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The Journal of Immunology, 2008, 180, 5963 -5972
Copyright © 2008 by The American Association of Immunologists, Inc.
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Plasmacytoid Dendritic Cells Migrate in Afferent Skin Lymph1

Florentia Pascale*, Vanessa Contreras*, Michel Bonneau{dagger}, Alexandre Courbet*, Stefan Chilmonczyk*, Claudia Bevilacqua{ddagger}, Mathieu Eparaud*, Violeta Niborski*, Sabine Riffault*, Anne-Marie Balazuc§, Eliane Foulon, Laurence Guzylack-Piriou||, Beatrice Riteau*, Jayne Hope#, Nicolas Bertho*, Bernard Charley2,* and Isabelle Schwartz-Cornil2,3,*

* Virologie et Immunologie Moléculaires, UR892 Institut National de la Recherche Agronomique, Jouy-en-Josas; {dagger} Centre de Recherche en Imagerie Interventionnelle, Jouy-en-Josas, France; {ddagger} Plateau d’instrumentation et de compétences en transcriptomique, Jouy-en-Josas, France; § Plate-forme de cytométrie, Institut Pasteur, Paris, France; Interactions hôte-pathogènes, École Nationale Vétérinaire de Toulouse-Institut National de la Recherche Agronomique, Toulouse, France; || Institute of Virology and Immunoprophylaxis, Mittelhausern, Switzerland; and # Institute for Animal Health, Compton, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Conventional dendritic cells enter lymph nodes by migrating from peripheral tissues via the lymphatic route, whereas plasmacytoid dendritic cells (pDC), also called IFN-producing cells (IPC), are described to gain nodes from blood via the high endothelial venules. We demonstrate here that IPC/pDC migrate in the afferent lymph of two large mammals. In sheep, injection of type A CpG oligodinucleotide (ODN) induced lymph cells to produce type I IFN. Furthermore, low-density lymph cells collected at steady state produced type I IFN after stimulation with type A CpG ODN and enveloped viruses. Sheep lymph IPC were found within a minor BnegCD11cneg subset expressing CD45RB. They presented a plasmacytoid morphology, expressed high levels of TLR-7, TLR-9, and IFN regulatory factor 7 mRNA, induced IFN-{gamma} production in allogeneic CD4pos T cells, and differentiated into dendritic cell-like cells under viral stimulation, thus fulfilling criteria of bona fide pDC. In mini-pig, a CD4posSIRPpos subset in afferent lymph cells, corresponding to pDC homologs, produced type I IFN after type A CpG-ODN triggering. Thus, pDC can link innate and acquired immunity by migrating from tissue to draining node via lymph, similarly to conventional dendritic cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Dendritic cells (DC)4 are professional APCs that collect and process Ags for presentation to naive T cells in lymph nodes. Two types of DC subsets, conventional DC (cDC) and plasmacytoid DC (pDC), have been described in several mammalian species, including mice (1, 2, 3), humans (1, 2, 3), rats (4), and pigs (5). These subsets differ in terms of markers and mechanisms of action. cDC are strategically located in peripheral tissues where they capture Ags, migrate in afferent lymph, process Ags, and educate T cells in lymph nodes. pDC, also called natural IFN-producing cells (IPC), are the most potent cell type for the rapid and strong production of IFN-{alpha} following TLR-7- or TLR-9-mediated interaction with viruses and bacteria (6). Type I IFN affects cDC function, maturation (7), and licensing for cross-priming (8). In addition, pDC directly prime naive CD8pos T cell populations when activated with influenza virus (9), they synergize with cDC through cell contact- dependent interaction for antitumor CD8pos T cell generation (10), and they can also cross-present lipopeptides and HIV Ags from apoptotic cells to specific CD8pos T cells (11). They can also induce regulatory T cells in the prevention of asthma (12) and in the tolerance of allogeneic cardiac grafts (13). Such immunoregulatory and stimulatory functions implicate pDC in the development of many diseases, such as cancer (14), infectious diseases (HIV (15), HCV (16), and autoimmunity (17, 18).

pDC are found in nodes (19), the lung (12), the liver (20), the skin (21), and the blood in low numbers (19). Most reports support that pDC reach lymph nodes from the hematogenous route via high endothelial venules, as do naive T cells (19, 22, 23). In addition, pDC are not detectable in rat intestinal and liver afferent lymph at steady state nor after feeding with resiquimod, a TLR-7 ligand (24). However, when fluorescent Ag was given intratracheally in mice, Ag-bearing pDC could be found both in the lung and in the tracheo-bronchial node (12). Furthermore, inactivated HSV injected intradermally in mice induced IPC in the subcapsular sinus of cervical lymph nodes (25). Both of these latter findings support the view that IPC may also migrate via afferent lymph. If pDC do not follow the tissue-to-lymph node axis but exclusively migrate via blood as generally accepted, the way by which they capture Ags for subsequent presentation is unclear. In this scenario, cDC and pDC would not be recruited from the same sites and they would thus interact in nodes only after some delay, a puzzling process for key cells involved in innate responses.

In this study, we used a surgical model of lymph duct catheterization in large mammals (pig and sheep) to investigate the possibility of pDC migration in skin lymph. Indeed, in these species, collection of huge amounts of afferent skin lymph (100– 200 ml of lymph per 12 h) can be achieved, allowing the sorting of a rare cell subset, what cannot be done in rodents. Sheep lymph duct cannulation is a classical model to study leukocyte migration, including cDC (26, 27). We have developed a novel technique for afferent lymph collection from mini-pig skin to determine whether the observation obtained in sheep can be extended to another large mammal. In this article, we used afferent lymph cannulation in sheep and pigs to demonstrate that pDC do migrate in afferent lymph. In sheep, we found that BnegCD11cnegCD45RBpos IPC, harboring bona fide pDC characteristics, migrate in afferent lymph as a minor cell population (0.7 ± 0.17% lymph cells), along with cDC (3.1 ± 0.7% lymph cells (28)). Furthermore, IPC were also detected in pig lymph and were characterized as SIRPposCD4pos cells, previously described as swine pDC (5). These results, obtained with a direct method for probing afferent lymph, provide further insight into pDC trafficking. Furthermore, modulation of pDC trafficking in lymph may be an important consideration in the development of vaccines and therapeutic strategies.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Surgical procedures

Prealpe female sheep (between 2 and 4 years old) were raised and housed in the Institut National de la Recherche Agronomique animal experimentation unit in Jouy-en-Josas, France. Mini-pigs (10 and 14 mo old) were housed in the Interventional Imaging Research Center (Jouy-en-Josas, France).

