Three populations of mouse lymph node dendritic cells with different origins and dynamics

of June 2, 2013.This information is current as

DynamicsDendritic Cells with Different Origins and Three Populations of Mouse Lymph Node

KlatzmannBenoi?t Salomon, José L. Cohen, Carole Masurier and David

http://www.jimmunol.org/content/160/2/7081998; 160:708-717; ;J Immunol

Referenceshttp://www.jimmunol.org/content/160/2/708.full#ref-list-1

, 37 of which you can access for free at: cites 53 articlesThis article

Subscriptionshttp://jimmunol.org/subscriptions

is online at: The Journal of ImmunologyInformation about subscribing to

Permissionshttp://www.aai.org/ji/copyright.htmlSubmit copyright permission requests at:

Email Alertshttp://jimmunol.org/cgi/alerts/etocReceive free email-alerts when new articles cite this article. Sign up at:

Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 1998 by The American Association of9650 Rockville Pike, Bethesda, MD 20814-3994.The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

by guest on June 2, 2013http://w

ww

.jimm

unol.org/D

ownloaded from

http://www.jimmunol.org/cgi/adclick/?ad=37767&adclick=true&url=http%3A%2F%2Fwww.biolegend.com%2Fbrilliantviolet

http://www.jimmunol.org/

Three Populations of Mouse Lymph Node Dendritic Cells withDifferent Origins and Dynamics1

Benoıt Salomon,* Jose L. Cohen,† Carole Masurier,* and David Klatzmann2*

We have identified three distinct populations of mouse lymph node dendritic cells (DC) that differ in their capacity to uptakeAg delivered by different routes, and in their dynamics. The “l-DCs” are large cells that resemble the interdigitating cells andhave a mature phenotype and a slow turnover. They derive from the regional tissues. The “sm-DCs” and “sl-DCs” are smaller(hence s-DC), have a more immature phenotype and a rapid turnover. The sl-DC phenotype, including CD8a expression,suggests a lymphoid-related origin. The sl-DC population is expanded 100-fold after an in vivo flt3 ligand treatment. The sm-DCphenotype suggests a myeloid-related origin. Interestingly, sm-DCs can acquire i.v. injected macromolecules in less than 30 minafter injection. They may, therefore, play an important role in the immune response against blood Ags. The Journal ofImmunology, 1998, 160: 708–717.

D endritic cells (DCs)3 have been shown to be very het-erogeneous with regard to their origin, maturationstate, and rate of turnover. There is general agreement

that DCs originate from a hemopoietic progenitor (1). However,they apparently have multiple pathways of differentiation.There are numerous arguments for a myeloid origin of someDCs (2, 3). Culture of bone marrow cell progenitors in semi-solid medium has allowed the characterization of mixed colo-nies that can differentiate into either DCs or myeloid cells (4).Human monocytes can also further differentiate into typicalDCs when cultured in the presence of IL-4 (5–7). The differ-entiation of these myeloid DCs from either immature precursorsor more differentiated monocytes always requires granulocyte-macrophage CSF. On the other hand, there is now substantialevidence for the existence of a pool of DCs with a lymphoid-related origin (8). A precursor that can differentiate into eitherT cells or DCs has been described in the mouse thymus. Thisprecursor has lost the ability to differentiate into either myeloidcells, B cells, or NK cells. DCs that develop from these thymicprecursors express the CD8a molecule (9). Granulocyte-mac-rophage CSF is not required for the development of these lym-phoid-related DCs (10). A common precursor for T cells, Bcells, NK cells, and DCs has also recently been identified in

human bone marrow (11). Langerhans cells may belong to athird DC lineage, and TGF-b1 is required for theirdevelopment (3, 12–14).

In addition to these distinct lineages, DCs differ in their matu-ration stage and functional properties. “Immature” DCs appearvery efficient for macromolecule capture and processing. Inflam-matory mediators induce their differentiation into more mature oractivated cells that have acquired the capacity to efficiently stim-ulate T lymphocytes (5, 15–17). Finally, DCs also appear hetero-geneous in terms of turnover (18). Some of them, such as theLangerhans cells (LCs), appear to be renewed very slowly in theabsence of inflammation (19, 20). Other DCs have a rapid turnoverwith a t1/2 , 1 wk (21–24).

DC heterogeneity has already been described in some lymphoidtissues. In the spleen, two DC populations with distinct phenotypesand localizations have been described: the DCs of the marginalzone and the DCs localized in the periarteriolar T cell region of thewhite pulp (25–28), that seem to be of myeloid- and lymphoid-related origin, respectively (9, 29). Similarly, DC heterogeneityhas also been described in the Peyer’s patches (30) andtonsils (31).

The characterization of these distinct populations of DCs in var-ious tissues or organs is important because they most probablyhave different functions in the immune system. Indeed, it has al-ready been suggested that in the spleen the myeloid-related DCs aplay major role in triggering immune responses, while the lym-phoid-related DCs participate in the regulation of these responses(32, 33). Surprisingly little is known about lymph node (LN) DCs,although these secondary lymphoid organs are major sites of im-mune response initiation. Until now, a single DC type has beendescribed, which is located in the paracortical zone and referred toas an interdigitating cell (34). It is believed that these cells arederived from immature DCs localized in regional tissues, whichmigrate to the draining LN via afferent lymph (Refs. 35–37; andreviewed in Ref. 38).

In this work, we present a detailed analysis of LN DCs. We haveidentified three distinct populations that can be distinguished onthe basis of their morphology and phenotype. Interestingly, thesecells also differ in their capacity to uptake Ag delivered by differentroutes and in their dynamics.

* Laboratoire de Biologie et Therapeutique des Pathologies Immunitaires, Uni-versite Pierre et Marie Curie, Centre National de la Recherche Scientifique(CNRS), Groupe Hospitalier Pitie-Salpetriere, and †Genopoıetic, Paris, France

Received for publication July 18, 1997. Accepted for publication October2, 1997.

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.1 Supported by: Universite Pierre et Marie Curie; Agence Nationale de Re-cherche contre le SIDA; Association de Recherche sur les Deficits ImmunitairesViro-Induits (ARDIVI); Genopoıetic; Assistance Publique-Hopitaux de Paris; andCentre National de la Recherche Scientifique. B.S. was supported by ARDIVI andSidaction, and J.L.C. was supported in part by ARDIVI.2 Address correspondence and reprint requests to Dr. David Klatzmann, CERVI,Groupe Hospitalier Pitie-Salpetriere, 83 blvd. de l’Hopital, 75651 Paris Cedex13, France. E-mail address: [email protected] Abbreviations used in this paper: DC, dendritic cell; LN, lymph node; PE,phycoerythrin; HSA, heat-stable antigen; GCV, ganciclovir; s- and l-cells, smalland large cells; sl-, small lymphoid; sm-, small myeloid; HSV, herpesvirus; TK,thymidine kinase.

Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00


ww

.jimm

unol.org/D

ownloaded from


Materials and MethodsMice

Inbred mouse strains DBA/2, C57BL/6, CBA/J, and FVB/N were obtainedfrom IFFA-Credo (L’Arbresle, France). Except where indicated, all exper-iments were performed with DBA/2 mice. Chimeric mice used to analyzeDC turnover were derived from LTR-TK transgenic mice expressing theHSV1-thymidine kinase (TK) in DCs as previously described (24, 39).They were generated by transplantation of 3 to 53 106 bone marrow cellsfrom LTR-TK transgenic mice into lethally irradiated (13 Gy) normal re-cipients with the same DBA/2 genetic background.

Enzymatic cell dissociation

LNs were cut into small fragments and incubated in RPMI 1640 supple-mented with 1.6 mg/ml (500 IU/mg) collagenase (type IV, Sigma ChemicalCo., Saint Quentin Fallavier, France) and 200mg/ml DNase I (BoehringerMannheim, Mannheim, Germany) at 37°C for 30 min. Cells were disso-ciated by repeated pipetting, reincubated at 37°C for 10 min, and washed.Cell suspensions were then incubated with 200mg/ml DNase I for 15 minat room temperature and resuspended in staining buffer (PBS, 3% FCS,0.02% azide) for further flow cytometric analyzes.

MHC class II/CD11c double staining

CD11c expression was analyzed with the unlabeled N418 mAb (hamsterIgG, HB224; American Type Culture Collection (ATCC), Rockville, MD)revealed by a PE-conjugated F(ab9)2 goat anti-hamster IgG (Caltag Labo-ratories, San Francisco, CA). Depending on the analysis performed, weused different mAbs against MHC class II molecules: the M5/114 mAb(ATCC TIB120) was revealed by a TriColor-conjugated F(ab9)2 goat anti-rat IgG (Caltag Laboratories) (see Figs. 1, 5,A andB, and 6); the 14.4.4SmAb (PharMingen, San Diego, CA), either FITC-conjugated or biotin-conjugated, was revealed by streptavidin TriColor (Caltag Laboratories)(see Figs. 3,A andB, and 7); the biotin-conjugated 2G9 mAb (PharMin-gen) was revealed by streptavidin TriColor (see Fig. 4); the 28-16-8S mAb(Caltag) was revealed by an allophycocyanin-conjugated goat anti-mouseIgM (Caltag) (see Figs. 3C and 5C).

Cell surface flow cytometric analysis

Panels of mAbs were selected to study the phenotype of LN DCs by three-color flow cytometry analysis. We used a panel of FITC-conjugated mAbsagainst B220 (RA3-6B2, Caltag Laboratories), mThy-1.2, mCD4 (CT-CD4, Caltag Laboratories), mCD8a (CT-CD8a, Caltag Laboratories), Gr-1(RB6-8C5, Caltag Laboratories), F4/80 (F4/80, Caltag Laboratories),Mac-1 (M1-70.15, Caltag Laboratories), heat stable Ag (HSA) (M1/69,PharMingen), MHC class II (M5/114, Boehringer Mannheim), CD44(IM7, PharMingen), and CD62L (Mel-14, PharMingen), a panel of biotin-conjugated mAbs against B7-1 (16.10. A1), B7-2 (GL-1), H2-Kd (SFI-1.1,PharMingen), and CD54 (KAT-1, Caltag Laboratories) and a panel of un-coupled rat mAbs against CD2 (AT37, Serotec, Oxford, U.K.), spleen DC(33D1, ATCC TIB 227), DEC 205 (NLDC145 (25)), CD40 (3/23, Serotec),and FcgII/IIIR (2.4G2, PharMingen).

To minimize nonspecific binding, cells were preincubated either with2.4G2 mAb and then analyzed using FITC- or biotin-conjugated thirdmAbs or with 10% mouse serum and analyzed using mAbs from the panelsof uncoupled mAbs. In all stainings, the N418 mAb was revealed by aPE-conjugated F(ab9)2 goat anti-hamster IgG (Caltag Laboratories). ForMHC class II, the staining differed according to the third mAb used. ForFITC-conjugated mAbs, we used a biotin-conjugated anti I-E mAb(14.4.4S) revealed by streptavidin TriColor. For mAbs from the panels ofbiotin-conjugated or uncoupled rat mAbs, revealed respectively by strepta-vidin TriColor (Caltag Laboratories) and a TriColor-conjugated F(ab9)2

goat anti-rat IgG (Caltag Laboratories), the anti I-E staining was performedwith the FITC-conjugated 14.4.4S mAb.

The following isotypic Ig controls were used: FITC-conjugated ratIgG2a (LODDNP-16, Immunotech, Marseille, France), FITC-conjugatedrat IgG2b (Cedarlane, Hornby, Ontario, Canada), and biotin-conjugated ratIgG2a, biotin-conjugated hamster IgG, unconjugated rat IgG2a, and un-conjugated rat IgG2b (Caltag Laboratories).

Cells were fixed in 1% formaldehyde, and analyses were performed ona FACScan (Becton Dickinson Co., Mountain View, CA).

Flow cytometry cell sorting

After collagenase digestion of inguinal, brachial and axillary LNs from 35DBA/2 mice, cells were fractionated on a discontinuous gradient of BSA(density5 1.082) as previously described (40). The recovered low-densitycells, preincubated with the 2.4G2 mAb to reduce nonspecific binding,

were stained with N418 revealed by PE-labeled anti-hamster Ig and FITC-labeled anti-I-E (14-4-4S) mAbs. Cells were sorted at a rate of 3000events/s on a FACStarPlus (Becton Dickinson) and were kept at 4°Cthroughout the procedure.

Immunostaining

Cells were cytocentrifuged for 5 min at 300 rpm on slides. Cells were fixedin 1% formaldehyde, 0.2% glutaraldehyde for 5 min at 20°C and conservedat 4°C in PBS until use. Slides were incubated for several min in TBS (50mM Tris, pH 7.6, 0.9% NaCl), then in TBS supplemented with 0.5% BSAand 0.02% Nonidet P-40 (staining buffer; Sigma Chemical Co.). Slideswere immunostained in staining buffer supplemented with saturating levelsof M5/114 hybridoma supernatant for 45 min at room temperature, washedtwice in TBS, incubated in the staining buffer with peroxidase-conjugatedrabbit anti-rat Ig (P 0450, Dako SA, Trappes, France), washed in 50 mMTris, colored by 3,39-diaminobenzidine hydrochloride (DAB tablets, SigmaFast, Sigma Chemical Co.) for 2 to 3 min, rinsed 5 min in H2O, counter-stained with hematoxylin, rinsed 5 min in H2O, fixed in 70% ethanol then100% ethanol, and mounted.

