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Originally published online as doi:10.1189/jlb.0307167 on July 18, 2007

Published online before print July 18, 2007
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(Journal of Leukocyte Biology. 2007;82:861-868.)
© 2007 by Society for Leukocyte Biology

Bidirectional MHC molecule exchange between migratory and resident dendritic cells

Magali de Heusch*, Didier Blocklet{dagger}, Dominique Egrise{dagger}, Bernard Hauquier{dagger}, Marjorie Vermeersch*, Serge Goldman{dagger} and Muriel Moser*,1

* Institut de Biologie et Médecine Moléculaires, Université Libre de Bruxelles, Gosselies, Belgium; and
{dagger} Hopital Erasme, Université Libre de Bruxelles, Brussels, Belgium

1 Correspondence: Laboratoire de Physiologie Animale, Université Libre de Bruxelles, Rue des Prof. Jeener et Brachet, 12, 6041 Gosselies, Belgium. E-mail: mmoser{at}ulb.ac.be


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dendritic cells (DCs) loaded extracorporeally with antigen can be used as an adjuvant in vivo. In this work, we analyzed the migration of transferred DC and monitored the phenotype of new migrants in the draining lymph nodes. It is surprising that we found that a majority of resident DCs expressed donor MHC molecules and that a proportion of injected DCs acquired host MHC molecules. These observations suggest that a bidirectional MHC molecule exchange occurs between migratory and resident DCs, a mechanism that may amplify antigen presentation in vivo.

Key Words: antigen presentation • expression • interaction • migration • lymph nodes


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The encounter between lymphocytes and antigen-bearing dendritic cells (DCs) in the specific microenvironments of secondary lymphoid organs is considered as a key, early event in acquired immunity [1 ]. T cell sensitization has been shown to require a sustained physical interaction between T cells and DCs, which involves TCR occupancy by antigen/MHC complexes and engagement of costimulatory molecules by their cognate receptors [2 3 4 ]. Among APCs, DCs have unique migratory properties in vivo [5 ] and upon infection or inflammation, migrate to the zone where T cells are located in the lymphoid organs. This movement is usually associated with changes in phenotype and function, a phenomenon called maturation, which converts antigen-capturing DCs into T cell-sensitizing cells [6 , 7 ]. We and others [8 , 9 ] have shown that DCs can be used as adjuvants in vivo: Adoptive transfer of DCs, pulsed extracorporeally with antigen, into syngeneic mice induces the activation of T and B lymphocytes.

There is some evidence that T cell-proliferative responses are proportional to the number of DCs, which reach the lymph nodes. Sallusto and colleagues [10 ] have shown that enhancing the number of DCs reaching the lymph nodes increases the number of CD4+ transgenic T cells, which proliferate and produce cytokines. However, the number of transferred DCs, which end up in the lymph nodes, draining the site of injection, is unexpectedly low, suggesting that indirect presentation of the antigen loaded on DCs by host APCs may be involved in T cell priming, although this phenomenon, often referred to as cross-priming, is still controversial (see Discussion below).

The aim of this study was to monitor the migration of splenic DCs and to characterize the phenotype of transferred DCs into the draining lymph nodes. It is unexpected that we found that when administered into MHC-disparate host, most transferred DCs acquired the host MHC in addition to their own MHC haplotype, suggesting a transfer of information between migrating and resident DCs in the lymphoid organs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
Balb/c and C57BL/6 mice, 6- to 9-weeks old, were purchased form Harlan (Nederland). All mice were bred in our pathogen-free facility and used at 6–9 weeks of age. All experiments were performed in compliance with the relevant laws and institutional guidelines and have been approved by the local committee from the Institut de Biologie et Médecine Moléculaires from the Université Libre de Bruxelles (Gosselies, Belgium). ICAM-1 knockout (KO) mice were kindly provided by Clotilde Théry and Sebastian Amigorena (Institut Curie, Paris France).

