at the surface of DCs and CD4⁺ T cells. DC-SIGN was primarily located on DC processes, and fewer gold particles were detected on the cell body (Fig. 3A). Similarly, ICAM-3 was expressed preferentially on the cellular extensions of the T cell shown in Fig. 3B. A quantitative analysis confirmed the concentration of DC-SIGN and ICAM-3 in microvilli of DCs and T cells (Fig. 3C). Thus, DC-SIGN appeared as a good candidate to initiate T-DC conjugate formation, which would begin with villi-villi interactions as previously suggested (8).

View larger version (67K): [in this window] [in a new window]   FIGURE 3. DC-SIGN and ICAM-3 are enriched in the cellular extensions of DCs and T cells. A, DCs were fixed and labeled with monoclonal anti-DC-SIGN Ab (AZN-D1). The Ab was detected with protein A coupled to 10-nm gold particles. B, CD4+ T resting T cells were stained with monoclonal anti-ICAM-3 Ab (TÜ41) after fixation. As above, the Ab was detected with protein A coupled to 10-nm gold particles. Bar, 1 µm. C, Quantitation of gold-labeled DC-SIGN and ICAM-1 in villi and cell body of DCs (left panel) and T cells (right panel). A total of 526 beads from six DCs and 210 beads from three T cells were scored. *, p < 0.05.

View larger version (25K): [in this window] [in a new window]   FIGURE 4. DC-SIGN is not involved in the T cell stimulatory function of DCs. A, Expression of DC-SIGN measured by flow cytometry in stably transfected Raji B cells. Thick line, DC-SIGN-labeled Raji transfectants. Thin line, Isotype control-labeled Raji transfectants. B, Percentage of Ca2+-responding CD4+ T cells induced by Raji B cells transfected with a control vector or with a DC-SIGN construct. Raji were loaded or not with 0.5 µg/ml superantigen, SEE. C, Percentage of CD69-positive T cells induced by Raji expressing or not DC-SIGN and loaded with increasing concentrations of SEE. CD69 expression was measured after 24 h of culture. D and E, Percentage of motile and Ca2+-responding CD4+ T cells induced by DCs pretreated with 10 µg/ml monoclonal anti-DC-SIGN Ab (AZN-D1) or an isotype control. F, Percentage of CD69-positive CD4+ T cells after 12–24 h of contact with DCs. A concentration of 10 µg/ml monoclonal anti-DC-SIGN Ab was present during the interaction time. Data are the mean of three experiments ± SD. Results in B, D, and E represent the means and SD of three experiments in which >30 cells were analyzed per experiment.

We next investigated the involvement of ICAM-1, which binds the ₂ integrin LFA-1 on T cells, in the T-DC interaction. The importance of ICAM-1 was first examined in adhesion experiments with a laminar flow chamber. T cells were left to adhere for 7 min in static conditions to DCs that were pretreated or not with a blocking anti-ICAM-1 Ab. T cells were then counted before and after increased fluxes (Fig. 5A, supplemental movies 5 and 6). In control conditions, 60% of T cells remained attached to DCs under shear stresses as high as 4 dyne/cm². By contrast, nearly all the T cells in contact with anti-ICAM-1-pretreated DCs were washed away by increasing shear stresses. In addition, blocking anti-ICAM-1 Ab markedly inhibited the T cell motility and Ca²⁺ response induced by DCs in the absence of exogenous Ag (Fig. 5, C and D). Anti-ICAM-1 Ab also reduced the percentage of CD69-positive T cells induced by DCs (Fig. 5E). These data highlight a major role of ICAM-1 in Ag-independent DC function. However, ICAM-1 cannot be the key molecule explaining the exceptional stimulatory efficiency of DCs, because Raji B cells that are poor T cell stimulators in the absence of Ag abundantly express ICAM-1 (data not shown). Therefore, ICAM-1 and additional DC-specific components, other than DC-SIGN, are involved in DC-induced T cell responses in the absence of exogenous Ag.

