P

Na

b

c

a

ARRAA

KDRAM

1

ottTaplf[asjDna

lo

PS

0d

Vaccine 29 (2011) 3655–3661

Contents lists available at ScienceDirect

Vaccine

journa l homepage: www.e lsev ier .com/ locate /vacc ine

henotypic localization of distinct DC subsets in mouse Peyer Patch

icolas Rochereaua, Bernard Verrierb, Jean-Jacques Pinc, Christian Genina, Stéphane Paula,∗

GIMAP, EA3064, Faculté de médecine, Universités de Lyon, 42023 Saint-Etienne, FranceFRE 3310 CNRS/UCBL, IBCP, 69000 Lyon, FranceDENDRITICS SAS, Bioparc Laennec, 69008 Lyon, France

r t i c l e i n f o

rticle history:eceived 20 September 2010eceived in revised form 25 February 2011ccepted 5 March 2011vailable online 23 March 2011

a b s t r a c t

Peyer’s patch have been extensively studied as a major inductive site for mucosal immunity within thesmall intestine. The intestinal mucosa contains numerous dendritic cells, which induce either protec-tive immunity to infectious agents or tolerance to innocuous antigens, including food and commensalbacteria. Although during the past few years, several subsets of human mucosal dendritic cells havebeen described, a precise characterization of the different mouse mucosal dendritic cells subpopulations

eywords:endritic cellsodentntibodiesucosa

remains to be achieved with regard to their phenotype and localization in Peyer’s patch. In this report, wehave investigated by immunofluorescence on cryosection and by flow cytometry, the phenotype and thelocalization of dendritic cells into Peyer’s patch of C57Bl/6 mouse intestine using dendritic cells markers.Positive and double staining for CD11c and BDCA-2, pDC/IPC, DC-LAMP, DC-SIGN, TLR8 and Langerinhave been observed revealing new mouse intestinal DC subsets. This study provides new insight in theunderstanding of mucosal immune responses induced by natural processes as infections but also new

ation

perspectives for the evalu

. Introduction

The mucosal surfaces of the gastrointestinal tract are continu-usly exposed to a large number of environmental antigens. Thus,he gut-associated lymphoid tissues (GALT) play a central role inhe induction of oral tolerance and immunity to infectious agents.he GALT is composed by an inductor site (Peyer Patches (PP)nd Isolated Lymphoid Follicles) and an effector site (Lamina Pro-ria). In PP, dendritic cells (DCs) are separated from the intestinal

umen by the follicle-associated epithelium (FAE). It allows trans-er of pathogens in lymphoid tissue through microfold (M) cells1]. Antigen is then captured by DCs, causing their maturationnd their migration to the intrafollicular areas [2]. DCs can alsoample antigen directly from the intestinal lumen by forming tight-

unction-like structures with intestinal epithelial cells [3]. So, PPCs are found both in the subepithelial dome that lies just under-eath the FAE, and in the interfollicular T cell regions. DCs arentigen-presenting cells that have a central role in the making

Abbreviations: PP, Peyer’s patch; DCs, dendritic cells; GALT, gut-associatedymphoid tissues; M cells, microfold cells; pDC, plasmacytoid DC; IF, immunoflu-rescence; PFA, paraformaldehyde.∗ Corresponding author at: Groupe Immunité des Muqueuses et Agents

athogènes (GIMAP, EA3064), Jean Monnet University, 15 rue Ambroise Paré 42023t Etienne, France. Tel.: +33 477828975.

E-mail address: stephane.paul@chu-st-etienne.fr (S. Paul).

264-410X/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2011.03.012

of oral vaccines.© 2011 Elsevier Ltd. All rights reserved.

of immune responses in the intestine. DCs have the capacity tostimulate naive T cells and may directly induce immunoglobulinswitching to IgA in B cell [4], or to produce high levels of IL-10 andthus may be also involved in promoting tolerance [5]. IntestinalDCs can either induce protective immunity to infectious agents orpromote tolerance to inoffensive antigens.