Reagents and antibodies
The mAb-to-mouse molecules used were 14.4.4 (anti-I-Ed), N418 (anti-CD11c), AF6-120.1 (anti-I-Ab, PharMingen, San Diego, CA, USA), and GL-1 (anti-CD86). CFSE and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) were purchased from Molecular Probes (Eugene, OR, USA). CpG 1826 was puchased from Eurogentec (Seraing, Belgium; T*C*C*A*T*G*A*C*G*T*T*TC*C*T*G*A*C*G*T*T*).

Purification of DC
Mice were injected i.p. with 10 µg recombinant human Fms-like tyrosine kinase 3 ligand (human Chinese hamster ovary cell-derived; produced at Amgen, Seattle, WA, USA) for 9 consecutive days. Splenic DC were purified by a modified procedure of a protocol described previously [9 ]. Briefly, spleen cells were digested with collagenase, further dissociated in Ca2+-free medium, and separated into a low- and high-density fraction on a Nycodenz gradient (Nycomed, Oslo, Norway). The low-density fraction was separated according to CD11c expression by incubation with anti-CD11c-coupled microbeads, followed by one passage over a MACS column (Miltenyi Biotec, Bergisch-Gladbach, Germany). The CD11c-positive cells were cultured in RPMI 1640 containing 2% Ultroser HY (Life Technologies, Paisley, Scotland) with GM-CSF (15 ng/ml) for 16 h at 37°C (referred to as mature as they undergo a phenomenon of maturation; ref. [9 ]; #611) or were used freshly (referred to as immature). When indicated, DCs were killed by freeze and thaw cycles.

Indium-111 labeling and homing of transferred DC
DCs (106) were incubated for 30 min at 20°C with 10 µCi 111In-oxine (Mallinckrodt Medical, Petten, Tyco Belgium) in RPMI with 2% HY and additives and washed three times. Labeled cells (106) were injected into the right hind and fore footpads, and the mice were analyzed at indicated time-points on a {gamma}-camera (DSX SMV, Buc, France, equipped with a medium energy, high resolution, parallel-hole collimator; energy detection: 173 KeV±10% and 247 KeV±10%; energy of the {gamma}-photons emitted by 111In). Thirty-minute images were acquired and read with a zoom factor of 2.

CFSE and/or DiI labeling
DCs were suspended at 107 cells/ml in RPMI incubated with CFSE (0.5 µM) and/or DiI (0.2 µM) at 37°C for 10 min and washed in RPMI containing 2% Ultroser HY and additives and then in PBS.

Anesthesia
Mice were treated i.p. with a mixture of 20 µl Imalgène 1000 (100 mg/ml ketamin, Merial, Lyon, France) and 10 µl Rompun 2% (Bayer, Brussels, Belgium).

Immunohistochemical studies
Lymph nodes were harvested and frozen in isopentane. Cryostat sections (10 µm) were prepared, and samples were fixed in neat acetone for 10 min and transferred to PBS containing 0.5% of blocking reagent (Boehringer, Germany) for 30 min. Sections were stained for CD4 expression using biotinylated RM4-5 (25 µg/ml, 1 h, 20°C), revealed by avidin-biotin-peroxidase complex (vectastain ABC kit, Vector Laboratories, Burlingame, CA, USA). The peroxidase activity was revealed with 3-amino-9-ethylcarbazole solution (Sigma Aldrich, St. Louis, MO, USA). The CFSE-stained cells were visualized by an alkaline phosphatase-linked anti-FITC Fab fragment (Boehringer; ref. [6 ]; #136).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Migration of transferred DCs to the T cell area of draining lymph nodes
Adoptive transfer of mature, splenic DCs, pulsed in vitro with antigen, has been shown to induce the priming of specific B and T lymphocytes in the lymph nodes draining the site of injection. To monitor the migration of DCs and in particular, of immature and mature, splenic DCs, 111In -labeled cells were injected into the footpads of syngeneic mice. The radioactivity was detected by a {gamma}-camera at various time-points. The data in Figure 1 show that 24 h after injection of 111In -labeled DCs (Fig. 1a and 1b) but not of free 111In (Fig. 1c) , 1–3% of the radioactivity injected was detected in the lymph nodes, although the highest proportion remained in the injected footpads. A kinetic study revealed that the amount of radioactivity detected in the draining lymph nodes increased with time after injection and reached a plateau at 24 h (Fig. 1e and 1f) . To test whether the 111In measured in the lymph nodes was associated with DCs, we purified CD11c+ cells by magnetic cell sorting and measured the radioactivity in the positive and negative fractions. The data show that 111In segregated with the cell fraction expressing CD11c (background=180 cpm; DC-enriched fraction: 26,000 cpm; DC-depleted fraction: 500 cpm). The transferred DCs, labeled with CFSE, localized in the T cell zone, as assessed by CD4 staining (Fig. 1g and 1h) . Similar results were obtained with freshly isolated (immature) and mature DCs, suggesting that the maturation did not affect the migratory capacity of splenic DCs (Fig. 1e and 1f) .