View larger version (71K): [in this window] [in a new window]   FIGURE 5. Blocking ICAM-1/LFA-1 interaction strongly inhibits DC-induced CD4+ T cell responses in the absence of Ag. A, CFSE-labeled CD4+ T cells (black) were allowed to adhere under static condition for 7 min to DCs pretreated with 10 µg/ml monoclonal anti-ICAM-1 Ab (HA58) or an isotype control. Frames were then captured before and after standardized flow. Time-lapse animations of control and anti-ICAM-1 Ab conditions are presented in supplemental movies 5 and 6. B, The percentage of T cells remaining adherent to DCs treated with an anti-ICAM-1 Ab or an isotype control was scored after increasing flow. Data are representative of three independent experiments. C–E, CD4+ T cell responses analyzed as in Fig. 4 except that a monoclonal anti-ICAM-1 Ab (10 µg/ml) was used. Data are the mean of three experiments ± SD in which >30 cells were analyzed per experiment. *, p < 0.05; ***, p < 0.001.

The above experiments were performed with primary bulk CD4⁺ T cells. We then used purified naive (CD45RA⁺) and memory (CD45RO⁺) CD4⁺ T cells and compared their ability to respond to immature DCs. For the three readouts studied, memory CD4⁺ T cells responded on average two to three times more frequently than naive CD4⁺ T cells (Table I). Because immune interactions depend on the ability of T cells and APCs to colocalize in vivo, namely, through the action of soluble chemoattractants, we used a Transwell assay to characterize the CD4⁺ T cells attracted by supernatant from immature DCs. For this experiment, primary unfractionated CD4⁺ input T cells were allowed to migrate during 3 h at 37°C, after which the expression of CD45RA and CD45RO was measured by flow cytometry within the input as well as the migrated cell populations. As shown in Fig. 6, DC supernatant produced a prominent enrichment of memory T cells (CD45RO⁺) and a reduction of naive (CD45RA⁺) T cells in the migrated population. Overall, these results indicate that immature DCs preferentially attract and induce a set of responses in CD4⁺ T cell with a memory phenotype.

View larger version (22K): [in this window] [in a new window]   FIGURE 6. Phenotype of CD4+ T cells attracted by DC culture supernatant. Input CD4+ T cells and cells that migrated to DC supernatant (diluted by half) over 3 h were incubated with anti-CD45RA, CD45RO, CCR7, and CD62L Abs, and analyzed by flow cytometry. CCR7 and CD62L expression were measured on CD45RO+ gated cells. Histograms are representative of three independent experiments.

CCL17 and CCL22 are important players in DC-induced attraction and migration of memory CD4⁺ T cells

At least some of the Ag-independent T cell responses described above could have been caused or influenced by DC-produced chemokines, which will be among the first molecules that T cells encounter before contacting the DCs. To address this point, we used PTX, which ADP-ribosylates and inactivates the _i subunit of the heterotrimeric G proteins used by chemokine receptors to transmit their intracellular signals. When memory CD4⁺ T cells were incubated with PTX overnight, Ag-independent T cell responses induced by DCs were inhibited (Fig. 7). No inhibitory effect was induced by the B subunit of PTX, which binds to the cell surface but does not possess a catalytic activity, and used here as a negative control. Collectively, these data suggest that DC-produced chemokines contribute to the Ag-independent T cell stimulatory ability of DCs.

View larger version (17K): [in this window] [in a new window]   FIGURE 7. In the absence of exogenous Ag, immature DC-induced T cell responses are inhibited by PTX. A and B, Percentage of motile and Ca2+-responding CD4+ memory T cells pretreated overnight with 50 ng/ml PTX or the B subunit of the PTX (suB) during their contact with DCs. Data are the mean of three to five experiments ± SD, in which >30 cells were analyzed per experiment. **, p < 0.01; ***, p < 0.001. Statistical significance was calculated between control (CTLR) and PTX conditions. C, Percentage of CD69-positive CD4+ memory T cells pretreated overnight with 10 ng/ml PTX or the B subunit of the PTX (suB) after 24 h of contact with DCs. Data represent the average value of duplicate samples ± SD and are representative of three independent experiments.

View larger version (39K): [in this window] [in a new window]   FIGURE 8. Chemokine production by DCs. A, mRNA expression was measured by RT-PCR at 25 and 30 cycles on immature DCs. Data are representative of eight independent experiments. B, Measurement by ELISA of CCL2, CCL17, and CCL22 produced by immature DC (1 x 106 cells/well) in 24 h. Data are the mean of three experiments ± SD.