Detection of CD4 transcription by RT-PCR

LN cells were lysed in an RNA extraction solution (RNA-BTM; Bioprobe,Montreuil, France). Cellular RNA was then reverse transcribed at 42°C for1 h in a 20-ml reaction containing: 1 mM of all four deoxynucleotidetriphosphates, 0.04 U of random primer P(dN)6, 40 U of RNase inhibitor(Pharmacia LKB Biotechnology, Uppsala, Sweden), and 200 U of MMLVreverse transcriptase (Life Technologies, Gaithersburg, MD). Five micro-liters of the reverse transcription product were used for amplification in a50-ml reaction containing: 1mM of murine CD4 orb-actin primers, 200mM of all four deoxynucleotide triphosphates, 1.5 mM MgCl2, and 1 UTaqDNA polymerase (Goldstar DNA polymerase; Eurogentec, Seraing, Bel-gium). The murine CD4 primers were: sense primer, 59-TGTGGCAGTGTCTGCTGAGTGA-39, in the D4 domain; antisense primer, 59-TGGCAGGTCTTCTTCTCACTGA-39, in the cytoplasmic region. There aretwo introns present between the genomic position of these primers, andthus they can only amplify CD4 cDNA. Reactions were performed in aDNA thermal cycler (Hybaid Ltd., Teddington, U.K.) as follow: an initialdenaturation cycle lasting 10 min at 94°C, followed by 35 cycles of am-plification each comprising denaturation for 30 s at 94°C, annealing for30 s at 62°C, and extension for 30 s at 72°C. The last cycle was followedby an extension cycle lasting 10 min at 70°C.

Allogeneic MLR

Nylon wool-passed T cells from mesenteric, brachial, axillary, and inguinalLN cells and graded numbers of FACS-sorted stimulator cells irradiated at20 Gy were cocultured in RPMI 1640 supplemented with 10% FCS (Flo-bio, Asnieres, France), 50mM 2-ME, and antibiotics in U-bottom 96-wellmicroplates. After 108 h of culture, cells were pulsed with 1mCi/well of[3H]TdR for an additional 12 h before harvesting and scintillation counting.

FITC skin painting

Mice were anesthetized with avertin (2.5% tribromoethanol) and the skinpainted at the level of the triceps muscle or on the abdomen at the level ofinguinal LN with 25 ml of 0.8% FITC (isomer 1, Sigma Chemical Co.)dissolved in a 1:1 mixture of acetone:dibutylphthalate just beforeapplication.

FITC-dextran

Mice were anesthetized with avertin and injected i.v. in the retro-orbitalsinus with 100ml of 50 to 100 mg/ml FITC-dextran, m.w. 40,000 (FD-40,Sigma Chemical Co.) or 150,000 (FD-150, Sigma Chemical Co.). Potentialsmall FITC contaminants were removed by dialysis through a dialysismembrane (membra-cell 24037, Polylabo France) with a m.w. cut-off ofunder 12,000 to 16,000. Dialysis was performed for 24 to 48 h at 4°C inPBS in the dark.

Ganciclovir and Flt3 ligand administration

Ganciclovir (GCV) was continuously administered at a dose of 50 to 55mg/kg/day for 7 days by using miniosmotic pumps (model 2001, AlzaCorp., Palo Alto, CA) implanted s.c. as previously described (24). Flt3ligand (Immunex, Seattle, WA) was injected i.p. once a day for 7 days ata dose of 10mg/day.

709The Journal of Immunology


ww

.jimm

unol.org/D

ownloaded from


ResultsMHC class II expression delineates two distinctCD11c-positive populations in lymph nodes

Double staining of LN cells, performed using the N418 mAb,which recognizes the murine CD11c (41), and a mAb againstMHC class II, allowed us to distinguish four cell populations. Be-sides double-negative and MHC class II1/CD11c2 cells, whichare mainly T and B cells, respectively, two distinct populations ofdouble-positive cells were detected (Fig. 1A). One expressed highCD11c levels and cell surface MHC class II at a level comparableto that of B cells (s-cells, Fig. 1A). The other expressed 5 to 10times more cell surface MHC class II and lower levels of CD11c(l-cells, Fig. 1A). These cells represented approximately 0.5 and1.5% of all LN cells, respectively. Size analysis of gated cellsshowed that l-cells were significantly larger than s-cells, hence thelabel s (small) and l (large) (Fig. 1B). Two MHC class II1/CD11c1 populations were identified in all of the LNs (brachial,inguinal, axillary, para-aortic, or mesenteric) and mouse strains(DBA/2, C57Bl/6, CBA/J, FVB) analyzed (not shown). However,in mesenteric LNs, l-cells expressed lower levels of cell surfaceMHC class II compared with peripheral LNs.

To analyze their T cell stimulatory capacity, s- and l-cells weresorted by flow cytometry and used as stimulators in mixed lym-phoid reaction. Both s- and l-cells had a strong T cell MLR-stim-ulating activity, similar to that of splenic DCs used as control (datanot shown).

Morphology and phenotype delineate three distinct LN DCpopulations

To observe the morphology of s- and l-cells, flow cytometry cellsorting was performed. Cytospins of sorted cells revealed for bothtypes of cells the typical dendritic cellular processes that charac-terize DCs (1). However, some differences could be observed: l-cells were larger and had longer and more numerous dendriticprocesses; s-cells contained intracellular clusters of MHC classII-rich compartments (Fig. 2).

To further characterize s- and l-cells, we performed three- andfour-color flow cytometry analysis (Fig. 3). The two populationsdid not express the classical markers of B cells (B220), T cells(Thy-1), or granulocytes (Gr-1). They did express a set of mole-cules associated with the Ag-presenting function of DCs such asMHC molecules and costimulatory and adhesion moleculesICAM-1, B7-1, B7-2, and CD40. l-Cells expressed intermediate tohigh levels of these molecules, while expression levels were lowerin s-cells.

l-Cells did not express CD4, CD8a, the myeloid marker F4/80,and the 33D1 marker of splenic DC; they expressed low levels ofthe interdigitating cell marker DEC-205 and heterogeneous levelsof HSA and Mac-1. In contrast, these molecules clearly definedtwo distinct populations of s-cells. One expressed CD4, F4/80,Mac-1, 33D1, and low levels of HSA and did not express DEC-205, while the other population expressed the lymphoid DCmarker CD8a and DEC-205, high levels of HSA, and low to zerolevels of CD4, F4/80, Mac-1, and 33D1 (Fig. 3 and data notshown). To verify that the detected CD4 expression was due toendogenous production, we analyzed CD4 transcription by RT-PCR on sorted cells. CD4 transcripts were indeed detected in s-DCusing primers that encompass an intron and thus cannot amplifygenomic DNA (data not shown). Altogether, based on their phe-notype, sm- and sl-DCs appear to be myeloid or lymphoid related,respectively (9, 29).