Pseudo-afferent prescapular and efferent lymph duct cannulations were performed in sheep as previously described (29). In pigs, the iliac and lombo-aortic nodes were removed and the left iliac lymph duct was catheterized. Lymph was collected twice a day. Thirty sheep and two pigs were used for the experiments described herein. All animal experiments were conducted under the authority of a license issued by the Direction des Services Vétérinaires of Versailles (accreditation numbers 78-93, 78-15, and A78-730).

Antibodies

All of the mAb primarily raised against bovine molecules cross-react with the corresponding sheep molecule. The anti-bovine CD45RB (CC76, IgG1) has been previously described (30). The anti-bovine CD1b (Th97A, IgG2a), bovine CD4 (ILA11, IgG1), bovine CD11c (BAQ153A, IgM), bovine CD14 (CAM36A, IgG1), ovine CD45RA (73B1, IgG1), ovine CD62L (DU1-29, IgG1), porcine CD172a (DH59B, IgG1), and bovine MHC class II (CAT82A, IgG1; TH14B, IgG2a) were bought from Veterinary Medical Research and Development. The anti-ovine CD11c (OM1, IgG1) was given to us by M. Pépin (Agence Française de Sécurité Sanitaire des Aliments, Maisons-Alfort, France), the anti-bovine CD86 (ILA190, IgG1) and anti-bovine CD172a (ILA24, IgG1) by J. Naessens (International Livestock Research Institute, Nairobi, Kenya), the anti-bovine IFN-{alpha} (1D10, IgG1; 1C6, IgG1; polyclonal rabbit IgG) by P. Griebel (Vaccine and Infectious Disease Organization, Saskatoon, Canada), the isotype control IgM (anticoronavirus) by J. Grosclaude (Institut National de la Recherche Agronomique, Jouy en Josas, France), the anti-bovine DEC205 (CC98, IgG2b) by C. Howard (Institute for Animal Health, Compton, U.K.), and the anti-ovine CD4 (ST4, IgG1) and anti-ovine B cell (DU2-104, IgM) by W. Hein (AgResearch, Palmerston North, New Zealand). The DU2-104 mAb labels sheep B cells but the recognized molecule is unknown (31). For clarity, DU2-104pos/neg cells will be called Bpos/neg cells. The anti-bovine IFN-{gamma} (CC330, IgG1 and CC302, Ig2b) and the anti-bovine IL-10 (CC318, IgG2b and CC320, IgG1) were bought from Serotec. The isotype control IgG1 (KP-53) was purchased at Sigma-Aldrich and isotype control IgG2a (NK-1.1) was purchased at BD Biosciences. The polyclonal rabbit serum (IgG fraction) anti-intracellular portion of human CD3 (DakoCytomation) labels sheep CD3pos T cells (32). The polyclonal rabbit serum (IgG fraction) anti-sheep surface Ig was bought from The Jackson Laboratory. The polyclonal rabbit IgG anti-porcine IFN-{alpha} was developed by us (33).

Lymph and blood cells

Afferent lymph was collected overnight in flasks containing 500 IU of heparin, 10,000 IU of penicillin, and 10 mg of streptomycin. Total lymph (TL) cells were enumerated with a hematocytometer and spun down at 700 x g. Low-density lymph (LDL) cells were obtained after centrifugation on a 1.065 density iodixanol gradient (Optiprep; Nycomed Pharma) as previously described (27).

PBMC were isolated by Percoll gradient purification as previously described (34).

In vitro induction of type I IFN

TL, LDL cells, or purified cell subsets (1.5 x 106/ml) were incubated overnight at 37°C in X-vivo 15 medium (BioWhittaker) containing 1% FCS, 100 IU/ml penicillin, and 100 µg/ml streptomycin (culture medium) either alone or with type I IFN inducers. The inducers used were the type A CpG oligonucleotide (ODN) D32-ODN (35), with the sequence ggT GCG TCG ACG CAG ggg gg (lower case letters for phosphorothioate linkages and upper case letters for phosphodiester linkages) (BioSource International), UV-irradiated transmissible gastroenteritis virus (UV-TGEV; stock titrating 2 x 107 PFU/ml before inactivation (36)) or PR8 influenza (stock titrating 109 PFU/ml). After overnight culture, cell supernatants were collected and kept frozen at –20°C for type I IFN titration.

In vivo induction of type I IFN

At least 10 days after surgery, cannulated sheep received 4 mg of CpG ODN in a total volume of 2 ml of endotoxin-free 0.9% NaCl, injected intradermally as 0.25 ml of multispots in the shoulder region with a 30-gauge needle. Lymph was collected before and after CpG ODN administration during specified collection periods, for up to 4 days. Samples were centrifuged at 700 x g. Lymph supernatants were kept frozen for type I IFN titration, and lymph cells were washed, resuspended in culture medium (see above) to a concentration of 107 cells/ml, and cultured overnight at 37°C for evaluation of their ability to secrete type I IFN.

Type I IFN bioassay

Type I IFN in cell supernatants, from lymph or sera, was quantified using a cytopathic effect reduction assay with bovine Madin-Darby bovine kidney cells challenged with vesicular stomatitis virus. An internal IFN-{alpha} reference was included as described elsewhere (37). Each supernatant was tested over eight serial dilutions. Data are expressed as type I IFN units per ml.

IFN{alpha}, IFN-{gamma}, and IL-10 ELISA

IFN-{gamma} was detected using a specific bovine/ovine IFN-{gamma} ELISA. Briefly, Immulon ELISA II plate (Dynatech) microplates were coated with anti-bovine IFN-{gamma} CC330 mAb (5 µg/ml; Serotec) in PBS. After washing, wells were saturated with PBS containing 0.05% Tween 20 and 20% FCS. Test supernatants (undiluted) were then incubated at 37°C for 2 h. After washing, the biotinylated anti-bovine/ovine IFN-{gamma} CC302 mAb (5 µg/ml; Serotec) was incubated for 1 h at room temperature. Following washing, peroxidase-conjugated streptavidin (1/1000; Beckman Coulter) was incubated at 4°C for 1 h. Positive reactions were determined with 2,2-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) in citrate buffer and OD were read at 405 nm. IL-10 detection was similarly performed using the anti-bovine/ovine IL-10 CC318 mAb and biotinylated CC320 mAb (Serotec).