Figure 1
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  		Figure 1. Immature and mature, splenic DCs migrate from the site of injection to the draining lymph node and home to the T cell zone. Splenic DCs from C57/BL6 mice were labeled with 111In and injected into the right footpads of syngeneic mice (106 DC/footpad). (a and b) Twenty-four hours after injection, mice were placed on a camera, which detects the {gamma}-rays emitted by the 111In. Arrows indicate the site of injection and the draining lymph nodes of mice injected with mature DCs (mDC; a) or immature DCs (iDC; b). (c) Mice were injected in the right footpad with free 111In (IND-111; corresponding to the activity contained in 106 DC) or (d) with 111In-labeled, mature, splenic DCs fixed for 10 min in glutaraldehyde, 0.1% (Gluta.), and placed 24 h later on a {gamma}-camera. (e) Mice treated as in a and b were placed on a {gamma}-camera at indicated time-points. Data indicate percent of radioactivity detected in lymph nodes relative to total amount of activity detected in mice. (f) Draining and nondraining lymph nodes of mice treated as above were harvested, and the radioactivity (in cpm) was counted in a {gamma}-counter 24 h and 48 h after injection. (g) Mature or (h) immature, splenic DCs were labeled with CFSE and injected into the hind footpads of syngeneic mice. After 24 h, draining lymph nodes were harvested, and cryosections were stained for CD4 expression in red and for fluorescein expression, in blue. (i and j) Mature, splenic DCs were labeled with 111In and injected into the right footpad of syngeneic mice (106 DC/footpad). The mice were anesthetized (anesth; i) or not (untr; j) with a mix of Rompun and ketamine for 5 h and placed on a {gamma}-camera. (k) Mice treated as above were placed on a {gamma}-camera at indicated time-points. Data indicate percent of radioactivity detected in draining or nondraining lymph nodes compared with total activity detected in mice. All scintigraphic data presented resulted from 30-min acquisitions on a {gamma}-camera. Similar results were obtained in four (a and b), three (c–h), or two (i and k) independent experiments with at least three mice per group tested individually.

 
In the course of these studies, we anesthesized some recipient mice (for 5 h after injection) and noticed that treatment with Imalgene (ketamine) and Rompun prevented the migration of 111In-labeled DCs to the draining lymph nodes (Fig. 1i 1j 1k) . In addition, no radioactivity was detected in the lymph nodes of mice injected with glutaraldehyde-fixed DCs (Fig. 1d) . These observations suggest that the migration of DCs is an active process and is dependent on the physiological status of the DCs and the recipient.

Injection of allogeneic DCs results in membrane display of donor and host MHC molecules by the same DCs
We next examined the phenotype of DCs within the draining lymph nodes 24 h after injection. To discriminate between donor and host DCs, we injected DCs from C57BL/6 mice (expressing I-Ab but not I-Ed, Fig. 2g and 2h ) into Balb/c recipients (H-2d) and monitored MHC class II expression. Our data in Figure 2b and c , clearly show that the majority of donor DCs present in the lymph nodes draining the site of injection displayed the host MHC (I-Ed) in addition to I-Ab. The analysis of costimulatory molecules revealed that migrant DCs expressed intermediate levels of CD86 (Fig. 2j) , as compared with the low and high expression by transferred, immature and mature DCs, respectively (Fig. 2i) .