Therefore, based on morphology, phenotype, and T cell-stimu-latory capacity, s- and l-cells can be considered as typical DCs (1)and will subsequently be referred to as s-DC and l-DC, accordingto their small and large size, respectively. Based on their putativelymphoid- or myeloid-related origin, the two s-DC populationswill subsequently be referred to as sl-DC and sm-DC, respectively.

l-DC acquire skin-painted Ags

The migration of DCs from extravascular compartments of non-lymphoid tissues to draining LNs via afferent lymph has been welldocumented (36–38). In this respect, 24 h after skin painting withFITC diluted in organic solvents, this tracer was detected in LNDCs, suggesting that epidermal LCs migrate to the draining LN(42, 43). We analyzed FITC staining of s-DC and l-DC at differenttime points after FITC skin painting. After 24 h, up to 60% ofl-DCs showed high levels of FITC fluorescence in the drainingbrachial LN, whereas no staining was observed in the contralateralone. FITC staining of l-DCs became detectable 12 h after appli-cation and peaked at 24 h (Fig. 4). Interestingly, in preliminaryexperiments, when we applied a 10-fold higher volume of FITC,we observed a significant increase in l-DCs proportion, suggestingthat new cells immigrated into the LN (data not shown). However,this procedure was not suitable for additional experiments due tostaining of the contralateral LN DCs. The proportion of FITC-stained cells then decreased, with only a few stained cells remain-ing at day 5 (Fig. 4); this could be due to either their migrationoutside the LN, their death in situ, or FITC degradation. During the5-day follow-up, no significant FITC staining could be detected inCD11c-negative cells, nor in either s-DC population (Fig. 4).

FIGURE 1. Two CD11c/MHC class II double-positive LN cell pop-ulations. After collagenase digestion of inguinal LN, cells were stainedwith mAbs against MHC class II (M5/114) and CD11c (N418) andanalyzed by flow cytometry. A, Analysis of whole LN cells (104 cells,left panel) and CD11c1 MHC class II1 cells (right panel) from 1.5 3105 cells gated as indicated on the left, showing the two populationsof s- and l-cells. B, Histograms representing the size (forward scatter) ofgated s- and l-cells and of all other LN cells. Similar results were ob-tained after saturation of FcgR or after exclusion of dead cells withpropidium iodide (not shown here).

710 THREE POPULATIONS OF LYMPH NODE DENDRITIC CELLS


ww

.jimm

unol.org/D

ownloaded from


sm-DC rapidly and efficiently acquire blood macromolecules

We similarly tested the capacity of the three populations of LNDCs to acquire blood Ag using i.v. injection of FITC-dextran.High m.w. dextrans are known to remain in the plasmatic com-partment (44). They have also been used as a marker to quantifythe endocytic capacity of DCs (17). Cells of peripheral LN wereanalyzed at different time points after i.v. injection of FITC-dex-tran. At 30 min, 17% of s-DCs were highly fluorescent in bothbrachial and inguinal LNs. In contrast, only 3% of l-DCs wereFITC1, and moreover, the fluorescence of these stained cells wasweak (Fig. 5). The proportion of FITC-stained s-DCs increasedrapidly during the first 30 min after injection, and then more slowlyduring the next 12 h to reach 30% of the cells. This percentage thenremained stable for 5 days. The percentage of fluorescent l-DCsincreased slowly, reaching the same proportion after 12 h as thatobserved at 30 min for s-DCs. At day 5, the percentages of fluo-rescent s- and l-DCs were similar. During this time period, onlytraces of CD11c2 cells were FITC1 (Fig. 5). Similar results wereobtained using FITC-dextran of 40,000 or 150,000 m.w. or afterdialysis to remove potential small m.w. FITC contaminants (datanot shown).

To analyze which of the s-DC acquire FITC-dextran, we per-formed four-color flow cytometry analysis. 30 min after FITC-dextran i.v. injection, 17% of the sl-DCs were stained, while.50% of the sm-DCs were FITC1 (Fig. 5C). However, sl- andsm-DCs populations overlap based on CD4 staining (Fig. 3A), and

it should be noticed that the FITC-stained sl-DCs are positionedclose to the bar separating sm- and sl-DC on the CD4 staining axis.Therefore, it can be assumed that most if not all stained cells aresm-DCs. Similar results were obtained at 12 h after FITC-dextraninjection (data not shown).

Careful analysis of FITC staining in s- and l-DCs revealed twointeresting features. First, there was a progressive increase in thecell surface expression of MHC class II in most FITC1 cells,which resulted in a shift from the s- to the l-DC population (Fig.6A). Furthermore, while CD11c expression was heterogeneousamong l-DCs, the l-DCs that became FITC1 had high CD11c ex-pression levels, comparable to that of s-DCs. Second, the intensityof FITC staining increased with time among s-DCs and also, but ina delayed manner, in l-DCs (Fig. 6B).

s-DCs and l-DCs have a different turnover

We next analyzed the in vivo turnover of l- and s-DCs. We useda model of transgenic mice allowing the conditional ablation ofdividing DC precursors expressing an HSV1-TK gene upon GCVtreatment (24, 39). This enzyme allows the conversion of the non-toxic GCV into GCV triphosphate, which can be incorporated intoelongating DNA, inducing elongation termination and ultimatelycell death. Because only those cells that express HSV1-TK andthat are dividing can be killed, size variation of a cell populationduring a GCV treatment reflects the turnover of these cells if theydivide or have a dividing precursor. However, this system will not

FIGURE 2. Morphology and MHC class II immunostaining of s-cells (left) and l-cells (right). After collagenase digestion of brachial, axillary, andinguinal LN from 35 mice, enrichment of low buoyant density cells, and double staining with a mouse mAb to I-E and a hamster mAb to CD11c,s- and l-cells were FACS sorted. Both populations were cytocentrifuged, and cytospins were further stained with a rat mAb against I-A/I-E (M5/114)revealed by peroxidase anti-rat IgG, slightly counter-colored with hematoxylin. Magnification: 31000.



ww

.jimm

unol.org/D

ownloaded from


FIGURE 3. Cell surface phenotype reveals three LN DC populations. After collagenase digestion of peripheral LN, the phenotype of s-cells (A,C) and l-cells (B) was determined by three (A, B)- and four-color (C) flow cytometry analysis. 300,000 to 500,000 events were acquired. (A, B)s- and l-cells gated based on I-E (TriColor) and CD11c (PE) staining were further analyzed for additional indicated markers (FITC). White histogramsshow negative controls labeled with the same amount of isotype-matched control mAbs. Each staining was performed two to four times withsimilar results, except for the mAb to B220 Ag, which sometimes labeled some of the l-cells. C, s-cells, gated based on I-A (allophycocyanin) andCD11c (PE) staining, were further analyzed for CD4 (TriColor) and additional indicated markers (FITC). The arrowheads on the left and right sidesof the dot plots indicate the mean fluorescence intensity for the two populations delineated by the dashed vertical line.



ww

.jimm

unol.org/D

ownloaded from


discriminate between the rapid turnover of a resident cell popula-tion that divides in situ or of a circulating cell population with adividing precursor. Using this model, we previously showed therapid turnover of spleen DCs, amounting to 10 to 15% renewal perday, in agreement with previous measurements obtained with othertechniques (21). Here, we found that a 7-day GCV treatment led tothe almost complete disappearance of both populations of s-DCs,while the l-DC population was only slightly reduced (Table I).These results reveal further differences between the three popula-tions of LN DCs, sm- and sl-DC having a rapid turnover, l-DC aslow turnover.