Intestinal mucosa represents an attractive target for oral deliv-ery of immunogens and for mucosal vaccination. Several oralstrategies, using biodegradable polymeric particles [6,7], liposome[8], bacterial ghost [9], plant lectins [10,11], adjuvanted vaccines[12] or transgenic plants [13,14], have been adopted to protect theantigens in the gastrointestinal tract and to increase uptake by DCs,causing their maturation and their migration to the intrafollicularareas. PLA (poly(lactic acid)) or PLGA (poly(lactic-co-glycolic acid))nanoparticles are suitable protein carriers offering antigen protec-tion, increased penetration across mucosal surface and controlledrelease of encapsulated antigen [15]. Furthermore, these systemsrevealed promising results as illustrated by their capacity to crossin vivo epithelial barriers or to be taken up by mucosal DCs [16].Transcutaneous immunization could also be used to induce activa-tion of specific IgA-secreting cells expressing CCR9 and CCR10 in thesmall intestine with a retinoic acid-dependent manner. This cross-

talk between the skin and gut immune systems might be mediatedby langerin+ dendritic cells in the mesenteric lymph nodes [17].Use of strong adjuvants for oral immunization in mice has beenalso tested to increase mucosal immune responses in the gastroin-testinal tract (enterotoxins such as cholera toxin (CT) or heat-labile

3 ccine

Ead

oGmCaiditeCiaiuaccPiPtaCi2ps(iil

oPGeaoiissfme

2

2

(pH68Ba(A

656 N. Rochereau et al. / Va

scherichia coli enterotoxin (LT)). CT is known to be endocytosednd transported by M cells and delivered into the subepithelialome region, thereby stimulating immature DCs [18].

Several DC subpopulations have been described in mice and areften classified on the basis of their surface receptors expression.enerally, mouse DCs express CD11c, CD11b, the interdigitating DCarker CD205, and the co-stimulator molecules CD80, CD86 and

D40. Expression of T-cell markers such as CD4 and CD8, whichre useful for segregating subtypes, has been also described. Theres a large diversity of DCs in the intestinal lamina propria (foundeep in the LP or associated to the epithelium) and more generally

n the GALT regarding their phenotypes, functions and localiza-ion. Peyer Patches DC subsets can be distinguished by the specificxpression of the CX3CR1, CCR6, and CCR7 chemokine receptors.CR7+ DCs are found mainly in T cell areas [19]. CX3CR1+ DCs are

n close contact with the FAE and their role consist to taking upntigens after M cell transcytosis [20]. This DCs subset is mainlymmature cells expressing low levels of MHC-I/MHC-II and costim-latory molecules with an highly macropinocytic and phagocyticctivities. CCR6+ DCs, located in the subepithelial dome, have theapacity to migrate to the FAE in response to infections and toontribute to antigen-specific T helper cell activation [20]. Peyeratches CD11b+ DCs are characterized by their Th2 polarizing abil-ty while CD8+ DCs possess rather a Th1 polarizing ability [21].lasmacytoid DCs (pDCs) characterized by their ability to produceype I IFNs, have also been described in Peyer Patches [22]. pDCs areble to induce differentiation of regulatory T cells (Treg) and uponpG-induced maturation retain their regulatory capacity [23]. PDCs

dentification in Peyer Patches can be made by the analysis of BDCA-, IL-3R� chain (CD123) and the lack of CD11c expression. MouseDCs are about 1.3% of the total PP cells [24], and display a cell-urface molecule recognized by the monoclonal antibody pDC/IPC120G8 clone) which is a useful tool to deplete this DC subtypen vivo [25]. Other DC subsets can also be identified and classifiedn the mouse lamina propria by the expression of CD11c (high andow), CD11b, CD103, CX3CR1, and CD70.

In this report, we have investigated by immunofluorescence (IF)n tissue cryosection and by flow cytometry analysis of cells fromeyer Patches, the phenotype and the localization of DCs into mouseut Associated Lymphoid Tissue (GALT). Our results revealed differ-nt phenotypes in which DCs are strategically positioned directlydjacent to mucosal epithelial cells, thus facilitating the acquisitionf antigens from luminal sites. Characterization of DC phenotypesnto the GALT is of great interest not only for a better understand-ng of mucosal immune responses induced by natural processesuch as infections, but also for the evaluation of oral vaccines. Inpite of that numerous reports describe human DC phenotypes andunctions into the GALT, our report is the first which describe new

ouse DC subsets into the GALT on the basis of their cell surfacexpression and localization.