A specific bovine/ovine IFN{alpha} ELISA was used as described by Nichani et al. (38) using the anti-bovine IFN-{alpha} 1C6 and 1D10 mAb and anti-bovine IFN-{alpha} rabbit IgG (provided by P. Griebel). A recombinant ovine IFN-{alpha}1 produced by a baculovirus expression system in SF9 cells was used as an internal standard so that the OD 405 nm values could be transformed in U/ml IFN.

Immunomagnetic cell selection

For isolation of type I IFN-producing cell fractions from lymph, LDL cells were incubated with primary Abs (2 µg/ml), washed in PBS plus 2% FCS, and further incubated with magnetic microbeads coated with goat anti-mouse (GAM) IgG H and L chain (GAM IgG microbeads, 10 µl beads/107 cells, MACS; Miltenyi Biotec); after wash, cells were applied onto a LS column (Miltenyi Biotec). The negative fraction was further processed through a CS column (Miltenyi Biotec). The purity of the resulting positive and negative fractions was controlled by staining with FITC-GAM IgG H and L chain secondary Abs (The Jackson Laboratory) and analyzed with a FACSCalibur using CellQuest software (BD Biosciences).

For isolation of BnegCD11cnegCD45RBpos lymph cells, LDL cells were first depleted of B cells and CD11cpos cells by passage through a CS column and incubated with the anti-CD45RB CC76 mAb followed by GAM IgG microbeads. CD45RB-labeled cells were applied on a LS column to obtain the CD45RBpos fraction. The flow-through fraction was subsequently applied onto a CS column to remove weakly labeled cells, giving the CD45RBneg fraction. The purity of the cell fractions was controlled by FACS after staining with FITC- GAM IgG (see Fig. 4).

For isolation of CD45RBpos cells from blood, PBMC or low-density PBMC were directly incubated with anti-CD45RB CC76 mAb and processed as described above.

For isolation of CD4pos T cells from blood, PBMC were incubated with the anti-CD4 ST4 mAb, washed in PBS containing 1% citrate and 2% FCS, and then incubated with GAM IgG-coated microbeads. After washing, cells were applied to a LS column, eluted, and checked for purity as described above.

Autologous transfer of LDL cells in skin

Autologous LDL cells (108) were labeled with CFSE (Molecular Probes) and were injected intradermally into 10 points (50 µl each) with a 26-gauge needle in the skin area drained by the prescapular lymph. The 16-h lymph collection before injection and the 3- to 22-h lymph collection after injection were processed for isolation of BnegCD11cnegCD45RBpos LDL cells. The purity was checked by labeling the sorted cells with PE-GAM IgG (Caltag Laboratories).

Electron microscopy

Cells were fixed for 1 h in 0.1 M sodium cacodylate buffer (pH 7.4) with 1.5% glutaraldehyde, postfixed in 1% osmium tetroxide, and embedded in Epon after dehydration. Thin sections stained with uranyl acetate and lead citrate were observed under an EMC12 Philips transmission electron microscope at 80 kV.

Immunophenotyping analysis of sheep blood and lymph IPC by flow cytometry

Low-density cells from lymph and blood were harvested from three sheep, mixed (40 x 106 cells in total), and preincubated in FACS medium (RPMI 1640 containing 4% horse serum) for 15 min on ice. The anti-CD45RB CC76 mAb and the phenotyping mAb are all IgG1, requiring a multistep labeling procedure. Cells were first labeled at 4°C with the anti-CD45RB mAb (hybridoma supernatant) followed by a saturating concentration (50 µg/ml) of FITC-donkey Fab anti-mouse IgG (The Jackson Laboratory). After two washes with FACS medium, cells were incubated with the second IgG1 primary Ab at 2 µg/ml (i.e., anti-CD45RA (73B1), or CD11c (OM1), or CD62L (DU1-29), or CD4 (ST4), or MHC class II (CAT82A), or CD86 (ILA190) or IgG1 isotype control). To exclude cDC and B cells, cells were further labeled with anti-CD11c (BAQ153A) or anti-CD1b (TH97A) (which was used in the case of CD11c analysis on CD45RBpos cells) and with anti-B cell mAb (DU2-104). Cells were then washed twice and incubated with a 1/200 dilution of PE-GAM IgM or PE-GAM IgG2a and tricolor-GAM IgG1 (Caltag Laboratories). After two washes, cells were treated with the Fix and Perm kit according to the manufacturer’s instructions (Caltag Laboratories) and intracellularly labeled with anti-CD3 rabbit IgG (1 µg/ml) that was revealed with biotinylated goat anti-rabbit IgG (1/ 200) followed by allophycocyanin-streptavidin (1/500; Caltag Laboratories). Irrelevant murine IgG1, murine IgM, and rabbit polyclonal IgG were used as negative controls.

Detection of pig lymph IPC by flow cytometry

Pig LDL cells (1.5 x 106/ml cells) were stimulated overnight with 20 µg/ml CpG in culture medium at 37°C. Cells were then processed for surface staining with the anti- bovine CD4 (ILA11) mAb that cross-reacts with pig CD4 (www.vmrd.com) and the anti-SIRP (DH59B) mAb followed by PE-GAM IgG2a and tricolor-GAM IgG1. They were subsequently permeabilized with the Fix & Perm kit (Caltag Laboratories). Intracellular porcine IFN-{alpha} was revealed using specific anti-swine IFN-{alpha} rabbit-purified IgG (36) followed by FITC-goat anti-rabbit IgG. The intracellular staining was controlled with purified IgG from a nonimmunized rabbit.

RNA isolation and real time RT-PCR

Total RNA was extracted using the Arcturus Pico-pure Kit. RNA concentration and quality were evaluated with Agilent RNA 6000 LabChips. All RNA preparation had a RNA integrity number above nine. RNA (100 ng) was reverse transcribed using the Multiscribe reverse transcriptase and random primers (Applied Biosystems). Quantitative real-time PCR was done in duplicate using 10 ng of cDNA with 300 nM primers in a final reaction volume of 25 µl of 1x SYBR Green PCR Master Mix (Applied Biosystems). The primers used for ovine GAPDH (39), TLR-7, and TLR-9 mRNA (40) quantification were published previously. The primers for ovine IFN regulatory factor 7 (IRF-7) were forward TGCCCCGGGACTGTGA and reverse CCCGGAACTCCACCAGTTCT. PCR cycling conditions were 95°C for 10 min, linked to 40 cycles of 95°C for 15 s and 60°C for 1 min. Real-time PCR data were collected by the Applied Biosystems 7900HT Sequence Detection System and 2–{Delta}{Delta}CT calculations for the relative expression of the different genes (arbitrary units) were performed with SDS 2.1 software (Applied Biosystems).