Rapid T cell polarization could be elicited not only by purified chemokines, but also, in 40% of CD4⁺ memory T cells, by DC culture supernatant (Fig. 9A). In addition, 15% of CD4⁺ memory T cells were attracted by DC supernatant in a Transwell assay (Fig. 9B). This response was abrogated when T cells were pretreated with PTX, confirming the involvement of DC-produced chemokines in memory T cell chemotaxis. Interestingly, neutralizing anti-CCL17 and anti-CCL22 Abs markedly inhibited T cell polarization as well as T cell attraction in a Transwell assay (Fig. 9, A and B). The synthetic compound TAK-779 that efficiently blocks CCR5 (receptor for CCL5, CCL3, and CCL4) and CCR2 (receptor for CCL2) (21) showed only a weak inhibitory effect (<20%, data not shown). Finally, pretreatment of immature DCs with anti-CCL17 and anti-CCL22, and simultaneous pretreatment of T cells with TAK-779 reduced by 60% the frequency of motile lymphocytes (Fig. 9C, left panel), but did not affect DC-induced Ca²⁺ responses (right panel). It should be noted that anti-CCL17 and anti-CCL22, used alone, produced a weaker but noticeable effect on the motility induced by DCs (up to 40% of inhibition, data not shown). These findings suggest that immature DCs polarize and attract memory CD4⁺ T cells mainly through the action of CCL17 and CCL22, two ligands of CCR4. In addition, CCL17 and CCL22 along with other DC-secreted chemokines transmit a motility signal to memory CD4⁺ T cells.

View larger version (18K): [in this window] [in a new window]   FIGURE 9. Anti-CCL17 and anti-CCL22 affect the early T cell responses induced by DCs. A, Percentage of polarized memory CD4+ T cells induced by DC supernatant pretreated with 10 µg/ml anti-CCL17 and anti-CCL22 or an isotype control. **, p < 0.01; ***, p < 0.001. B, Percentage of DC supernatant-attracted memory CD4+ T cells measured with a Transwell chamber. DC supernatants diluted by half were pretreated with 10 µg/ml anti-CCL17, anti-CCL22, or an isotype control. T cells were treated or not with 50 ng/ml PTX 12 h before the experiment. Error bars represent SD from duplicate data sets. Data are representative of two independent experiments. C, Percentage of motile (left panel) and Ca2+-responding (right panel) memory CD4+ T cells induced by DCs pretreated with 10 µg/ml monoclonal anti-CCL17 and anti-CCL22 Abs, or an isotype control. T cells were treated or not with 1 µg/ml TAK-779. Data are the mean of three experiments ± SD, in which >30 cells were analyzed per experiment.

To determine whether ICAM-1 and DC-secreted chemokines could mimic immature DCs in their ability to stimulate memory CD4⁺ T cells, we have tracked the motility pattern of T cells on an ICAM-1 layer before and after the addition of DC culture supernatant. As shown in Fig. 10A and supplemental movie 7, DC supernatant rapidly triggered T cell polarization and motility, clearly evoking the crawling behavior induced by immature DCs. Unstimulated T cells, on the contrary, remained round and showed only Brownian-like movements easily distinguishable from the several micrometer-long excursions observed upon DC supernatant treatment (Fig. 10B). CCL17 and CCL22 proved to be important for triggering motility on the ICAM-1 layer: the percentage of CD4⁺ memory T cells showing a motile behavior in the presence of DC supernatant was reduced when the supernatant was preincubated with anti-CCL17 and anti-CCL22 (from 27.6 ± 7.8% to 17.7 ± 1.6%; n = 3 experiments, in which >30 cells were analyzed per experiment; p < 0.05). In addition, anti-CCL17 and anti-CCL22 significantly decreased the average velocity of motile T cells. Whereas in control conditions responding T cells migrated with an average velocity of 8.4 ± 3.2 µm/min (n = 13 cells), in the presence of neutralizing Abs, motile lymphocytes presented a reduced average velocity of 5.1 ± 2 µm/min (n = 12 cells; p < 0.02). Thus, the combination of immobilized ICAM-1 and DC-derived chemokines seems to be sufficient to support active T cell crawling. In contrast, no Ca²⁺ increases could be detected under these conditions (data not shown), indicating that the minimum molecular requirements for this signal must comprise additional DC-specific components.