Dramatic increase in the sl-DC population after Flt3 ligandtreatment

The in vivo administration of Flt3 ligand, a stimulator of hemo-poietic progenitor cells, has recently been described to induce hy-pertrophy of lymphoid tissues and to dramatically increase the pro-portion of spleen DC (45, 46). The effects of Flt3 ligand on thedistinct subpopulations of LN DCs were analyzed by three-colorflow cytometry after a 7-day treatment. The l-DCs did not appearto be affected by Flt3 ligand. In contrast, the proportion of s-DCsreached 30% of total LN cells vs 0.5% in controls (Fig. 7A). Theexpanded population did not express CD4, F4/80, or Mac-1, butexpressed low levels of CD8a and high levels of HSA, indicatingthat Flt3 ligand treatment affected mostly if not exclusively thesl-DC subpopulation (Fig. 7B). A moderate increase in the otherDC populations could be masked by the dramatic increase of thesl-DCs.

DiscussionIdentification of three distinct populations of lymph nodeDCs

In mouse lymphoid tissues, N418 is the only mAb that stronglyreacts with DCs but not with freshly isolated macrophages or lym-

phoid cells (29, 41). We looked for a possible heterogeneity ofDCs in LN within CD11c1, as already described in the spleen (27,29, 32, 47) and in Peyer’s patches (30). We used double-staininganalysis of whole LN cells without any purification procedure orculture, which could have modified the cell characteristics or re-sulted in the loss of a DC subpopulation. Three distinct CD11c1/MHC class II1 populations were clearly disclosed by four-colorflow cytometry analysis. Given their overall heterogeneity, it isadmitted that identification of DCs is based on a combination ofcharacteristics including: 1) their morphology, with the existenceof dendrites; 2) their phenotype, showing the presence of mole-cules involved in T cell activation; and 3) their T cell stimulatorycapacity (1). The three CD11c1/MHC class II1 cell populationsfulfill all of these criteria and can thus be considered astypical DCs.

Due to their high level cell surface expression of MHC class IIand costimulatory molecules, their size, and their dendritic mor-phology with numerous and long cell processes, l-DCs resemblemature DCs. On the other hand, due to the mainly intracellularlocation of MHC class II molecules, their smaller size, and theirsmaller dendrites, both populations of s-DCs resemble more im-mature DCs (15, 17, 48). Despite differences in MHC class IIexpression, all of these cells strongly stimulate in vitro allogeneicresponses to similar levels. This is probably due to a rapid in vitromaturation of s-DCs during the culture, as suggested by prelimi-nary experiments (data not shown).

Expression of the Mac-1 and F4/80 myeloid markers on thesm-DC population supports a myeloid origin of these cells. CD4expression does not argue against such a hypothesis, since CD4 isnot considered a marker of lymphoid-related DCs (49). Nonethe-less, sm-DC are the only identified DCs that clearly express CD4at levels similar to that of CD4 T cells. In addition, based on 33D1expression and low HSA expression levels, sm-DCs resemble mar-ginal zone splenic DCs (26, 29), a subpopulation considered to bemyeloid related (9, 50). In contrast, sl-DCs expressed CD8a, amarker considered to define lymphoid-related DCs (9). sl-DCs alsoexpress DEC-205 and high levels of HSA, thus resembling splenicDCs of the T cell zone that have been considered lymphoid related(9, 26). Finally, the observation that Flt3 ligand treatment inducedthe expansion of only sl-DCs reinforces the hypothesis that the twos-DCs populations have distinct lineages.

Different turnover of the lymph node DCs

In addition to these phenotypic differences, we showed that theseDC subpopulations have a different turnover by using an animalmodel of conditional ablation of HSV1-TK-expressing DC uponGCV treatment. Using this model, we previously showed that a

FIGURE 4. A fraction of l-DCs is derived from epi-dermal Langerhans cells. At different time points af-ter skin painting with FITC, cells from the drainingbrachial LN of painted mice and cells from a controlmouse were stained with mAbs to MHC class II andCD11c. They were analyzed by flow cytometry fortheir size (forward scatter) and FITC fluorescence.Results are from the analysis of 1.2 3 105 LN cellsfor s- and l-DCs, gated as previously described, andof 4 3 104 LN cells for “other cells.” One represen-tative of three experiments is shown.

Table I. Turn-over of s-DC and I-DC a

s-DC I-DC

Control mice 0.60 6 0.10 1.60 6 0.15TK mice 0.05 6 0.04 1.25 6 0.25

a Turn-over of s- and 1-DC was evaluated using lethally irradiated mice re-constituted with bone marrow cells derived from transgenic mice expressingherpes simplex virus type 1-thymidine kinase in DCs (TK mice) as previouslydescribed (24, 39). In this system, only the dividing DC precursors are killed byganciclovir treatment, allowing the assessment of DC turnover. After a 7-dayganciclovir treatment of control and chimeric mice, s- and 1-DCs were analysedas described in Figure 1. Results are given as the percentage of total LN cellnumber (mean 6 SD of four independent experiments).



ww

.jimm

unol.org/D

ownloaded from


FIGURE 5. sm-DCs rapidly uptake a plasmatic tracer. A, Size (forward scatter) and FITC fluorescence of inguinal LN cells analyzed by flowcytometry 30 min after i.v. injection of 5 mg of FITC-dextran (m.w. 40,000). s-DCs and l-DCs were gated as described in Figure 1. Controlsrepresent the LN staining of a noninjected mouse. B, Same analysis performed at different times postinjection with brachial or inguinal LN cells.FITC-positive cells correspond to cells with a fluorescence intensity at least 10 times higher than that of unlabeled cells. Data from brachial andinguinal LNs were similar and were pooled, as were experiments using 5 or 10 mg of FITC-dextran (m.w. 40,000). Data show the means 6 SDof independent experiments. Except for the analysis at day 5 (one experiment), each time point was performed at least three times (18 times forthe 30-min time point). C, FITC-dextran staining at 30 min after i.v. injection among s-DCs gated based on I-A (allophycocyanin) and CD11c (PE).The vertical dashed line separates the sm- and sl-DCs based on CD4 expression (see Fig. 3). This staining was performed twice, 30 min and 12 hafter i.v. injection.



ww

.jimm

unol.org/D

ownloaded from


7-day GCV treatment led to complete disappearance of splenicDCs of the white pulp marginal zone, while LCs that also ex-pressed HSV1-TK were minimally affected (24, 39). Since GCVkills only HSV1-TK-expressing cells that are dividing (51), theseexperiments indicate that the splenic DCs have a very rapid turn-over, whereas LC have a slow turnover, in agreement with previ-ous observations (19–21). Using the same experimental system,we show here that sm- and sl-DC populations have a rapid turn-over, since a 7-day GCV treatment resulted in the disappearance ofalmost all s-DCs. This rapid turnover could be due to either a highdivision rate of the s-DC themselves or to a rapid transit into LNof cells that have a rapidly dividing precursor. Additional experi-ments will be needed to address this issue. On an other hand, GCVtreatment induced only a slight depletion of l-DCs (1.2 vs 1.6% incontrol mice). This result is compatible with the persistence of theLC-derived l-DC and the disappearance of the sm-DC-derivedl-DC (see below).