Mixed lymphocyte reaction

CD11cpos and BnegCD11cnegCD45RBpos cells were selected with immunomagnetic beads from LDL cells and were irradiated (3000 rad). Irradiated cells were plated in 96-well plates in culture medium at different ratios with 3 x 105 allogeneic CD4pos T cells in triplicates. After 72 h, 100 µl of supernatant was harvested and frozen at –70°C for IFN-{gamma} and IL-10 ELISA testing and 100 µl of fresh medium was added. After 96 h, the cultures were pulsed for 18 h with 1 µCi of [3H]thymidine.

Induction of pDC differentiation

Isolated BnegCD11cnegCD45RBpos (1.5 x 106/ml) LDL cells were plated alone or with 1.33 PFU/cell UV-TGEV in culture medium for 72 h. They were subsequently observed by contrast-phase microscopy and processed for labeling with anti-MHC class II (TH14B) and CD86 (ILA190) mAb followed by FITC-GAM IgG. Cells were analyzed by gating on live cells by exclusion of 7-aminoactinomycin D staining.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Production of type I IFN by sheep afferent lymph cells in response to type A CpG ODN in vivo

Type A unmethylated CpG ODN induces huge production of type I IFN by IPC in many species via TLR9 engagement (4, 41, 42) and stimulates type I IFN production in sheep PBMC (43). To determine whether IPC migrate in sheep afferent skin lymph, type A CpG ODN (4 mg) were injected intradermally and skin-draining afferent lymph was sequentially collected. Production of type I IFN was detected in lymph fluid early after injection; it lasted for 2–3 days and reached maximal levels (100–350 U/ml) 5–20 h after in vivo stimulation (Fig. 1A).


Figure 1
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  		FIGURE 1. Intradermal injection of CpG ODN induced type I IFN production in sheep afferent lymph cells. A, CpG ODN (4 mg) was injected intradermally into four sheep and type I IFN was measured (U/ml) from skin afferent lymph collected at different times after injection. Data from two of the four sheep are represented. B, Injection of CpG induced type I IFN production in migrating lymph cells. Lymph cells from two sheep were collected at different time points after CpG ODN injection, washed, and cultured overnight (107 cells/ml). The supernatants were collected and type I IFN was measured by bioassay (U/ml).

 
To better assess whether type I IFN can be produced by cells migrating in lymph, skin afferent lymph was sequentially collected from two sheep injected with CpG ODN and lymph cells were examined for type I IFN production. Type I IFN was detected in overnight lymph cell culture supernatants of both sheep (Fig. 1B). The highest level of type I IFN was produced by lymph cells collected within 4 h after injection, whereas cells collected before CpG ODN administration did not secrete any detectable quantity. These data showed that type I IPC were migrating in afferent skin lymph after CpG ODN injection.

In vitro induction of type I IFN synthesis in sheep lymph cells by type A CpG ODN and viruses

We then determined whether afferent skin lymph cells that migrate at steady state were able to produce type I IFN after in vitro stimulation with either type A CpG ODN, UV-TGEV, coronavirus, or influenza virus. Skin afferent lymph cells from two sheep (TL cells) were stimulated with D32 CpG ODN (42, 44), UV-TGEV, coronavirus (36), or live PR8 influenza virus (45) (Fig. 2A). Whatever the stimulus, low amounts of type I IFN synthesis were induced in TL cells (Fig. 2A). To investigate whether this type I IFN production was the fact of a lymph cell subset, LDL were obtained and were cultured with these same stimuli (Fig. 2A). As compared with what was obtained with TL cells, higher amounts of type I IFN were found with LDL cells, reaching 250 and 800 U/ml in the two different sheep, respectively. Important variations in type I IFN response were observed between outbred sheep, but all of the tested LDL cells (20 sheep in total) displayed type I IFN response after in vitro stimulation with CpG ODN (data not shown). These intersheep variations in type I IFN response might be attributable to individual genetic characteristics, such as single nucleotide polymorphism in the TLR or in the signaling molecules of the TLR cascade (46). Interestingly, when a same sheep was cannulated for afferent and efferent lymph collection on contralateral sides, LDL cells collected from efferent lymph and cultured with CpG did not produce any detectable IFN, whereas LDL cells from afferent lymph produced 500 UI/ml type I IFN (Fig. 2B).


Figure 2
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  		FIGURE 2. CpG ODN, UV-TGEV, and PR8 influenza virus triggered type I IFN production in afferent lymph cell cultures. A, Type I IFN was measured following overnight in vitro stimulation of TL and LDL cells using CpG ODN, UV-TGEV virus, and PR8 influenza. The experiment was done using lymph cells from two different sheep. Type I IFN was measured by bioassay (U/ml). B, Afferent and efferent lymph LDL cells from the same sheep were stimulated with 20 µg/ml CpG and type I IFN was measured by bioassay.

 
Altogether the results show that IPC that produce type I IFN after activation with influenza, coronavirus, or CpG ODN are migrating in afferent skin lymph in sheep and not in efferent lymph.

Sheep IPC were not found among cDC, monocyte-macrophages, and Bpos LDL cells and were CD45RBpos cells

Since no Ab is available to stain for intercellular sheep IFN-{alpha}, the phenotype of the sheep lymph IPC was determined by cell separation experiments. We used immunomagnetic bead cell selection of LDL cells to determine the IPC phenotype collected from different sheep. The type I IFN bioactivity was found in the DEC205negCD11cneg fractions, showing that IPC were not included among the cDC subset (Fig. 3A). In addition, IPC were not detected within the B or the minor CD14pos subset (Fig. 3A). The sorting experiments were performed on different outbred sheep that showed very variable levels of type I IFN responses to CpG.