View larger version (64K): [in this window] [in a new window]   FIGURE 10. A, Representative CFSE-loaded memory CD4+ T cell (black) plated on ICAM-1 and stimulated with DC supernatant diluted by half. Images captured at different times after supernatant addition were overlaid. Red dots mark the position of the T cell at successive 20-s intervals. Bar, 10 µm. B, Paths of 10 individual memory CD4+ T cells plated on ICAM-1 and stimulated (right panel) or not (left panel) with DC supernatant (DC-sup). Tracks have been overlaid and normalized to their starting coordinates.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

ICAM-1 but not DC-SIGN is involved in Ag-independent T cell responses induced by DCs

An essential participation of DC-SIGN during the initial interaction between DCs and human resting T cells has been proposed (13). The authors showed that blocking Abs had an inhibitory effect on the proliferation of resting T cells induced by allogeneic DCs. However, the inhibitory effect observed in a MLR does not allow us to conclude that DC-SIGN is involved in an early step of immunological synapse formation, as proposed. Indeed, the only early response for which a clear effect of DC-SIGN could be demonstrated was a reduction of conjugates formed between DCs and ICAM-3-transfected K562 cells.

Our results do not confirm this hypothesis. By using a reconstitution approach as well as examining the consequences of blocking the ICAM-3/DC-SIGN interaction, we have shown that DC-SIGN does not play an important role in immature DC-induced early T cell responses. However, the responding T cells in our conditions were mainly memory T cells, and a difference between naive and memory T cells for the use of DC-SIGN during the initial T-DC interaction could be envisioned.

In contrast to DC-SIGN, ICAM-1 played an essential function in the Ag-independent T cell responses induced by DCs. Our data are consistent with studies demonstrating an important participation of LFA-1/ICAM-1 during the Ag-independent adhesion of human DCs to T cells (22). However, ICAM-1 cannot be considered as the key DC molecule, because Raji B cells, despite the expression of very high levels of ICAM-1 (data not shown), are poor T cell stimulators in the absence of Ag. In addition, because LFA-1 in resting T cells has a low affinity for ICAM-1 and must receive a first signal before being able to bind its ligand efficiently, a molecule able to trigger this avidity enhancement still needs to be identified. DC-SIGN, through its activation of ICAM-3, was a good candidate for such a function, but we have shown that it does not fulfill the necessary requirements.

Immature DCs trigger CD4⁺ memory T cell motility

T cell motility and migration are emerging as important components of the process of immune recognition. Indeed, two-photon experiments have shown that, in the absence of exogenous Ag, naive T cells are extremely motile in intact LNs (23, 24, 25). This motility most likely enables T cells to search for rare Ags expressed at the DC surface. In the complex environment of the LN, several cells and factors could affect T cell migration. For instance, stromal cells secrete CCL19, which is known to attract CCR7⁺ T cells (26). In addition, the extracellular matrix may be considered as a path on which lymphocytes can move. The importance of such paths has been underlined in an in vitro study where T cell migration was observed in the presence of DCs in a three-dimensional collagen gel (27).

In the present work, we have used a simple assay to demonstrate that immature DCs alone, without any additional cells or proteins from the extracellular matrix, are able to transmit a migration signal to primary nonstimulated CD4⁺ T cells, and in particular to memory T cells. By contrast, autologous monocytes were unable to trigger such responses. The migratory behavior of T cells observed in this study is very similar to that described in intact LNs. In particular, in our in vitro conditions, T cells displayed a pattern of alternating high and low velocities with random paths as recently reported in vivo (23, 24). This suggests that the T cell motile behavior in intact LNs is not only that of naive T cells but also of memory T cells. In addition, it indicates that DCs probably provide essential signals that promote the T cell motility observed in intact LNs, although it is likely that other factors present in LNs might also influence the T cell behavior.