Figure 2
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  		Figure 2. Transferred DCs acquire and/or give MHC class II to host cells in the lymph node. Immature and mature, splenic DCs from C57BL/6 mice (donor, expressing I-Ab; g) were injected into the footpads of BALB/c mice (host, expressing I-Ed). Twenty-four hours later, lymph nodes were harvested, pooled (three to five mice), and enriched in DCs by positive selection of CD11c+ cells. (a–f) DC were enriched from lymph nodes of Balb/c mice, untreated (a and d) or injected with immature (b and e) or mature (c and f) DCs 24 h previously, and double-stained in red for I-Ab and in green for I-Ed (a–c) or CD11c (d–f). (g–j) I-Ab (g), I-Ed (h), or CD86 (i and j) expression on immature (thin line) or mature (thick line) splenic DCs from C57BL/6 mice before injection (g–i) or purified from lymph nodes 24 h later (j). Controls include immature DC from Balb/c mice (filled histogram) and unstained DCs (dotted line). Similar results were obtained in three independent experiments.

 
We next tested whether cell types distinct from DCs may also acquire the donor MHC in injected animals. The data in Figure 2e and f , show that the majority of cells, which display donor MHC molecules, was CD11c+, suggesting that the acquisition of MHC molecules from the DC vaccine is a property of DC populations in the lymphoid organs.

DCs acquire MHC molecules in vitro
To test whether membrane exchange occurs in vitro, we cultured Balb/c DCs with DCs purified from the spleens of C57BL/6 mice and labeled with CFSE, a stable cytoplasmic dye. The analysis of I-Ab expression on CFSE-negative cells 24 h later clearly indicates that Balb/c DC displayed high levels of membrane I-Ab molecules (Fig. 3 ) and that the acquisition increases in the presence of CpG.


Figure 3
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  		Figure 3. In vitro MHC transfer. Immature DCs from BALB/c mice (expressing I-Ed) were cultured (thin and thick line) or not (dotted line) with CFSE-labeled DCs from C57BL/6 mice (I-Ab) in the presence (thick line) or absence (thin line) of CpG. Twenty-four hours later, cells were harvested and stained for I-Ab. The data show I-Ab expression on Balb/c DCs (gated for CFSE– expression). Negative controls include Balb/c DCs cultured alone (dotted line) and unstained (plain histogram). The results are representative of three independent experiments.

 
The exchange of MHC molecules between host and donor DCs is bidirectional
To identify donor DCs in the recipient, we labeled C57BL/6 DCs with CFSE before transfer into Balb/c mice. The analysis of MHC expression revealed that in addition to the acquisition of donor MHC by recipient DCs (Fig. 4I , f vs. e), a significant proportion (65%) of CFSE+ donor DCs acquired host MHC (Fig. 4I 4c) , suggesting that a bidirectional transfer of MHC molecules occurred between donor and host DCs. The FACS analysis of doublets (using propidium iodide and CFSE staining) showed that 3–15% of CFSE+ cells were doublets, confirming that at least 50% of host cells acquired donor MHC.


Figure 4
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  		Figure 4. Transfer of MHC class II between donor and host is bidirectional. CFSE-labeled, immature DCs from C57BL/6 mice (expressing I-Ab) were injected into the footpads of BALB/c mice (host, expressing I-Ed) or CB6 F1 mice (Balb/cxC57BL/6; II, b, c, g, h; host, expressing I-Ab and I-Ed). Twenty-four hours later, lymph nodes were harvested, pooled (three to five mice), and enriched for DC by positive selection of CD11c+ cells. DC-enriched cells were unstained (left panels) or stained for I-Ed (I, b and c, and II, b–e) or I-Ab (I, e and f, and II, g–j). Results are representative of 15 (I) and three (II) experiments. Similar results were obtained with mature DCs. CTRL, Control.

 
To evaluate the effect of MHC disparity, we injected CFSE-labeled C57BL/6 DCs into (C57BL/6xBalb/c) F1 or Balb/c recipients and monitored MHC expression by FACS. The results in Figure 4II , c and e, show that 64% of DCs injected in Balb/c, as well as in F1 recipients, acquired host MHC molecules, excluding alloreactivity as a cause of MHC transfer.