Figure 3
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  		FIGURE 3. Sheep lymph IPC are BnegCD11cnegCD14neg and CD45RBpos cells. A, The CD11cpos/neg, DEC205pos/neg, Bpos/neg, and CD14pos/neg fractions from LDL cells originating from different cannulated sheep were selected by indirect coupling to GAM-IgG-coated magnetic beads using specific primary mAb directed against CD11c (BAQ153A), DEC205 (CC98), B marker (DU2-104), or CD14 (CAM36). The purity of the positively and negatively selected populations used in the assays was >80% and varied with markers (>95% for CD11c, >80% for DEC205, >92% for B marker, and >84% for CD14). Blood CD45RBpos/neg cell fractions were selected using the anti-CD45RB mAb (CC76). The positive fraction was recovered with an LS-MACS column (>85% purity). The negative cells were recovered by passing the flow-through fraction into a CS-MACS column (>85% purity). For separating CD45RBpos/neg lymph cells, LDL cells were depleted of CD11cpos and B cells by indirect coupling to GAM-IgG- coated magnetic beads using the anti-CD11c (BAQ153A) and B mAb (DU2-104) followed by CS-MACS column. The depleted fraction was further separated in CD45RBpos cells with LS-MACS selection using indirect coupling to GAM-IgG- coated magnetic beads with the anti-CD45RB (CC76) mAb. The flow-through of the LS-MACS column was passed through a CS-MACS to obtain the CD45RBneg cells. The purity of the CD45RBpos and CD45RBneg fractions was >90% after FACS control (Fig. 4A). For type I IFN induction, the positive and negative cell fractions from different cell selection experiments (1.5 x 106/ml, black diamonds; 5 x 105/ml, gray diamonds) were cultured with 20 µg/ml CpG for 18 h, and the supernatants were analyzed for type I IFN with a bioassay. B, Type I IFN production of BnegCD11cnegCD45RBpos cells induced by CpG ODN (one sheep, three cultures at different cell dilutions, purity of selected cells >90%) and tested with a bioassay ({blacksquare}, duplicated wells) and a specific IFN-{alpha} ELISA ({square}, mean of duplicated wells ± SEM). C, BnegCD11cnegCD45RBpos and BnegCD11cnegCD45RBneg (1.5 x 106/ml, purity of selected cells >90%) were cultured overnight with UV-TGEV (1.3 PFU/cell) or PR8 influenza (6.6 PFU/cell) and the supernatants were assayed for type I IFN using a bioassay (duplicated wells). Results are representative of one of three sheep.

 
In the next experiments, LDL cells depleted of CD11cpos and Bpos cells were used to refine the sheep IPC phenotype. Because no Ab to surface CD3 was available in the sheep at the time of the study, depletion of CD3pos T cells could not be performed. Consistent to what is observed in mice (47), elevated concentrations of type I IFN (25,000, 35,000, and 48,000 IU/ml with 1.5 x 106 cells/ml from three different sheep) were detected in the culture supernatant of the CD45RBpos fractions (>90% purity; Fig. 4A) and none of the CD45RBneg fraction (>90% purity; Fig. 4A) obtained from BnegCD11cneg LDL cells (Fig. 3A). Furthermore, IPC from blood were also found in a CD45RBpos subset in sheep (Fig. 3A). Thus, selection with an anti-CD45RB mAb resulted in a strong enrichment in IPC. Because T cells could not be depleted with specific mAbs, we labeled the MACS-selected cells with a rabbit IgG anti-intracellular motif of human CD3. CD3pos T cells were the major cell type among BnegCD11cnegCD45RBneg cells (65%), whereas they were rare in the BnegCD11cnegCD45RBpos fraction (~6%; Fig. 4B). Surface Ig could not be found on the selected subsets, consistent with the efficient depletion of B cells using the DU2-104 mAb (Fig. 4C). Given the relative paucity of these cells in lymph (see below), the recovery of CD45RBpos cells from LDL cells was ~1–5 x 105 cells per overnight collection.


Figure 4
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  		FIGURE 4. The purity of the selected CD45RBpos and CD45RBneg sheep LDL cells. A, MACS-selected BnegCD11cnegCD45RBpos and BnegCD11cnegCD45RBneg LDL cells were incubated with FITC- GAM IgG. B, MACS-selected BnegCD11cnegCD45RBpos and BnegCD11cnegCD45RBneg LDL cells were permeabilized and stained with rabbit anti-CD3 and rabbit control IgG (control). C, MACS-selected BnegCD11cnegCD45RBpos and BnegCD11cnegCD45RBneg LDL cells were incubated at 37°C for 1 h to promote internalization of potential FcR-bound IgG. Cells were stained with FITC-rabbit IgG directed to sheep H and L chains for detection of surface Ig and with FITC-rabbit IgG to mouse H and L chains (control).

 
To establish that type I IFN produced by the BnegCD11cnegCD45RBpos cells included IFN-{alpha}, we processed CpG ODN-induced cell supernatants in a bovine/ovine IFN-{alpha} ELISA using recombinant ovine IFN-{alpha}1 as a reference. We found that CpG ODN-stimulated CD45RBpos cells produced large amounts of IFN-{alpha} (Fig. 3B); the IFN-{alpha} ELISA may be specific of some certain IFN-{alpha} isoforms, resulting in lower values obtained in ELISA than in the bioassay.

BnegCD11cnegCD45RBpos LDL cells were directly activated with influenza and UV-TGEV. Both viruses induced type I IFN in BnegCD11cnegCD45RBpos LDL cells, whereas the BnegCD11cnegCD45RBneg lymphocyte fraction was unresponsive (Fig. 3C).

Thus, the phenotype of sheep IPC can be defined as BnegCD11cnegCD14neg and CD45RBpos. CD45RBpos cells isolated from CD11cneg and Bneg LDL cells will be called IPC-CD45RBpos cells in the rest of the study.

IPC-CD45RBpos cells deposited in skin migrated in afferent lymph

To further document that IPC-CD45RBpos cells have the capability to migrate from skin to lymph duct, we injected autologous CFSE-labeled LDL cells (108 cells, containing ~2 x 106 IPC-CD45RBpos cells) in the dermis of the prescapular zone. We discarded the first 3 h of lymph collection to eliminate the CFSE-positive cells that may have passively transferred into lymph due to the pressure of injection; indeed, we previously showed that the passive translocation of particles ends up within 1 h after injection (48). Whereas no fluorescent cells were detectable among the BnegCD11cnegCD45RBpos LDL cells collected before injection, 1.5% of the BnegCD11cnegCD45RBpos LDL cells were CFSE positive after 22 h after transfer (Fig. 5), representing ~0.2% of the injected IPC-CD45RBpos cells. This rate of migration corresponds to the rate of CD8neg DC migration over 22 h after s.c. injection in mice (49). This experiment indicates directly in vivo that IPC-CD45RBpos cells can translocate from skin to lymph.