. Materials and methods

.1. Materials

PE anti-mouse CD11c, PE anti-mouse CD45R/B220, PE rat IgG1isotype control), and PE hamster IgG (isotype control) wereurchased from Becton Dickinson (San Jose, California, USA).uman product list: A488-TLR8 (clone 307D3), A488-TLR3 (clone19F7), A488-TLR7 (clone 66H3), A488-Langerin (clones 929F3,

08E10), A488-DC-SIGN (clones 109H12, 103G2, 102E11), A488-DCA-2 (clones 101C9, 104C12), A488-DORA (clone 104A10)nd A488-IL3R� (clone 107D2) were purchased from DendriticsLyon, France). Mouse product list: A488-Langerin (clones 205C1),488-pDC/IPc (clones 102H4.16, 120G8, 102F11, 122A1), A488-

29 (2011) 3655–3661

DCLamp (clone 1006F7) and A488-CD205 (clone HB290Dec205)were purchased from Dendritics (Lyon, France). C57BL/6 micewere purchased from Charles River Laboratories (Lyon, France) andhandled following national guidelines in accordance with Euro-pean guidelines for animal care (Journal Officiel des CommunautésEuropéennes, L358, 18 Decembre 1986).

2.2. Preparation of intestine cryosection

Six weeks-old C57Bl/6 mice were starved overnight. A pieceof intestine containing a PP was isolated, extensively washed andfixed for 2 h in 3% paraformaldehyde (PFA). Pieces of intestine werecryogenized with OCT embedding matrix (CML, Nemours, France),and cryosectioned using a Leica cryostat model CM3050 S (LeicaMicrosystems, Wetzlar, Germany). Sections of 7 �m were capturedon superfrost microscope slides (VWR International, Fontenay-sous-Bois, France) before staining.

2.3. Immunolabelling on intestine cryosections

Slides were washed in PBS to eliminate residual OCT, incu-bated for some of them in 95% acetone for 15 min, and blockedwith PBS containing 5% FCS for 30 min at room temperature.PE-B220, PE-CD11c, A488-TLR8, A488-TLR3, A488-TLR7, A488-Langerin, A488-DC-SIGN, A488-BDCA-2, A488-pDC/IPc, A488-DCLamp, A488-DORA, A488-IL3R� diluted at 0.5 �g/ml wereincubated for 1 h at room temperature. The slides were then washedin PBS, and for some of them incubated for 1 h at room tempera-ture with PE-B220 and PE-CD11c. After two washes, slides wereair-dried, mounted with Fluoprep (BioMerieux) and observed byImmunofluorescence microscopy (Eclipse Ti, Nikon, France).

2.4. Isolation of Peyer’s Patches cells

Animals were sacrificed, and then the intestine has beenremoved, opened and flushed with PBS. The PP were isolated fromthe non-follicular tissue. Cells were separated from tissue by crush-ing on a cell strainer then washed in PBS.

2.5. Flow cytometry analysis

Isolated Peyer’s Patches cells were labeled with the followingspecific antibodies PE-CD11c, A488-TLR8 (307D3), A488-Langerin(808E10), A488-DC-SIGN (109H12), A488-BDCA-2 (104C12), A488-pDC/IPc (120G8 and 102F11), A488-DCLamp (1006F7) at 5 �g/mland were incubated for 30 min at room temperature in FACS buffer(FCS 5%, sodium azide 0,1%, DMEM w/o phenol red(Gibco)). Afterwashing, cells were analyzed on a FACS Calibur cytometer (BD Bio-sciences) using CellQuest software (BD Biosciences).

3. Results

In this report, we have performed an analysis of DC phenotypeand localization in PP. For this study, we have used a large num-ber of human DC specific antibodies. The cells were fixed by thePFA to see a membrane labeling or by acetone to permeabilize cellsand to see a potential intracellular staining. For positive staining,co-localization were then realized with CD11c antibodies, found athigh levels on most human dendritic cells, but also on monocytes,macrophages, neutrophils, and some B cells, and B220 antibodies,which is expressed on B cells, on activated T cells, on a subset of

dendritic cells and other antigen presenting cells. The results areshown in Fig. 1 and summarized in Table 1. We observe specificstaining for the following antibodies: 1006F7 DC-LAMP (Fig. 1B),120G8 and 102F11 pDC/IPC (Fig. 1C and D), 307D3 TLR8 (Fig. 1E),104C12 BDCA-2 (Fig. 1F), 109H12 DC-SIGN (Fig. 1G), and 808E10