Injection of CpG increases the acquisition of donor MHC molecules by host DCs
We next tested whether TLR ligands might influence this phenomenon. Recipient mice were injected with CFSE-labeled DCs, mixed or not with CpG. The data in Figure 5b and 5c , clearly show a two- to threefold increase in the number of DC displaying MHC after CpG coinjection. A similar effect was observed in wild-type mice injected with MyD88 KO DCs (Fig. 5e and 5f) and was abrogated when CpG-treated DCs were washed before transfer (Fig. 5d and 5g) , suggesting that CpG acted selectively on recipient DCs and increased their capacity to acquire donor MHC. We next tested whether dead cells could similarly transfer their MHC. The results (Fig. 6 ) show that host cells acquired MHC II molecules from live and dead cells in the same proportion and that this acquisition increased in the presence of CpG. As expected, only live, transferred DCs (CFSE+) were detected in the lymph nodes draining the site of injection.


Figure 5
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  		Figure 5. The MHC class II exchange between donor and host cells is increased in the presence of CpG. CFSE (FL-1)-labeled, immature DCs from C57BL/6 (b–d) or MyD-88 KO (e–g) mice (donor, expressing I-Ab) were injected into the footpads of BALB/c mice (host, expressing I-Ed). Donor DCs were left untreated (b and e), mixed with CpG before injection (c and f), or treated with CpG for 1 h at 37°C and washed before injection (d and g). Twenty-four hours later, the draining lymph nodes were harvested, pooled (three to five mice), and enriched for DCs by positive selection of CD11c+ cells. The data show I-Ab (FL-3) staining and CFSE expression of DC-enriched cells from untreated (a) or treated mice (b–g). Similar results were obtained in four independent experiments. WT, Wild-type.

 

Figure 6
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  		Figure 6. Host cells acquire MHC class II from dead, injected DCs. Splenic, immature CFSE (FL-1)-labeled DCs from C57BL/6 mice (donor, expressing I-Ab) were untreated (b and c) or subjected to freeze/thaw cycles (d and e) and injected into the footpads of BALB/c mice (host, expressing -Ed) with (c and e) or without (b and d) CpG. Twenty-four hours later, lymph nodes were harvested, pooled (three to five mice), enriched for DCs by positive selection of CD11c+ cells, and analyzed by FACS for CFSE (FL-1) and I-Ab (FL-3) staining. Similar results were obtained in four independent experiments.

 
Role of lipid membranes, exosomes, and cell fusion in MHC class II molecule transfer
In an attempt to identify the mechanism of MHC exchange, we tested whether lipids were transferred to host cells. Splenic DCs from C57BL/6 mice were labeled with CFSE and DiI, a fluorescent dye, which stains membrane lipids, and injected into the footpads of Balb/c mice. The results in Figure 7I show that stained lipids were expressed on host DCs, illustrating that lipid membranes were transferred together with MHC molecules.


Figure 7
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  		Figure 7. (I) Lipids are transferred with MHC molecules. Immature DCs from C57BL/6 mice were labeled with CFSE and DiI, a fluorescent label that stains lipid membranes, and injected into the footpads of BALB/c mice, with (c) or without (b) CpG. Twenty-four hours later, lymph nodes from injected (b and c) and untreated mice (a) were harvested, pooled (three to five mice), enriched for DC by positive selection of CD11c+ cells, and analyzed by FACS for CFSE (FL-1) and DiI (FL-2) staining. Similar results were obtained in five independent experiments. (II) MHC class II exchange does not require ICAM-1 expression by donor cells. CFSE (FL-1)-labeled DCs from C57BL/6 (b and c) or ICAM-1 KO (d and e) mice (donor, expressing I-Ab) were injected into the footpads of BALB/c mice (host, expressing I-Ed). Transferred DCs were untreated (b and d) or mixed with CpG before injection (c and e). Twenty-four hours later, lymph nodes from injected (b–e) and untreated mice (a) were harvested, pooled (three to five mice), enriched for DCs by positive selection of CD11c+ cells, and analyzed by FACS for CFSE (FL-1) and I-Ab (FL-3) staining. Similar results were obtained in three independent experiments.