Origin of the l-DCs

The majority of l-DCs come from the peripheral territories drainedby the regional LN. Indeed, at 3, 12, 24, and 120 h after FITC skinpainting, l-DCs but not s-DCs were FITC stained. Because therewas a progressive increase in the proportion of l-DC-stained cellsfrom 12 to 24 h after skin painting, without any detectable staineds-DCs throughout this time period, it is unlikely that we could havemissed a transition from stained s-DCs to l-DCs. This suggests thatneither sm- nor sl-DCs represent an intermediate differentiationstage from LCs to l-DCs and that LCs have completed their phe-notypic maturation when they arrive in the draining LN. Thesel-DCs were thus derived from LCs, which themselves are believedto derive from the CFU-DC progenitors yielding pure DC colonies

in semisolid medium (3, 12). A maximum of 60% of all l-DCs wasstained after skin painting. This may indicate that not all the ter-ritory drained by the analyzed LN was painted and/or that somel-DCs have a different origin. In this respect, there is some evi-dence that a fraction of the l-DC population is derived from sm-DCs. First, sm-DCs were the first to be stained after i.v. injectionof FITC-dextran, followed by a delayed staining in l-DCs, whichappeared with kinetics compatible with a maturation of sm-DC tol-DC. Second, although CD11c expression in l-DCs was quite het-erogeneous, those l-DCs stained by the plasmatic tracer had aCD11c expression that was more homogeneous and was similar tothat of s-DCs. Finally, preliminary experiments suggest that somes-DCs can evolve toward an l-DC-like phenotype upon culture(data not shown).

Altogether, these results suggest that LNs contain DCs from thethree DC lineages that have been proposed so far, originating from:1) a pure DC progenitor (for most of l-DC) (3, 12); 2) a commonmyeloid/DC precursor (for sm-DC) (3, 4); and 3) a common lym-phoid/DC precursor (for sl-DC) (10).

Capture of blood Ag by sm-DCs

Intravenous injection of FITC-dextran led to a remarkably rapidstaining of sm-DCs. This staining was observed with both 40,000and 150,000 m.w. FITC-dextran. These molecules are known toremain in the blood circulation and are actually used in humans toreplenish the plasma compartment. Therefore, it is unlikely thatthey diffused directly from the blood to the LN parenchyma. Onthe contrary, the staining was specific in many ways. First, onlysm-DCs, representing 0.2% of all LN cells, were stained by FITC-dextran 30 min after i.v. injection, whereas all of the LN cells werestained when incubated with FITC-dextran in vitro (data notshown). Second, contamination with plasma molecules present inthe LN blood vessels and occurring during preparation of LN cells

FIGURE 6. MHC class II/CD11c phenotype and FITC fluorescenceintensity of FITC-positive cells at different time points after i.v. injec-tion of FITC-dextran. At the different indicated time points after i.v.injection of FITC-dextran, A, FITC-positive cells (as determined in Fig.5) were analyzed for cell surface MHC class II and CD11c expression;and B, s- and l-DCs, gated as previously described, were analyzed forFITC fluorescence intensity. Results are from the analysis of 1.2 3 105

axillary LN cells. One representative experiment of three is shown.

FIGURE 7. sl-DC numbers dramatically increase after in vivo ad-ministration of Flt3 ligand. A, Peripheral LN cells of a control mouse(left) and a mouse treated for 7 days with Flt3 ligand at 10 mg/day(right) were analyzed for cell surface MHC class II and CD11c expres-sion. Gated cells that correspond to the s-DC of the control mouse(0.4% of LN cells) represent 30% of total LN cells of the Flt3 ligand-treated mouse. B, The phenotype of gated cells of this mouse wasdetermined by three-color flow cytometry analysis for the third indi-cated Ag. White histograms show labeling performed with the sameamount of isotype-matched controls.



ww

.jimm

unol.org/D

ownloaded from


was ruled out; no staining could be observed when LN cells froman untreated mouse were prepared in the presence of the plasma ofa FITC-dextran treated mouse (data not shown). Finally, staineds-DCs were only detected in peripheral but not in mesenteric LN.These observations also indicate that the staining of sm-DCs wasnot due to passive diffusion of FITC-dextran by blood vessel leak-age. Therefore, these results suggest that FITC-dextran was ac-quired within the blood compartment by sm-DCs; this could bedue to either the existence of DCs within the blood vessel endo-thelium layer of peripheral LNs or to a translocation of blood DCsto LNs. In this respect, we observed that the intensity of FITCstaining in sm-DCs increased with time, suggesting that some sm-DCs were exposed several times to the plasmatic tracer. Thesecells may therefore be extremely mobile, migrating from the bloodto LNs and back to the blood. Such a recirculation has not beenobserved for spleen or lymph-borne DCs (52–54). Altogether, ourresults suggest a possible circulation of DC from blood to LN.Further careful analysis will be required to clarify this importantpoint, which conflicts with the current paradigm for DCcirculation (38).

Until now, Ags delivered through the blood supply have beenassumed to be trapped mainly by APCs in the spleen, as well as inthe liver for particulate Ags (38, 55). Our results demonstrate thatregardless of the mechanism, previously unidentified LN DCs canspecifically uptake a plasmatic molecule. These cells, therefore,might play an important role in the control of blood pathogens. Inthis line, this cell population seems to play a role in the transportof HIV from the blood to the LNs (our manuscript in preparation).

In conclusion, we show for the first time the existence of a DCheterogeneity in the LN. The three DC populations identified ap-pear to belong to distinct lineages and to differ in their capacity touptake Ags administrated by different routes. These results, to-gether with further analysis of the functional properties of theseDCs, may have important implications for a better understandingof the relationship between the route of Ag introduction and thenature of the subsequent immune response.

AcknowledgmentsWe thank Drs. Genevieve Milon, Polly Matzinger, Olivier Boyer, andPieter Leenen for helpful discussions, Drs. Jean Claude Gluckman andMichelle Rosenzwajg for critical reading of the manuscript, Sylvie Brueland Catherine Pioche for their contribution to some of the experiments,Dr. Eugene Maraskovsky, Immunex Corp., for providing us with the flt3ligand, and Genevieve Milon and Jeffrey Bluestone for providing some ofthe mAbs.

References1. Steinman, R. M. 1991. The dendritic cell system and its role in immunogenicity.

Annu.Rev.Immunol. 9:271.2. Inaba, K., M. Inaba, N. Romani, H. Aya, M. Deguchi, S. Ikehara, S. Muramatsu,

and R. M. Steinman. 1992. Generation of large numbers of dendritic cells frommouse bone marrow cultures supplemented with granulocyte/macrophage colo-ny-stimulating factor.J. Exp.Med. 176:1693.