N. Rochereau et al. / Vaccine 29 (2011) 3655–3661 3657

Fig. 1. Immunostainings of intestine cryosections. Stainings were made for 2 h with the following antibodies: 1006F7 DC-LAMP (Fig. 1B), pDC/IPC 102F11 and 120G8 (Fig. 1Cand D), TLR8 307D3 (Fig. 1E), BDCA-2 104C12 (Fig. 1F), DC-SIGN 109H12 (Fig. 1G), and Langerin 808E10 (Fig. 1H). Fig. 1A shows a staining of B220 and CD11c alone, a negativecontrol (PBS) and isotype controls (hamster and rat). Intestine cryosection were fixed with Acetone or PFA and were colocalized with CD11c and B220 marker. LI: IntestinalLumen; LT: Lymphoid Tissue.

3658 N. Rochereau et al. / Vaccine

Table 1Staining obtained with different DC antibodies on mouse PP cryosections. Two fix-ation methods have been compared (PFA and Aceton). Double staining with CD11cand B220 cells is also mentioned.

Ab used (clone) PFA fixation Aceton fixation CD11c B220

BDCA2 (101C9) − − − −BDCA2 (104C12) + + + −pDC/IPC (102H4.16) − − − −pDC/IPC (120G8) + + + +pDC/IPC (102F11) − + − −pDC/IPC (122A1) − − − −DCLamp (1006F7) + + − −DORA (104A10) − − − −DCSign (109H12) + − − −DCSign (103G2) − − − −DCSign (102E11) − − − −IL3R� (107D2) − − − −TLR3 (619F7) − − − −TLR8 (307D3) + + + −TLR7 (66H3) − − − −Langerin (929F3) − − − −

L1Iwt

lpd

iFicpt

adWi

fiCi

iird

gle

ct(1ospo

thus Langerin could become a natural barrier to HIV-1 infection

Langerin (205C1) − − − −Langerin (808E10) − + + −Langerin (DEC205) − − − −

angerin (Fig. 1H). All other clones (101C9 and 102H4.16 BDCA-2,22A1 pDC/IPC, 104A10 DORA, 103G2 and 102E11 DC-Sign, 107D2

L3R�, 619F7 TLR3, 66H3 TLR7, 929F3, 205C1 and DEC205 Langerin)ere tested under different conditions of time and/or concentra-

ion without any specific staining.Fig. 1B shows a very specific staining of DC-LAMP molecules,

ocated mainly in the dome, with both acetone (intracellular com-artments), and PFA fixation (cell surface). We did not observeouble-staining with CD11c and B220 antibodies.

Fig. 1C shows a specific labeling with 120G8 pDC/IPC mAb bothn acetone and PFA fixation. In contrast, 102F11 pDC/IPC clone inig. 1D stains only with acetone fixation. In addition, a double stain-ng with CD11c and with B220 is observed with 120G8 pDC/IPClone but not with 102F11 pDC/IPC clone. 120G8+ and 102F11+

DC/IPC cells are located in the dome and spread in the lymphoidissues.

As described in Fig. 1E with acetone fixation, TLR8+ cellsre located predominantly in intracellular vacuoles, but are alsoetected at the membrane level as shown in the PFA fixation panel.e observe double labeling between CD11c and TLR8 distributed

n the lymphoid tissue.Fig. 1F clearly shows a specific staining in both acetone and PFA

xation condition. In addition, we can observe a co-expression withD11c staining. BDCA-2+ cells are located in the dome and spread

n the lymphoid tissues.Fig. 1G shows a very specific staining of DC-SIGN (DC-specific

ntracellular adhesion molecule 3 grabbing nonintegrin) expressionn inter-follicular areas. Moreover, marking appears by PFA fixationevealing that DC-SIGN is expressed at the membrane of DCs. Weid not observe double-staining with DC-SIGN and CD11c or B220.

As shown in Fig. 1H, acetone fixation allows the detection of Lan-erin molecules in Birbeck granules intracellular compartments,ocated mainly in the dome. In addition, we can observe a co-xpression with CD11c staining.

Observations made by microscopy were confirmed by flowytometry analysis (Fig. 2). PP cells suspensions were stained forhe positives markers observed by immunofluorescence in Fig. 1A488-307D3, A488-808E10, A488-109H12, A488-104C12, A488-20G8, A488-102F11, and A488-1006F7). A positive staining was

bserved for all markers except for 1006F7 DC-Lamp clone. Double-taining with the CD11c DC marker were also observed for TLR8,DC/IPC and more weakly for DC-SIGN confirming the resultsbtained by immunofluorescence. In contrast, no double-staining

29 (2011) 3655–3661

were observed between CD11c and 104C12 BDCA2 and 808E10Langerin antibodies by flow cytometry.