Figure 5
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  		FIGURE 5. Sheep IPC-CD45RBpos LDL cells translocate from skin to lymph. Autologous CFSE-labeled LDL (108) were injected intradermally. BnegCD11cnegCD45RBpos were selected from LDL cells collected before injection and 3–22 h after injection and then analyzed by FACS.

 
The afferent lymph sheep IPC-CD45RBpos subset showed a plasmacytoid morphology and high levels of TLR-7, TLR-9, and IRF-7 mRNA expression

The skin lymph IPC-CD45RBpos subset was isolated from CD11cnegBneg LDL cells as described above (Fig. 3). When examined under transmission electron microscopy (Fig. 6A), most IPC-CD45RBpos cells exhibited a plasmacytoid morphology, with an excentered nucleus and well-developed endoplasmic reticulum (Fig. 6A, left). In comparison, lymph cDC (CD11cpos) presented a typical DC morphology with extensive dendritic formations (Fig. 6A, right).


Figure 6
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  		FIGURE 6. The sheep IPC-CD45RBpos subset harbored typical features of pDC. A, IPC-CD45RBpos cells (>90% purity) were selected from LDL cells with immunomagnetic beads after depletion of CD11cpos and Bpos cells (left panel) and CD11cpos classical DC (>95% purity) were positively selected from LDL cells using immunomagnetic sorting (right panel). Cells were pelleted and processed for transmission electron microscopy. Bar, 1 µm. B and C, TLR-7 and -9 and IRF-7 mRNA levels in CD11cpos and IPC-CD45RBpos cells. CD11cpos cells (CD11c+, >95% purity), IPC-CD45RBpos (CD45RB+, >90% purity), and BnegCD11cnegCD45RBneg (CD45RB–, >85% purity) cells were selected from different LDL preparations of afferent lymph or of blood (B, triplicates are from independent collections but two come from the same animal; C, triplicates are from different animals). TLR-7, TLR-9, IRF-7, and GAPDH mRNA expression was determined for each lymph cell collection by real-time quantitative RT-PCR using cDNA duplicates. B, The {Delta}{Delta}CT method was used with a BnegCD11cnegCD45RBneg sample as a calibrator and GAPDH as an endogenous control. C, The {Delta}{Delta}CT method was used with a CD11cpos sample as a calibrator ({circ}) and GAPDH as endogenous control. Arbitrary units are reported. Significant differences between groups (Student’s t test) are indicated (*, p < 0.05; **, p < 0.01).

 
TLR-7 and -9 are important receptors linking virus signaling to type I IFN induction in human and mouse IPC/pDC. Expression of TLR-7 and -9 mRNA was analyzed by real-time RT-PCR in the IPC-CD45RBpos, CD11cpos, and BnegCD11cnegCD45RBneg fractions as a relative control (essentially T lymphocytes; Fig. 4B). Fig. 6B shows that TLR-7 and TLR-9 mRNA were expressed at higher levels in IPC-CD45RBpos cells than in the BnegCD11cnegCD45RBneg control fraction (12- and 4-fold, respectively; p < 0.01, Student’s t test) and than in CD11cpos (30- and 6-fold, respectively; p < 0.01). Thus, TLR-7 and -9 mRNA are clearly more abundant in sheep lymph IPC/pDC than in cDC.

The hallmark of pDC/IPC in mice and humans is the constitutive expression of IRF-7 that allows rapid production of type I IFN even in the absence of positive feedback signaling via the type I IFN receptor. The average IRF-7 mRNA level measured in the IPC-containing CD45RBpos subsets from three different animals at steady state was 7.5-fold higher than that in CD11cpos cells (p < 0.05; Fig. 6C). Furthermore, the IRF-7 mRNA mean level was also found 15-fold higher in blood IPC-CD45RBpos cells than in CD11cpos cells; however, differences in IRF-7 mRNA levels between lymph and blood IPC-CD45RBpos cells from different animals were not statistically different.

Thus, IPC/pDC from blood and lymph have high basal levels of IRF-7 mRNA.

Sheep afferent lymph and blood IPC-CD45RBpos cells share similar phenotypes

To investigate whether the phenotypes of blood IPC-CD45RBpos cells differed from those of the lymph compartment, the IPC-CD45RBpos were analyzed for the expression of several cell surface molecules. The CD45RBpos cells were gated after exclusion of the BposcDCpos and CD3pos cells from low-density cells (Fig. 7) or from total cells (Table I). Using this method, IPC-CD45RBpos cells represented 0.7 ± 0.17% of skin lymph cells (four sheep; Table I) and they represented 0.15% of PBMC (2 sheep; data not shown). The ratio of IPC-CD45RBpos cells to erythrocytes was >104-fold higher in lymph than in blood, strongly arguing against the possibility that IPC-CD45RBpos cells in lymph could result from blood contamination. Phenotypic analysis for lymph and blood cells was performed on low-density cells to analyze enough cell numbers. There was no clear difference in CD4, CD62L, CD11c, CD45RA, MHC class II, and CD86 expression between sheep afferent lymph and blood IPC-CD45RBpos cells (Fig. 7). CD4 was found expressed on 24% lymph and 15% blood IPC-CD45RBpos cells and CD62L was found expressed on 62% lymph and 70% blood IPC-CD45RBpos cells, respectively. CD11c, CD45RA, MHC class II, and CD86 were expressed at very low levels (Fig. 7). Consistent with these staining, we found that type I IFN was induced by CpG ODN both in the CD4pos and CD4neg LDL cell fractions (data not shown), and it was only induced in the CD45RAneg (data not shown) and CD11cneg selected cells (Fig. 3A). Thus, a significant proportion of sheep blood and lymph IPC-CD45RBpos cells expresses surface CD4 and CD62L and little to no CD11c, CD45RA, MHC class II, and CD86. The blood IPC phenotype established with available markers was not modified by transmigration of the cells into the afferent lymph compartment.


Figure 7
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  		FIGURE 7. Sheep lymph and blood IPC-CD45RBpos cells expressed similar phenotypes. A, LDL cells were stained with anti-B cell (DU2-104), anti-CD11c (BAQ153A) or anti-CD1b (TH97A), anti-CD3 (rabbit polyclonal IgG, intracellular staining), and anti-CD45RB (CC76) as well as with specific Abs to CD45RA (73B1), CD4 (ST4), CD62L (DU1-29), CD11c (OM1), MHC class II (CAT82A), or CD86 (ILA190) and analyzed in multicolor flow cytometry. An electronic gate excluding cDCpos, Bpos, and CD3pos cells was used to analyze the expression of CD45RA, CD4, CD11c, and CD62L relative to an IgG1 isotype control. Exclusion of cDC was generally performed with anti-CD11c (BAQ153A) except for the analysis of CD11c expression on IPC that required the use of anti-CD1b (TH97A). B, Low-density blood cells were analyzed as in A. The experiment was conducted with two different pools of cell collections from three animals and gave similar results.