3. Caux, C., B. Vanbervliet, C. Massacrier, C. Dezutter-Dambuyant,B. de Saint-Vis, C. Jacquet, K. Yoneda, S. Imamura, D. Schmitt, andJ. Banchereau. 1996. CD341 hematopoietic progenitors from human cord blooddifferenciate along two independent dendritic cell pathways in response toGM2CSF1TNFa. J. Exp.Med. 184:695.

4. Inaba, K., M. Inaba, M. Deguchi, K. Hagi, R. Yasumizu, S. Ikehara,S. Muramatsu, and R. M. Steinman. 1993. Granulocytes, macrophages, and den-dritic cells arise from a common major histocompatibility complex class II-neg-ative progenitor in mouse bone marrow.Proc. Natl. Acad.Sci USA 90:3038.

5. Sallusto, F., and A. Lanzavecchia. 1994. Efficient presentation of soluble antigenby cultured human dendritic cells is maintained by granulocyte/macrophage col-ony-stimulating factor plus interleukin 4 and downregulated by tumor necrosisfactor alpha.J. Exp.Med. 179:1109.

6. Romani, N., S. Gruner, D. Brang, E. Kampgen, A. Lenz, B. Trockenbacher,G. Konwalinka, P. O. Fritsch, R. M. Steinman, and G. Schuler. 1994. Prolifer-ating dendritic cell progenitors in human blood.J. Exp.Med. 180:83.

7. Zhou, L. J., and T. F. Tedder. 1996. CD141 blood monocytes can differentiateinto functionally mature CD831 dendritic cells.Proc. Natl. Acad.Sci USA 93:2588.

8. Ardavin, C., L. Wu, C. L. Li, and K. Shortman. 1993. Thymic dendritic cells andT cells develop simultaneously in the thymus from a common precursor popu-lation. Nature 362:761.

9. Wu, L., C.-L. Li, and K. Shortman. 1996. Thymic dendritic cell precursor: rela-tionship to the T lymphocyte lineage and phenotype of the dendritic cell progeny.J. Exp.Med. 184:903.

10. Saunders, D., K. Lucas, J. Ismaili, L. Wu, E. Maraskovsky, A. Dunn, andK. Shortman. 1996. Dendritic cell development in culture from thymic precursorcells in the absence of granulocyte/macrophage colony-stimulating factor.J. Exp.Med. 184:2185.

11. Galy, A., D. Travis, D. Cen, and D. Chen. 1995. Human T, B, natural killer anddendritic cells arise from a common bone marrow progenitor subset.Immunity3:459.

12. Young, J. W., P. Scabolcs, and M. A. S. Moore. 1995. Identification of dendriticcell colony-forming units among normal human CD341 bone marrow progeni-tors that are expanded by c-kit-ligand and yield pure dendritic cell colonies in thepresence of granulocyte/macrophage colony-stimulating factor and tumor necro-sis factora. J. Exp.Med. 182:1111.

13. Strobl, H., E. Riedl, C. Scheinecker, C. Bello-Fernandez, W. F. Pickl,K. Rappersberg, O. Majdic, and W. Knapp. 1996. TGF-b1 promotes in vitrodevelopment of dendritic cells from CD341 hematopoietic progenitors.J. Im-munol. 157:1499.

14. Borkowski, T. A., J. J. Letterio, A. G. Farr, and M. C. Udey. 1996. A role forendogenous transforming growth factorb1 in langerhans cell biology: the skin oftransforming growth factorb1 null mice devoid of epidermal langerhans cells.J.Exp.Med. 184:2417.

15. Schuler, G., and R. M. Steinman. 1985. Murine epidermal Langerhans cells ma-ture into potent immunostimulatory dendritic cells in vitro.J. Exp.Med. 161:526.

16. Romani, N., S. Koide, M. Crowley, M. Witmer-Pack, A. M. Livingstone,C. G. Fathman, K. Inaba, and R. M. Steinman. 1989. Presentation of exogenousprotein antigens by dendritic cells to T cell clones: intact protein is presented bestby immature, epidermal Langerhans cells.J. Exp.Med. 169:1169.

17. Sallusto, F., M. Cella, C. Danieli, and A. Lanzavecchia. 1995. Dendritic cells usemacropinocytosis and the mannose receptor to concentrate macromolecules in themajor histocompatibility complex class II compartment: downregulation by cy-tokines and bacterial products.J. Exp.Med. 182:389.

18. Fossum, S. 1989. Dendritic leukocytes: features of their in vivo physiology.Res.Immunol. 140:883.

19. Katz, S. I., K. Tamaki, and D. H. Sachs. 1979. Epidermal Langerhans cells arederived from cells originating in bone marrow.Nature 282:324.

20. Chen, H. D., C. L. Ma, J. T. Yuan, Y. K. Wang, and W. K. Silvers. 1986.Occurrence of donor Langerhans cells in mouse and rat chimeras and their re-placement in skin grafts.J. Invest.Dermatol. 86:630.

21. Steinman, R. M., D. S. Lustig, and Z. A. Cohn. 1974. Identification of a novel celltype in peripheral lymphoid organs of mice. III. Functional properties in vivo.J.Exp.Med. 139:1431.

22. Pugh, C. W., G. G. MacPherson, and H. W. Steer. 1983. Characterization of nonlymphoid cells derived from rat peripheral lymph.J. Exp.Med. 157:1758.

23. Holt, P. G., S. Haining, D. J. Nelson, and J. D. Sedgwick. 1994. Origin andsteady-state turnover of class II MHC-bearing dendritic cells in the epithelium ofthe conducting airways.J. Immunol. 153:256.

24. Salomon, B., P. Lores, C. Pioche, P. Racz, J. Jami, and D. Klatzmann. 1994.Conditional ablation of dendritic cells in transgenic mice.J. Immunol. 152:537.

25. Kraal, G., M. Breel, M. Janse, and G. Bruin. 1986. Langerhans’ cells, veiled cells,and interdigitating cells in the mouse recognized by a monoclonal antibody.J.Exp.Med. 163:981.

26. Crowley, M., K. Inaba, M. Witmer-Pack, and R. M. Steinman. 1989. The cellsurface of mouse dendritic cells: FACS analyses of dendritic cells from differenttissues including thymus.Cell. Immunol. 118:108.

27. Agger, R., M. Witmer-Pack, N. Romani, H. Stossel, W. J. Swiggard, J. P. Metlay,E. Storozynsky, P. Freimuth, and R. M. Steinman. 1992. Two populations ofsplenic dendritic cells detected with M342, a new monoclonal to an intracellularantigen of interdigitating dendritic cells and some B lymphocytes.J. LeukocyteBiol. 52:34.