4. Discussion

Interestingly, we have shown that DCLamp+ cells are locatedmainly in the dome, suggesting that they could participate to thesampling of the antigen. DC-LAMP belongs to the LAMP family thathas been identified as the most discriminative marker of humanbut not murine DC [26]. In human DC, DC-LAMP is functionallycoupled with the intracellular MHC II compartment, by playing arole in the transfer of MHC class II molecules to the cell surface[27]. The definite function of DC-LAMP has not yet been estab-lished.

Human and mouse type 1 interferon-(�, �, �)-producing cellsare a rare subtype of circulating DCs found in the blood as wellas in peripheral lymphoid organs. Consistently with the workof Asselin-Paturel et al. showing pDC-specific staining with thepDC/IPC 120G8 mAb in the T cell area of spleen, lymph nodes, andPP [25], we have observed an important proportion of 120G8+

DCs in the dome and spread in the lymphoid tissues. PP local-ization of DCs may facilitate the cellular cooperation during anantiviral immune response through the secretion of type 1 IFNs[28].

DCs play a major role in the immune response during viral infec-tion because they have the ability to recognize single-stranded RNAthrough TLR7 and TLR8 intracellular innate receptor. They pro-duce signals which are known to dramatically impact the cytokinemilieu, thus leading to distinct patterns of naive CD4+ T cell differ-entiation. As shown in Figs. 1E and 2E, we observe a lymphoid tissuedistribution of double positive CD11c+/TLR8+ DCs which couldhave a potent role in the induction of specific immune responses[29].

In human, BDCA-2 is a type II C-type lectin expressed by pDCsspecialized in the targeting of antigens into the antigen processingand peptide-loading compartment. BDCA-2 is also involve in theIFN-synthesis inhibition. BDCA-2 lectin may have a central func-tion in the immune activation by promoting Th2 responses [30]. Inthis report, we describe for the first time the presence of immatureBDCA-2+ pDCs in the dome of mouse PP [31].

As shown in Fig. 1G, and also as previously described in human,DC-SIGN+ DCs are present in the dome and inter-follicular regionsof mouse PP [32]. DC-SIGN is a C-type lectin expressed on the DCsurface which is involved in the cross-talk with lymphocytes. DCsalso express the high affinity receptor for ICAM-3, which is alsoexpressed on naive T cells. Interactions between ICAM-3 and DC-SIGN stabilize the initial step of contact between DC and naive Tcell [33]. Human DC-SIGN has been shown to bind all HIV-1, HIV-2,and SIV viruses and plays an important role in virus adhesion toDCs [34].

Langerin (CD207), a type II Ca2+-dependent C-type lectin recep-tor, is known to capture and to route antigen into the organelles,providing access to a non-classical antigen-processing pathway[35]. Langerin is mainly found in Birbeck granules as well as onthe cell surface [36]. Langerin expression is predominant in skinDCs, but Langerin-expressing DCs also exist in mouse mucosal tis-sue [37]. As shown in Fig. 1H, CD11c+/Langerin+ DCs are present inmouse Peyer Patches. Langerin+ DCs are located mainly in the dome,confirming the function of this subtype in the uptake of antigen. deWitte et al. have shown that Langerin mediates HIV-1 degradation,

[38].Positive staining observed by immunofluorescence were also

confirmed by flow cytometry with the exception of DC-Lamp(1006F7 clone). Percentages of each DC populations measured by

N. Rochereau et al. / Vaccine 29 (2011) 3655–3661 3659

Fig. 2. Flow cytometry analysis of Peyer’s Patches cells. PP cells were isolated from the non-follicular tissue, and labeled for 30 min with the following antibodies: 1006F7DC-LAMP (Fig. 2B), pDC/IPC 102F11 and 120G8 (Fig. 2C and D), TLR8 307D3 (Fig. 2E), BDCA-2 104C12 (Fig. 2F), DC-SIGN 109H12 (Fig. 2G), and Langerin 808E10 (Fig. 2H).Double staining were obtained after incubation with PE-CD11c antibody. Fig. 2A shows a FSC/SSC acquisition dot plot with 300,000 events.