 
At least two mechanisms have been described, which may favor membrane exchange: release of exosomes and cell fusion. Exosomes are membrane vesicles formed in late, endocytic compartments, which bear functional, MHC-peptide complexes. They activate antigen-specific, naïve T cells, only after their recapture by APCs, a process that requires ICAM-1 expression [11 ]. To assess the role of exosomes in MHC exchange, we injected DC from ICAM-KO mice into wild-type animals. The data in Figure 7II , show that recipient DC similarly acquired allo-MHC from transferred DC, expressing ICAM-1 or not.

To test whether CFSE+-injected cells acquired MHC molecules by fusing with host cells, we measured the DNA content of donor and host DCs displaying both MHC. Our results show that between 4% and 10% of the CFSE+ cells (4%, 5%, and 10% in three independent experiments) and 2% of the host cells expressed double DNA content, suggesting that fusions may occur but are rare events (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The data presented herein clearly demonstrate that MHC exchange occurs at high frequency in vivo between transferred and resident lymph node DCs. Indeed, more than 60% of injected DCs acquired host MHC in the lymph nodes, and 4–8% of host DCs expressed MHC of donor-type in all experiments performed. This proportion reached 15–30% when CpG was coinjected with DCs. Our observations suggest that transfer of MHC and associated peptide between migrant and resident DCs may enhance and amplify antigen presentation in vivo. This property seems to be restricted to cells of the dendritic family, as B lymphocytes do not efficiently acquire or give MHC molecules in the same conditions.

Our observations are in agreement with a few reports showing that two populations of DCs may synergize to sensitize, naive T cells. In a MLR in vitro, a secondary presentation of alloantigens (acquired from stimulator DC) by DC of responder type has been described [12 ]. Of note, Kleindienst and Brocker [13 ] have reported that DC vaccine was responsible only for partial CD4+ T cell activation and that participation of resident DCs was crucial to obtain optimal expansion of T cells in vivo. They showed that three times more T cells were activated in recipients with targeted expression of restricting MHC on DCs. Finally, Herrera et al. [14 ] have demonstrated the capacity of DCs to acquire intact MHC/peptide complexes from other cells and present them to T cells. Our results extend their studies and show that CpG increases the acquisition of MHC molecules by host cells and that the exchange is bidirectional.

Our results showing that host cells acquired MHC II molecules from migrant and not migrant (dead) DCs in the same proportion suggest that the membrane exchange may take place in the lymph node as well as in the periphery.

Although the mechanism of MHC transfer is still elusive, several possibilities may be envisaged, which include cell fusion, secretion of exosomes, and tunneling nanotubes. Exosomes have been shown to promote the exchange of functional peptide/MHC complexes between DCs and become competent for naive T cell activation only after uptake by other DCs [15 ]. Therefore, a role of exosomes would be compatible with the observation that the synergy occurs exclusively between DCs. However, we found that DCs from ICAM-KO mice similarly exchanged their MHC with host DCs, suggesting that exosomes, which bind on DCs through interaction of ICAM-1 with its ligands, play only a minor role. We have shown further that cell fusion may occur but at low frequency and would not account for the acquired MHC expression. Onfelt et al. [16 ] have reported an unexpected mechanism for intercellular communication between immune cells. They showed that nanotubes can traffic cell surface proteins between immune cells, but this mechanism seems unlikely, as we have never been able to detect a MHC patch on the surface of DCs acquiring MHC molecules. A possible mechanism may rely on the capacity of DCs to acquire with high efficiency antigens expressed on the membrane of apoptotic cells [17 , 18 ], which would be consistent with the fact that host DCs similarly acquire MHC from dead, injected cells. Another study [19 ] shows that simple interactions between DCs and other healthy cells lead to substantial acquisition of plasma membrane and cytoplasmic proteins by DC. More recently, Dolan et al. [20 , 21 ] have demonstrated that DCs may be "cross-dressed", i.e., may acquire peptide MHC complexes directly from other cells and activate CD4+ and CD8+ T cells. Of note, triggering by CpG has been shown to enhance cross-priming [22 ] but may also have an inhibitory effect on cross-presentation [23 ].