 

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  		Table I. IPC-CD45RBpos cell representation in lymph

 
The lymph IPC-CD45RBpos cell subset stimulated IFN-{gamma} production in allogeneic CD4pos T cells

Fresh lymph IPC-CD45RBpos cells were used as stimulator cells in direct allogeneic MLR, and their activation capacity was compared with that of CD11cpos cells. As shown in Fig. 8A, freshly isolated IPC-CD45RBpos cells stimulated resting CD4pos T cell proliferation, although much less efficiently than CD11cpos cells. We also tested the capacity of sheep lymph IPC-CD45RBpos to drive an IFN-{gamma} response in allogeneic resting CD4pos T cells. IFN-{gamma} production was measured in the culture supernatant of the allogeneic stimulation after 3 days. IPC-CD45RBpos cells stimulated a weaker production of IFN-{gamma} than lymph CD11cpos cells (Fig. 8B). We did not detect IL-10 in the supernatants of IPC-CD45RBpos and CD4pos T cell cocultures or in the supernatants of IPC-CD45RBpos cells cultured alone or with LPS (20 µg/ml; data not shown). Thus, sheep lymph IPC promoted allogeneic T cell proliferation and IFN-{gamma} response, as do other species of IPC/pDC (45, 50), indicating that sheep lymph IPC have T cell stimulatory properties.


Figure 8
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  		FIGURE 8. Lymph IPC-CD45RBpos cells displayed Ag presentation abilities and matured into DC-like cells following coronavirus stimulation. A, CD4pos T cells (>97% purity) were cultured with allogeneic irradiated IPC-CD45RBpos LDL (>85% purity) or CD11cpos cells (>95% purity) at a 10:1, 100:1, or 1000:1 ratio (CD4:APC ratio) for 96 h and pulsed with [3H]thymidine for 18 h. Stimulation indexes (SI; mean and SD from duplicated wells) are reported on the y axis. The star indicates significant differences in [3H]thymidine incorporation relative to CD4 T cells alone (p < 0.05; Student’s t test). One experiment is shown of two that gave similar results. B, Allogeneic CD4pos T cells were cultured with IPC-CD45RBpos LDL and CD11cpos cells at a 10:1, 100:1, or 1000:1 ratio (CD4:APC ratio) for 72 h. The supernatants were tested for IFN-{gamma} by ELISA (OD at 405 nm on the y axis). The dashed line indicate OD signal with CD4pos T cells alone. Two independent experiments gave similar results. C, IPC-CD45RBpos LDL cells (>90% purity) were observed under contrast-phase microscopy before culture and after 72 h of culture with 1.3 PFU/cell UV-TGEV. Bar, 1 µm. The micrographs were taken with a Leica Leitz DMBR microscope, x63/1.32-0.6, Olympus DP50 camera, using the View Finder Lite 1.0 software. D, IPC-CD45RBpos LDL cells (>90% purity) cultured alone (control) or with UV-TGEV were analyzed for MHC class II (CAT82A) and CD86 (ILA190) expression on the live cells (7-aminoactinomycin D exclusion).

 
Lymph IPC-CD45RBpos cells mature into DC-like cells following coronavirus stimulation

The IPC-CD45RBpos selected cells cultured for 72 h with UV-TGEV increased in size and developed extensive dendrite formations (Fig. 8C). This change in morphology was also observed after stimulation with PR8 influenza but not with CpG ODN (data not shown). In addition, >50% of the IPC-CD45RBpos selected LDL cells were dead after 72 h of culture without stimulation, whereas the majority (>80%) of the IPC-CD45RBpos cells survived after 72 h of culture with UV-TGEV or influenza (data not shown). MHC class II and CD86 cell surface expression levels were higher on cells cultured with UV-TGEV than on IPC-CD45RBpos LDL cells cultured alone (Fig. 8D), which however appeared lower than what we described previously on sheep lymph cDC (28). These results show that the lymph IPC-CD45RBpos subset can differentiate into DC-like cells following maturation with virus.

pDC migrate in pig afferent lymph

To assess whether the presence of IPC/pDC in afferent lymph was linked to the ovine species or whether IPC/pDC from other large mammals behaved similarly, we developed a new model of lymph duct cannulation in out bred mini-pigs. Afferent skin pig lymph cells were enriched in low-density cells on an Optiprep cushion and cultured overnight with CpG ODN. Type I IFN was detected in the supernatant (Fig. 9A). Furthermore, cells positive for IFN-{alpha} staining (1% of the LDL cell fraction) were detected in CD4posSIRPpos cells, consistent with the swine pDC phenotype (5) (Fig. 9, B–D). This result provides further evidence that IPC/pDC cells do migrate in afferent lymph in large mammals.


Figure 9
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  		FIGURE 9. IFN-{alpha} production in pig afferent lymph IPC/pDC. A, TL and LDL cells (1.5 x 106/ml cell) from two pigs were stimulated with 20 µg/ml CpG and the supernatant was analyzed for type I IFN in a bioassay. B–D, Pig LDL cells were cultured for 18 h with 20 µg/ml CpG, surface-stained with anti SIRP (DH59B) and anti-CD4 (ILA-11) mAb, and intracellular IFN-{alpha} was detected with anti-IFN-{alpha} polyclonal rabbit IgG. B, The isotype control for IFN-{alpha} detection is shown (FL-1/FL-4 plot). C, IFN-{alpha}-expressing cells (1% LDL cells, FL-1/FL-4 plot) are gated in R1. D, R1-positive cells were analyzed for SIRP and CD4 expression and 92% IFN-{alpha}pos cells were expressing both markers.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We show herein that cells presenting phenotypic and functional features of bona fide IPC/pDC migrate in afferent lymph in two large mammal species, sheep and pigs. We showed in sheep that CpG ODN injection elicits type I IFN production in lymph and that it triggers lymph IPC/pDC to produce type I IFN. The quantity of type I IFN (100–350 U/ml) detected in the sheep lymph in our study has been shown by others to be sufficient to activate cross-priming in cDC (51). In addition, early pDC-cDC interaction might possibly license cDC for Ag presentation before they interact with naive T cells. Finally, this migration pathway enables pDC to optimally sense invading pathogen and to capture Ags at peripheral sites for subsequent Ag presentation in lymph nodes. Altogether our data indicate that pDC can follow the tissue-to-lymph node axis via lymph vessels, a routing that contributes to their role in bridging innate to acquired immunity.