3 ccine

flq

(6hmlt[ettfioP

PTslalavaDa

A

dacR

R

R

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

660 N. Rochereau et al. / Va

ow cytometry analysis are consistent and correlated with theuantity of positive cells observed on cryosections.

No specific staining has been obtained with the following mAb101C9, 102H4.16, 122A1, 104A10, 103G2, 102E11, 107D2, 619F7,6H3, 929F3, 205C1, and DEC205), raising several non-exclusiveypothesis as the absence of cross-reactivity between human andouse, the fact that antibodies are inappropriate to IF studies, the

ow of negative expression of the target molecule in the analyzedissue. TLR3 has been found to be specifically expressed in human39] and canine [40] DCs. DORA (Down Regulated by Activation)xpression was also observed in human DC [41]. Human plasmacy-oid pre-DC strongly express TLR7 [29] and a large subset of DC inhe T cell-dependent areas of human lymphoid organs were identi-ed by high levels of IL-3R� expression [42]. However, we did notbserve any specific staining of these specific markers on murineP cryosections.

In this study, new phenotype and localization of mouse DC intoP have been described (BDCA-2+, pDC/IPC+, DC-LAMP+, DC-SIGN+,LR8+, and Langerin+ DCs). These DC populations could have apecific role in the functions of the GALT as the maintenance ofocal tolerance or in the induction of pro-inflammatory responsegainst mucosal pathogens. Specific interactions between particu-ar intestinal DCs and Ag-experienced T cells could initiate eithern immunogenic or a tolerogenic pathway. This study can pro-ide important informations about antigen uptake by local DCsfter mucosal delivery. Specific-targeting of particular intestinalCs could improve drastically the efficacy of mucosal vaccinationpproaches.

cknowledgments

We would like to thank the “Unité Hospitalo-Universitaire’expérimentation animale” technical platforms of IFR143. Theuthors acknowledge Dendritics for their generous gift of dendriticells antibodies, Dr. Diane Razanajaona-Doll and Dr. Marie-Clotildeissoan for critical reading of the manuscript.

This work was financed from fundings to GIMAP, by Regionhône-Alpes (Cluster 10 “Infectiologie”) and SIDACTION.

eferences

[1] Alpan O, Rudomen G, Matzinger P. The role of dendritic cells, B cells, and Mcells in gut-oriented immune responses. J Immunol 2001;166:4843–52.

[2] Stagg AJ, Hart AL, Knight SC, Kamm MA. The dendritic cell: its role in intestinalinflammation and relationship with gut bacteria. Gut 2003;52:1522–9.

[3] Rescigno M, Urbano M, Valzasina B, Francolini M, Rotta G, Bonasio R, et al.Dendritic cells express tight junction proteins and penetrate gut epithelialmonolayers to sample bacteria. Nat Immunol 2001;2:361–7.

[4] Mora JR, Iwata M, Eksteen B, Song S, Junt T, Senman B, et al. Genera-tion of gut-homing IgA-secreting B cells by intestinal dendritic cells. Science2006;314:1157–60.

[5] Dillon SM, Rogers LM, Howe R, Hostetler LA, Buhrman J, McCarter MD, et al.Human intestinal lamina propria CD1c+ dendritic cells display an activatedphenotype at steady state and produce IL-23 in response to TLR7/8 stimulation.J Immunol 2010;184:6612–21.

[6] Katz DE, DeLorimier AJ, Wolf MK, Hall ER, Cassels FJ, van Hamont JE, et al.Oral immunization of adult volunteers with microencapsulated enterotoxi-genic Escherichia coli (ETEC) CS6 antigen. Vaccine 2003;21:341–6.

[7] Mishra N, Tiwari S, Vaidya B, Agrawal GP, Vyas SP. Lectin anchored PLGAnanoparticles for oral mucosal immunization against hepatitis B. J Drug Target2011;19:67–8.

[8] Butts C, Murray N, Maksymiuk A, Goss G, Marshall E, Soulières D, et al. Random-ized phase IIB trial of BLP25 liposome vaccine in stage IIIB and IV non-small-celllung cancer. J Clin Oncol 2005;23:6674–81.

[9] Mayr UB, Haller C, Haidinger W, Atrasheuskaya A, Bukin E, Lubitz W, et al. Bacte-rial ghosts as an oral vaccine: a single dose of Escherichia coli O157:H7 bacterial

ghosts protects mice against lethal challenge. Infect Immun 2005;73:4810–7.