CCL17 and CCL22 are key players in immature DC-induced polarization and attraction of CD4⁺ T cells

We have performed a thorough analysis of the chemokines produced by immature DCs that could control Ag-independent T cell responses. Our data indicate that immature DCs constitutively express transcripts for CCL17, CCL22, and CCL18, confirming previous findings obtained on monocyte-derived DCs (28). In addition, other transcripts including CCL2, CCL3, and CCL5 were detected, although less abundantly.

We have shown that DC-produced chemokines play an important role in DC function, as judged by the inhibitory effect of PTX on several Ag-independent T cell responses induced by DCs. Other groups have examined the participation of chemokines in the initial murine T-DC initial interaction. Thus, PTX has been shown to largely inhibit the adhesion between murine Ag-activated T cells and bone marrow DC, and CCL22 appeared as the most important chemokine controlling this process (14). However, no effect of PTX was observed on the adhesion of murine naive T cells to bone marrow-derived DCs (15). Altogether, these data and our findings indicate that murine Ag-activated T cells and human CD4⁺ memory T cells present several chemokine-dependent, DC-induced responses.

Among the different chemokines produced by immature DCs, two of the most abundant ones, CCL17 and CCL22, both acting on CCR4, proved to be important for the earliest Ag-independent responses induced by DCs in memory T cells. Neutralizing Abs against CCL17 and CCL22 largely inhibited the T cell attraction and polarization that precede the interaction with DCs. In addition, the motile phenotype that T cells rapidly acquire after their contact with DCs was also reduced by a combination of Abs against CCL17 and CCL22 plus the synthetic compound TAK-779 that antagonizes CCR2 and CCR5. However, other downstream responses that were partially dependent on chemokines appeared to have a more complex regulation. Indeed, neither the Ca²⁺ responses nor the CD69 up-regulation were inhibited by neutralization of CCL17 and CCL22 with specific Abs and by blocking CCR2 and CCR5 with TAK-779. This result implies the existence of additional, unidentified DC-produced chemokines that are important for T cell stimulation.

DC-produced chemokines could act at multiple levels before and during T-DC interactions. By attracting T cells, chemokines promote the encounter with DCs and therefore increase the proportion of responding T cells. However, in our experiments, T cell responses were inhibited by PTX even when the DC layer was close to confluency, suggesting that chemokines may be acting via additional mechanisms.

Another mode of action of chemokines might reside in their ability to increase LFA-1 avidity for ICAM-1 (29). This mechanism is known to be important for leukocytes to adhere to endothelial cells. However, in our conditions, PTX showed only a modest inhibitory effect (<20%) on T-DC adhesion as measured with a laminar flow chamber. This suggests that multiple redundant mechanisms participate in the formation of Ag-independent conjugates between T cells and DCs. Abrogating the contribution of chemokines was not sufficient to inhibit the adhesion process.

Chemokines also affect T cell morphology, inducing a polarized phenotype with a leading edge and a uropod, and sustained cytoskeletal dynamics that are necessary for lymphocyte migration. Cellular shape changes are accompanied by the polarization of cell surface and signaling molecules that are involved in early T cell responses. As a result, a T cell contacting an APC with its leading edge is much more responsive than a T cell that makes contact through its uropod (19). The molecular basis of this polarized sensitivity is still unclear. Chemokines could contribute to this phenomenon by promoting the translocation from the leading edge to the uropod of molecules with an antiadhesive function such as CD43 (30). If this view is correct, formation of an immune synapse enriched in adhesion and signaling molecules could be preceded and prepared by molecular reorganizations mediated by DC-produced chemokines.

Finally, it has been proposed that chemokines could activate intracellular signaling pathways that would potentiate the signals delivered by synaptic molecules (31).