28. De Smedt, T., B. Pajak, E. Muraille, B. Lespagnard, E. Heinen, P. De Baetselier,J. Urbain, O. Leo, and M. Moser. 1996. Regulation of dendritic cell numbers andmaturation by lipopolysaccharide in vivo.J. Exp.Med. 184:1413.

29. Witmer-Pack, M. D., M. T. Crowley, K. Inaba, and R. M. Steinman. 1993. Mac-rophages, but not dendritic cells, accumulate colloidal carbon following admin-istration in situ.J. Cell Sci. 105:965.

30. Kelsall, B. L., and W. Strober. 1996. Distinct populations of dendritic cells arepresent in the subepithelial dome and T regions of the murine Peyer’s patch.J.Exp.Med. 183:237.

31. Grouard, G., I. Durand, L. Filgueira, J. Banchereau, and Y. J. Liu. 1996. Den-dritic cells capable of stimulating T cells in germinal centres.Nature 384:364.

32. Suss, G., and K. Shortman. 1996. A subclass of dendritic cells kills CD4 T cellsvia Fas/Fas-ligand-induced apoptosis.J. Exp.Med. 183:1789.

33. Kronin, V., K. Winkel, G. Suss, A. Kelso, W. Heath, J. Kirberg, H. von Boehmer,and K. Shortman. 1996. A subclass of dendritic cells regulates the response ofnaive CD8 T cells by limiting Il-2 production.J. Immunol. 157:3819.

34. Witmer, M. D., and R. M. Steinman. 1984. The anatomy of peripheral lymphoidorgans with emphasis on accessory cells: light-microscopic immunocytochemicalstudies of mouse spleen, lymph node, and Peyer’s patch.Am.J. Anat. 170:465.



ww

.jimm

unol.org/D

ownloaded from


35. Hoefsmit, E. C., A. M. Duijvestijn, and E. W. Kamperdijk. 1982. Relation be-tween Langerhans cells, veiled cells, and interdigitating cells.Immunobiology161:255.

36. Bujdoso, R., J. Hopkins, B. M. Dutia, P. Young, and I. McConnell. 1989. Char-acterization of sheep afferent lymph dendritic cells and their role in antigen car-riage.J. Exp.Med. 170:1285.

37. Liu, L. M., and G. G. MacPherson. 1993. Antigen acquisition by dendritic cells:intestinal dendritic cells acquire antigen administered orally and can prime naiveT cells in vivo.J. Exp.Med. 177:1299.

38. Austyn, J. M. 1996. New insights into the mobilization, and phagocytic activityof dendritic cells.J. Exp.Med. 183:1287.

39. Salomon, B., C. Pioche, P. Lores, J. Jami, P. Racz, and D. Klatzmann. 1995.Conditional ablation of dendritic cells in mice: comparison of two animal models.Adv.Exp.Med.Biol. 378:485.

40. Coligan, J., A. Kruisbeeck, D. Margulies, E. Shevach, and W. Strober. 1995.Current Protocols in Immunology.J. Wiley & Sons, New York.

41. Metlay, J. P., M. D. Witmer-Pack, R. Agger, M. T. Crowley, D. Lawless, andR. M. Steinman. 1990. The distinct leukocyte integrins of mouse spleen dendriticcells as identified with new hamster monoclonal antibodies.J Exp Med. 171:1753.

42. Macatonia, S. E., S. C. Knight, A. J. Edwards, S. Griffiths, and P. Fryer. 1987.Localization of antigen on lymph node dendritic cells after exposure to the con-tact sensitizer fluorescein isothiocyanate: functional and morphological studies.J.Exp.Med. 166:1654.

43. Kripke, M., G. Munn, A. Jeevan, J. Tang, and C. Bucana. 1990. Evidence thatcutaneous antigen-presenting cells migrate to regional lymph nodes during con-tact sensitization.J. Immunol. 145:2833.

44. Buchweitz-Milton, E., and H. R. Weiss. 1988. Perfused microvascular morphom-etry during middle cerebral artery occlusion.Am.J. Physiol. 255:623.

45. Brasel, K., H. McKenna, P. Morrissey, K. Charrier, A. Morris, C. Lee,D. Williams, and S. Lyman. 1996. Hematologic effect of flT 3 ligand in vivo inmice.Blood 88:2004.

46. Maraskovsky, E., K. Brasel, M. Tepee, E. R. Roux, S. D. Lyman, K. Shortman,and H. J. McKenna. 1996. Dramatic increase in the number of functionally ma-

ture dendritic cells in flT3 ligand-treated mice: multiple dendritic cell subpopu-lations identified.J. Exp.Med. 184:1953.

47. Crowley, M. T., K. Inaba, M. D. Witmer-Pack, S. Gezelter, and R. M. Steinman.1990. Use of the fluorescence-activated cell sorter to enrich dendritic cells frommouse spleen.J. Immunol.Methods 133:55.

48. Larsen, C. P., P. J. Morris, and J. M. Austyn. 1990. Migration of dendritic leu-kocytes from cardiac allografts into host spleens: a novel pathway for initiationof rejection.J. Exp.Med. 171:307.

49. Wu, L., D. Vremec, C. Ardavin, K. Winkel, G. Suss, H. Georgiou,E. Maraskovsky, W. Cook, and K. Shortman. 1995. Mouse thymic dendritic cells:kinetics of development and changes in surface markers during maturation.Eur.J. Immunol. 25:418.

50. Shortman, K., L. Wu, D. Vremec, G. Suss, and K. Lucas. 1996. Development andfunction of lymphoid-related dendritic cells [abstract].Fourth International Sym-posium on Dendritic Cells in Fundamental and Clinical Immunology.RicercaScientifica ed Educazione Permanente, Milan, Italy.

51. St. Clair, M. H., C. U. Lambe, and P. A. Furman. 1987. Inhibition by ganciclovirof cell growth and DNA synthesis of cells biochemically transformed with her-pesvirus genetic information.Antimicrob.Agents Chemother. 31:844.

52. Kupiec-Weglinski, J. W., J. M. Austyn, and P. J. Morris. 1988. Migration patternsof dendritic cells in the mouse: traffic from the blood, and T cell-dependent and-independent entry to lymphoid tissues.J. Exp.Med. 167:632.

53. Fossum, S. 1988. Lymph-borne dendritic leucocytes do not recirculate, but enterthe lymph node paracortex to become interdigitating cells.Scand.J. Immunol.27:97.

54. Morikawa, Y., K. Tohya, H. Ishida, N. Matsuura, and K. Kakudo. 1995. Differentmigration pattern of antigen-presenting cells correlate with Th1/Th2-type re-sponses in mice.Immunology 85:575.

55. Matsuno, K., T. Ezaki, S. Kudo, and Y. Uehara. 1996. A life stage of particle-laden rat dendritic cells in vivo: their terminal division, active phagocytosis, andtranslocation from the liver to the draining lymph.J. Exp.Med. 183:1865.



ww

.jimm

unol.org/D

ownloaded from


Documents

Three populations of mouse lymph node dendritic cells with different origins and dynamics