10] Lavelle EC, Grant G, Pfuller U, O’Hagan DT. Immunological implications of theuse of plant lectins for drug and vaccine targeting to the gastrointestinal tract.J Drug Target 2004;12:89–95.

11] Lavelle EC, Grant G, Pusztai A, Pfüller U, O’Hagan DT. Mucosal immunogenicityof plant lectins in mice. Immunology 2000;99:30–7.

[

[

29 (2011) 3655–3661

12] Tokuhara D, Yuki Y, Nochi T, Kodama T, Mejima M, Kurokawa S, et al. SecretoryIgA-mediated protection against V. cholerae and heat-labile enterotoxin-producing enterotoxigenic Escherichia coli by rice-based vaccine. Proc Natl AcadSci U S A 2010;107:8794–9.

13] Zhou B, Zhang Y, Wang X, Dong J, Wang B, Han C, et al. Oral administrationof plant-based rotavirus VP6 induces antigen-specific IgAs, IgGs and passiveprotection in mice. Vaccine 2010;28:6021–7.

14] Tacket CO, Pasetti MF, Edelman R, Howard JA, Streatfield S. Immunogenicityof recombinant LT-B delivered orally to humans in transgenic corn. Vaccine2004;22:4385–9.

15] Samstein RM, Perica K, Balderrama F, Look M, Fahmy TM. The use of deoxy-cholic acid to enhance the oral bioavailability of biodegradable nanoparticles.Biomaterials 2008;29:703–8.

16] Primard C, Rochereau N, Luciani E, Genin C, Delair T, Paul S, et al. Traffic ofpoly(lactic acid) nanoparticulate vaccine vehicle from intestinal mucus to sub-epithelial immune competent cells. Biomaterials 2010;31:6060–8.

17] Chang S, Cha H, Igarashi O, Rennert PD, Kissenpfennig A, Malissen B, et al.Cutting edge: langerin + dendritic cells in the mesenteric lymph node set thestage for skin and gut immune system cross-talk. J Immunol 2008;180:4361–5.

18] Anosova NG, Chabot S, Shreedhar V, Borawski JA, Dickinson BL, Neutra MR.Cholera toxin, E. coli heat-labile toxin, and non-toxic derivatives induce den-dritic cell migration into the follicle-associated epithelium of Peyer’s patches.Mucosal Immunol 2008;1:59–67.

19] Iwasaki A, Kelsall BL. Localization of distinct Peyer’s patch dendritic cell sub-sets and their recruitment by chemokines macrophage inflammatory protein(MIP)-3alpha, MIP-3beta, and secondary lymphoid organ chemokine. J Exp Med2000;191:1381–94.

20] Salazar-Gonzalez RM, Niess JH, Zammit DJ, Ravindran R, Srinivasan A, MaxwellJR, et al. CCR6-mediated dendritic cell activation of pathogen-specific T cells inPeyer’s patches. Immunity 2006;24:623–32.

21] Iwasaki A, Kelsall BL. Unique functions of CD11b+, CD8 alpha+, and double-negative Peyer’s patch dendritic cells. J Immunol 2001;166:4884–90.

22] Contractor N, Louten J, Kim L, Biron CA, Kelsall BL. Cutting edge: Peyer’s patchplasmacytoid dendritic cells (pDCs) produce low levels of type I interferons:possible role for IL-10, TGFbeta, and prostaglandin E2 in conditioning a uniquemucosal pDC phenotype. J Immunol 2007;179:2690–4.

23] Bilsborough J, George TC, Norment A, Viney JL. Mucosal CD8alpha+ DC, with aplasmacytoid phenotype, induce differentiation and support function of T cellswith regulatory properties. Immunology 2003;108:481–92.

24] Duriancik DM, Hoag KA. The identification and enumeration of dendritic cellpopulations from individual mouse spleen and Peyer’s patches using flow cyto-metric analysis. Cytometry Part A 2009;75A:951–9.

25] Asselin-Paturel C, Brizard G, Pin J, Brière F, Trinchieri G. Mouse strain differ-ences in plasmacytoid dendritic cell frequency and function revealed by a novelmonoclonal antibody. J Immunol 2003;171:6466–77.

26] Salaun B, de Saint-Vis B, Clair-Moninot V, Pin J, Barthélemy-Dubois C,Kissenpfennig A, et al. Cloning and characterization of the mouse homologue ofthe human dendritic cell maturation marker CD208/DC-LAMP. Eur J Immunol2003;33:2619–29.