Functional importance of DC-induced memory T cell responses in the absence of exogenous Ag

Our data show that, by producing the CCR4 ligands CCL17 and CCL22, immature DCs preferentially attract and stimulate a fraction of CD4⁺ memory T cells. The fact that the memory CD4⁺ T cells that respond to immature DCs are both CCR7⁺ and CCR7^– suggests that in vivo such interaction could occur both in LNs and in the peripheral tissues. Further characterization of the cell populations attracted by DC culture supernatant indicates that these are enriched in T cells that express CCR4, cutaneous lymphocyte-associated Ag, and CD25 (E. Real and E. Donnadieu, unpublished data). CCR4 and cutaneous lymphocyte-associated Ag are largely expressed by a subset of memory T cells recruited to the skin (32), whereas CD25 is constitutively expressed by a population of CD4⁺ regulatory T cells (Treg) that have been implicated in the induction and maintenance of peripheral immunological tolerance. Interestingly, all three cell surface markers were recently found associated in a large fraction of CD4⁺CD25⁺ Treg cells (33). Therefore, it is possible that these Ag-independent early responses triggered by immature DCs maintain Treg cells in an optimal functional state, ultimately favoring tolerance induction. This would fit with recent data showing that CD4⁺CD25⁺ T cells can survive for long periods in vivo in the absence of specific Ag, while retaining their suppressive function (34).

In contrast, Ag-independent signals transmitted by DCs could represent an important priming mechanism that would favor Ag detection and responsiveness. Indeed, the qualitative nature of the signals induced by immature DCs in the absence of Ag shows some resemblance to the early events that follow Ag recognition, suggesting that they may contribute to T cell priming. According to such an interpretation, skin-homing memory CD4⁺ T cells would be recruited by skin-resident DCs that, through the triggering of an extensive crawling behavior, would allow them to collect Ag-independent signals and remain in a state of functional alertness. In contrast, naive and central memory T cells would have access to such stimuli at the time of their passage through LNs, where they might colocalize with resident DCs. Such a scenario would fit data from our lab showing that simple adhesion to DCs is sufficient to prime murine T cells for anti-CD3 responses in vitro (35). Interestingly, two recent studies have shown that MHC class II binding in a resting animal can heighten the responsiveness of both naive and memory T cells to future encounters with foreign Ag (36, 37). Although these studies did not directly implicate DCs, it is quite possible that this sensitization is related to the signaling events reported in this study.

In conclusion, our results suggest that immature DCs continuously communicate with T cells in an Ag-independent manner. This might be yet another mechanism used by DCs to accomplish their powerful role of APC, possibly by preparing T cells for Ag encounter. We propose that DCs owe part of their outstanding Ag presentation talent to their ability to trigger T cell adhesion and motility, leading to efficient scanning of the APC surface. These phenomena do not seem to involve DC-SIGN, but they require both the ICAM-1/LFA-1 interaction and DC-released chemokines. In the case of memory T cells, the relevant chemokines are CCL17 and CCL22, both acting on CCR4. Thus, chemokines and chemokine receptors stand out as important factors in the setting up of immune interactions, not only by permitting cell-cell encounters to take place, but possibly by affecting the architecture of the interacting surfaces.

	Acknowledgments

 
We thank Yvette van Kooyk and Carl Figdor for providing the blocking anti-DC-SIGN Ab (AZN-D1); Fernando Arenzana-Seisdedos for helpful advice and the gift of TAK-779; Jean-Edouard Corbière for his help with the RT-PCR experiments; and Jean-Pierre Abastado, Clotilde Randriamampita, Patrick Revy, Jérôme Delon, and Georges Bismuth for their critical comments on the manuscript.

	Footnotes

 
¹ This work was supported by grants from the Centre National de la Recherche Scientifique, the Région Ile-de-France, and the Ligue Nationale contre le Cancer. E.R. is a recipient of a fellowship from the Portuguese Foundation for Science and Technology.

² Address correspondence and reprint requests to Dr. Emmanuel Donnadieu, Département de Biologie Cellulaire, Institut Cochin, 22 rue Méchain, 75014 Paris, France. E-mail address: donnadieu{at}cochin.inserm.fr

³ Abbreviations used in this paper: DC, dendritic cell; LN, lymph node; Treg, CD4⁺CD25⁺ regulatory T cell; DC-SIGN, DC-specific ICAM-3-grabbing nonintegrin; [Ca²⁺]_i, intracellular Ca²⁺ concentration; PTX, pertussis toxin; SEE, bacterial staphylococcal enterotoxin E; T_CM, central memory T cell; T_EM, effector memory T cell.

⁴ The on-line version of this article contains supplemental material.

Received for publication December 22, 2003. Accepted for publication April 20, 2004.

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