The analysis of T cell/DC interactions within intact, explanted lymph nodes revealed that T cells formed long-lived associations with a single APC and showed evidence of activation only after 36-48 h [2 ]. In the case of DC vaccines, the formation of stable couples is difficult to reconcile with the low percentage of DCs reaching the area where T cells are located. The membrane exchange between transferred and endogenous DCs would solve this problem by increasing the number of antigen-bearing DCs, thereby favoring the encounter of T cells with the DC-bearing, stimulatory ligand. We calculated that the number of DCs presenting the donor antigen/MHC complexes in the lymph nodes would be increased by four- to 15-fold in the absence or presence of CpG, respectively (from 1% to 2% of donor DC reaching the draining lymph nodes to an additional 4–8% or 15–30% upon CpG treatment of resident DCs acquiring donor MHC molecules). Of note, the increase in DCs presenting a given antigen will also help to avoid the competition between T cells for access to antigen-bearing APCs [24 ].

There is evidence that inter-DC antigen transfer may be required for the induction of immune responses in physiological conditions. Two recent reports suggest that skin DCs play a minor role in priming T cells in response to skin pathogens. Allan et al. [25 , 26 ] showed that infection of murine epidermis by HSV did not result in priming of virus-specific CTL by Langerhans cells but required a distinct CD8{alpha}+ DC subset. They postulate that initial transport of antigen by migrating DCs is followed by its transfer to the lymphoid-resident DCs for presentation and CTL priming. Similarly, Iezzi and colleagues [27 ] have characterized the cell population responsible for initial activation of Leishmania major-specific T cells in mice. Antigen presentation in lymph nodes peaked as early as 24 h after infection and was mediated mainly by a population of CD11chighCD11bhigh DCs residing in lymph nodes. Collectively, these observations highlight the critical role of DCs, which are already present in the lymph nodes at the time of infection.

Our results further show that the migration of transferred DCs is an active process, which requires physiologically intact DCs and host, and immature and mature DCs similarly migrate to the lymph nodes draining the site of injection. Recent data indicate that the magnitude of the immune response induced by the adoptive transfer of antigen-pulsed DCs correlated with the number of DCs reaching the draining lymph nodes. In particular, injection of CCR7-deficient DCs, which are incapable of migrating to the lymph node, failed to induce immunity [10 ]. Accordingly, we have shown previously that immature and mature DCs induce similar expansion of antigen-specific T cells, when adoptively transferred into syngeneic recipients [28 ] Therefore, the similar migration of antigen-loaded DCs at either stage of maturation translates into similar activation of specific T cells. Although the expression of CCR7 is lower on immature, splenic DCs, as compared with mature DCs (ref. [29 ] and our data not shown), the migration of immature DCs may result from up-regulation of CCR7 expression on immature DCs, reaching levels sufficient for migration, as shown in ref. [30 ], and/or that from the involvement of other molecules, such as CCR2, which appears as an important determinant of DC/Langerhans cell migration and localization [31 ].

In conclusion, the unique property of DCs to induce primary immune responses may not only rely on their capacity to migrate to areas where T cells reside and to provide stimulatory signals but may also depend on their unique capacity to synergize with other cells of the same family.


   ACKNOWLEDGEMENTS
 
The Laboratory of Animal Physiology is supported by grants of the Fonds National de la Recherche Scientifique (FNRS)/Télévie, by the Fonds de la Recherche Fondamentale Collective, by the Belgian Program on Interuniversity Poles of Attraction initiated by the Belgian State, by European Grants, and by the Belgian Cancer Foundation. M. d. H. had a fellowship from the Fonds pour la Formation à la Recherche dans l'Industrie et l'Agriculture. M. M. is Research Director from the FNRS. The authors are grateful to Clotilde Théry and Sebastian Amigorena for interesting discussions and for providing ICAM-1-KO mice, to Nicolas Kesteman for valuable help, and to Philippe Veirman for animal care.

Received March 16, 2007; revised June 5, 2007; accepted June 25, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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