27] Barois N, de Saint-Vis B, Lebecque S, Geuze HJ, Kleijmeer MJ. MHC class II com-partments in human dendritic cells undergo profound structural changes uponactivation. Traffic 2002;3:894–905.

28] Colonna M, Trinchieri G, Liu Y. Plasmacytoid dendritic cells in immunity. NatImmunol 2004;5:1219–26.

29] Kadowaki N, Ho S, Antonenko S, de Waal Malefyt R, Kastelein RA, Bazan F, et al.Subsets of Human Dendritic Cell Precursors Express Different Toll-like Recep-tors and Respond to Different Microbial Antigens. The Journal of ExperimentalMedicine 2001;vol. 194:863–70.

30] Dzionek A, Sohma Y, Nagafune J, Cella M, Colonna M, Facchetti F, et al. BDCA-2, a novel plasmacytoid dendritic cell-specific type II C-type lectin mediatesantigen capture and is a potent inhibitor of interferon �/� induction. J ExpMed 2001;194:1823–34.

31] Asselin-Paturel C, Boonstra A, Dalod M, Durand I, Yessaad N, Dezutter-Dambuyant C, et al. Mouse type I IFN-producing cells are immature APCs withplasmacytoid morphology. Nat Immunol 2001;2:1144–50.

32] Jameson B, Baribaud F, Pohlmann S, Ghavimi D, Mortari F, Doms RW, et al.Expression of DC-SIGN by dendritic cells of intestinal and genital mucosae inhumans and Rhesus Macaques. J Virol 2002;76:1866–75.

33] Geijtenbeek TBH, Torensma R, van Vliet SJ, van Duijnhoven GCF, Adema GJ, vanKooyk Y, et al. Identification of DC-SIGN, a novel dendritic cell-specific ICAM-3receptor that supports primary immune responses. Cell 2000;100:575–85.

34] Baribaud F, Pohlmann S, Sparwasser T, Kimata MTY, Choi Y, Haggarty BS, et al.Functional and antigenic characterization of human, Rhesus Macaque, PigtailedMacaque, and Murine DC-SIGN. J Virol 2001;75:10281–9.

35] Kissenpfennig A, Ait-Yahia S, Clair-Moninot V, Stossel H, Badell E, Bordat Y, et al.Disruption of the langerin/CD207 gene abolishes birbeck granules without amarked loss of langerhans cell function. Mol Cell Biol 2005;25:88–99.

36] Valladeau J, Ravel O, Dezutter-Dambuyant C, Moore K, Kleijmeer M, Liu Y, et al.Langerin, a novel c-type lectin specific to langerhans cells, is an endocytic recep-

tor that induces the formation of birbeck granules. Immunity 2000;12:71–81.

37] Chang S, Kweon M. Langerin-expressing dendritic cells in gut-associated lym-phoid tissues. Immunol Rev 2010;234:233–46.

38] de Witte L, Nabatov A, Pion M, Fluitsma D, de Jong MAWP, de Gruijl T, et al.Langerin is a natural barrier to HIV-1 transmission by Langerhans cells. NatMed 2007;13:367–71.

ccine

[

[

N. Rochereau et al. / Va

39] Matsumoto M, Funami K, Tanabe M, Oshiumi H, Shingai M, Seto Y, et al. Sub-cellular localization of toll-like receptor 3 in human dendritic cells. J Immunol2003;171:3154–62.

40] Bonnefont-Rebeix C, Marchal T, Bernaud J, Pin J, Leroux C, Lebecque S, et al. Toll-like receptor 3 (TLR3): a new marker of canine monocytes-derived dendriticcells (cMo-DC). Vet Immunol Immunopathol 2007;118:134–9.

[

[

29 (2011) 3655–3661 3661

41] Bates EEM, Dieu M, Ravel O, Zurawski SM, Patel S, Bridon J, et al. CD40Lactivation of dendritic cells down-regulates DORA, a novel member of theimmunoglobulin superfamily. Mol Immunol 1998;35:513–24.

42] Olweus J, BitMansour A, Warnke R, Thompson PA, Carballido J, Picker LJ, et al.Dendritic cell ontogeny: a human dendritic cell lineage of myeloid origin. ProcNatl Acad Sci U S A 1997;94:12551–6.