IPC/pDC migration in lymph is demonstrated using two different mammal species, ruling out the hypothesis that the phenomenon might be a species singularity. Under basal situations, IPC/pDC representation in lymph cells (0.7% TL) is around 5-fold lower than that of cDC (28) and is in the same order of magnitude in PBMC. This suggests that afferent lymph is a major route for cDC migration and that IPC/pDC can also follow this path but to a lesser extent. However, in mice, it is accepted that pDC reach lymph nodes via high endothelial venule. Indeed in L-selectin-deficient mice, pDC numbers were reduced by 85% in lymph nodes (52), suggesting pDC extravasation via high endothelial venule. However, mouse pDC exposure to HSV up-regulated a CCR7-dependent migration in vitro; in addition, pDC were occasionally found close to lymph vessels in HSV-induced lesions, suggesting that a CCR-7- mediated lymph migration may occur (53). Expression of CCR7 has also been demonstrated on human pDC (54) and it could be involved in pDC trafficking in lymph due to CCL19/CCL21 production by lymphatic endothelial cells (55). Nonetheless, in inflammatory conditions, pDC recruitment is clearly occurring from blood via high endothelial venules in mice (19, 22, 23) and in humans (47, 54), involving L-and E-selectin-mediated adhesion (19, 22), C3a- and C5a-mediated chemotaxis (56) and engagement of CCR5 (19), CCR7 (44), and CXCR3 associated to CXCR4 (57). Thus, pDC recruitment in lymph nodes probably occurs via two nonexclusive routes, blood and lymph. It is possible that species differences and hygiene status (conventional for sheep and specific pathogen free for rodents) modulate the relative contribution of lymph and blood in pDC recruitment in nodes.

Our data are not in accordance with Yrlid et al. (24), who could not detect IPC in rat mesenteric afferent lymph. This discrepancy may be due to differences in the methodologies. Indeed, Yrlid et al. (24) used FACS sorting to purify pDC before exposure to CpG ODN, and we found that this sorting procedure was detrimental to type I IFN synthesis on sheep lymph cells, possibly due to a peculiar sensitivity of sheep pDC to the flow pressure. Indeed, the simple passage of sheep LDL and low-density blood cells in the flow reduced their capacity to produce type I IFN by 85% (data not shown). Yrlid et al. (24) also used ~10 times fewer cells than us per culture condition and they did not enriched for LDL cells, what could preclude detectability of type I IFN production. Finally, it is possible that IPC/pDC are less abundant in mesenteric lymph. Actually, IPC/pDC frequency was ~5 times lower in sheep oronasal than in skin lymph (data not shown), suggesting that IPC/pDC migration in lymph may differ between tissue types. Finally, although characteristics of cDC subsets and T cell migration in lymph are largely similar among rats, sheep, and pigs (28, 58, 59), it remains possible that IPC/pDC migration in the rat differs from that in large mammals. Taken altogether, the combination of these various technical and biological factors may explain why IPC could not be detected in rat lymph.

We did not detect increased pDC migration in lymph following injection of CpG or the sheep dsRNA bluetongue virus. CpG injection induced type I IFN in lymph but did not induce measurable IPC/pDC nor cDC influx in afferent lymph (B. Hemati, F. Pascale, and I. Schwartz-Cornil, unpublished data). In addition, whereas cDC were potently recruited in lymph following intradermal injection of attenuated bluetongue virus, IPC/pDC migration was unmodified (manuscript in preparation). These observations demonstrate that pDC and cDC migration does not involve the same molecular pathways and that type I IFN induction is not coupled to IPC/pDC recruitment in lymph.

pDC are key cells at the interface of innate and acquired immunity. Their role has been underlined in several immunopathological disorders (17, 18), allograft tolerance (13), infectious diseases (15), and cancers (14). How pDC patrol tissues, accumulate, and reach decisional lymph node is crucial to be considered and understood to target and manipulate these powerful cells for developing novel vaccine strategies and therapeutic approaches.


   Acknowledgments
 
We are very grateful to Dominique Buzoni-Gatel, Artur Summerfield, Melissa Nicholson, and Agnès Petit-Camurdan for great help in improving this manuscript. We thank the technicians of the Animal Experimentation Unit for maintaining the animals and the technicians of Interventional Imaging Center for involvement in organizing the surgery. We are grateful to Philip Griebel for providing the Abs for IFN-{alpha} detection. We thank Catherine Dubuquoy for technical assistance with the IFN-{alpha} ELISA, C. Devaureix for preparing vesicular stomatitis virus, Behzad Hemati for providing lymph cells, and J. J. Leplat for cell irradiation.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 F.P. performed the largest part of research, analyzed data, and prepared figures. V.C. performed research, analyzed data, prepared figures, and wrote small sections. M.B. performed surgery. A.C. performed initial research. S.C., C.B., N.B., and A.-M.B. contributed to analytical tools. M.E., V.N., J.H., E.F., B.R., L.G.-P., and S.R. provided critical reagents. B.C. designed initial research, analyzed data, and supervised experiments. I.S.-C. designed research, analyzed data, supervised experiments, and drafted the manuscript and figures. Back

2 B.C. and I.S.-C. share senior authorship. Back

3 Address correspondence and reprint requests to Dr. Isabelle Schwartz-Cornil, Unité de Virologie et Immunologie Moléculaires, UR892 Institut National de la Recherche Agronomique, Domaine de Vilvert, 78352 Jouy-en-Josas Cedex, France. E-mail address: isabelle.schwartz{at}jouy.inra.fr Back

4 Abbreviations used in this paper: DC, dendritic cell; TL, total lymph; LDL, low-density lymph; IPC, IFN-producing cells; ODN, oligodinucleotide; UV-TGEV, UV-irradiated transmissible gastroenteritis virus; cDC, conventional DC; pDC, plasmacytoid DC; GAM, goat anti-mouse; CT, cycle threshold. Back

Received for publication November 19, 2007. Accepted for publication March 1, 2008.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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