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DCs are ‘professional’ antigen-presenting cells (APCs) capable of acti-vating naive CD4 and CD8 T cells. DCs act as sentinels in vivo, andthey transport antigens from nonlymphoid peripheral tissues todraining lymph nodes. In nonlymphoid tissues such as skin, DCs existin an immature form, highly proficient in acquiring and processingantigen. After antigen acquisition, DCs undergo maturation, duringwhich they lose their ability to acquire antigens and transform intohighly potent APCs by up-regulating expression of the major histo-compatibility complex (MHC) class I and class II molecules and Tcell–stimulatory surface molecules such as CD40, CD80 and CD86.Mature DCs also secrete immune stimulatory cytokines that influencethe type of immune response generated (T helper cell type 1 or 2).Maturation also induces DCs to migrate from nonlymphoid tissues todraining lymph nodes, where they present antigens as processed pep-tides to naive CD4 and CD8 T cells, thus inducing rapid and robust Tcell responses1–7.

The migration of DCs from the skin to the draining lymph nodes hasbeen studied by immunization with a gene gun or by epicutaneousapplication of fluorescent contact sensitizers. Immunization with DNAvaccines by gene gun represents a new approach in which gold particlescoated with mammalian expression plasmid DNA encoding genes frompathogenic organisms are bombarded into the skin7,8. This bombard-ment transfects plasmid DNA directly into the extensive network ofDCs present in the skin. Transfected DCs express the encoded antigenicgene products and present the processed peptides to rare antigen-specific T cells to initiate an immune response in draining lymphnodes9,10. Immunization by gene gun with DNA vaccine plasmids

containing reporter molecules such as lacZ, which encodes β-galactosi-dase (β-gal), has been used to track antigen-specific DCs from the skininto the draining lymph nodes8. With this approach, it was shown thatthe DCs that migrate from the skin to the draining lymph nodes arerare (about 50–100 per lymph nodes) and short-lived (lifespan, 48 h;ref.8). DC migration has been studied by epicutaneous administrationof fluorescent contact sensitizers. These experiments showed that thefluorescent DCs that migrated to the draining lymph nodes disap-peared in 3–4 d (refs. 11–13).

T cell receptors (TCRs) are generated by stochastic gene-segmentrearrangements that produce a large repertoire of antigen-bindingspecificities. In the T cell repertoire, the frequency of T cells specific fora particular antigen-MHC complex is very low and it has been esti-mated to be less than 1 in 100,000. How such small numbers of short-lived DCs1,8,14 seek out and activate rare antigen-specific T cells indraining lymph nodes is unclear. One possibility is that the frequencyand lifespan of antigen-bearing mature DCs that migrate to the drain-ing lymph nodes are substantially greater than previously estimated.The approaches used so far for tracking DCs (epicutaneous adminis-tration of contact sensitizers and immunization with reporter plas-mids by gene gun) have been expedient and informative, but themarking of DCs has been transient because of dilution of fluo-rochrome or silencing of gene expression by the reporter-encodingDNA plasmid. Here, we used a highly sensitive in vivo system to per-manently mark antigen-bearing DCs through a Cre/loxP recombina-tion strategy15. Using this system, we tracked the migration,magnitude, phenotype and duration of the dendritic cell response.

1Department of Microbiology and Immunology, Vaccine Research Center, Yerkes National Primate Research Center, Emory University, 954 Gatewood Road, Atlanta, Georgia 30322, USA. 2Department of Ophthalmology, Emory University, Atlanta, Georgia 30322, USA. 3Department of Bioregulation, Leprosy ResearchCenter, National Institute of Infectious Diseases, 4-2-1 Aoba-cho, Higashimurayama, Tokyo 182-0002, Japan. Correspondence should be addressed to J.J. (jjacob3@emory.edu).

Published online 10 August 2003; doi:10.1038/ni962

Genetic tagging shows increased frequency andlongevity of antigen-presenting, skin-deriveddendritic cells in vivoSanjay Garg1, Alp Oran1, Janine Wajchman2, Shin Sasaki3, Charles H Maris1, Judith A Kapp2 & Joshy Jacob1

Dendritic cells (DCs) are key regulators of immune responses that activate naive antigen-specific T lymphocytes. In draininglymph nodes, antigen-bearing DCs are reported to be rare and short-lived. How such small numbers of short-lived DCs canactivate rare antigen-specific T cells is unclear. Here we show that after immunization of mouse skins by gene gun, the numberof antigen-bearing DCs that migrate to draining lymph node is 100-fold higher than previously estimated and that they persist forapproximately 2 weeks. The substantial frequency and longevity of DCs in situ ensures ample antigen presentation andstimulation for the rare antigen-specific T cells in draining lymph nodes.

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After immunizing mice with plasmid DNA delivered by a gene gun, wefound that the number of antigen-bearing DCs in the draining lymphnode was 100-fold higher than previously estimated8 and that theypersisted in the draining lymph nodes for approximately 2 weeks. TheDCs that migrated from the skin to the lymph nodes wereCD11c+CD11b+CD80+CD86+DEC205+MHC class IIhi Langerhanscell–derived DCs that were capable of stimulating cytokine productionand proliferation in antigen-specific T cells directly ex vivo.

RESULTSIrreversible tagging of DCsTo develop a method for permanently marking antigen-bearing DCswith high efficiency in vivo, we used the Cre-indicator transgenicmouse strain ROSA26R16. In this strain of mice, a loxP-flankedneomycin cassette followed by the bacterial lacZ gene has been targeteddownstream of the ubiquitous ROSA promoter17. The ROSA promoteris highly active in all tissues, but the expression of the downstream dor-mant lacZ occurs only after Cre-mediated recombination and excisionof the intervening loxP-flanked neomycin cassette (Fig. 1). We used agene gun to deliver gold bullets coated with the eukaryotic expressionplasmid containing the cytomegalovirus immediate early promoter/enhancer (pCMV-Cre) to the skin of ROSA26R mice. DCs, epithelial

cells and keratinocytes at the site of immunization are directly trans-fected10 with the Cre plasmid, which causes efficient recombination ofthe loxP-flanked neomycin cassette and expression of β-gal. Transfectedcells express β-gal for the remainder of their lifespan without the needfor continuous expression of the Cre transgene. This provides a moreaccurate method to track DCs to the draining lymph nodes than meth-ods previously used, which have been limited by transient expression ofthe reporter. At 2.5 d after immunization, we hypotonically loaded cellsfrom draining inguinal lymph nodes with the β-gal substrate fluores-cein-β-D-di-galactopyranoside (FDG), and quantified fluorescent cellsby flow cytometry18. We found β-gal+ cells exclusively in the CD11c+

fraction from mice immunized with pCMV-Cre. In the lymph nodes,CD11c+ DCs account for only a small fraction (2–4%) of the totalnumber of cells in the draining lymph nodes, and we routinelyenriched CD11c+ DCs by magnetic sorting to >95% purity. At 60 hafter immunization, approximately 12% of the purified CD11c+ DCs inthe lymph nodes from mice immunized with pCMV-Cre were β-gal+

(Fig. 1d), whereas we found no β-gal+ cells in mice that were notimmunized (Fig. 1b) or in ROSA26R mice immunized with controlDNA plasmid encoding influenza hemagglutinin (Fig. 1c). None of theCD11c– cells (Fig. 1c) residing in the draining lymph nodes, whichincludes CD8 T cells, CD4 T cells, macrophages and B cells, expressed

908 VOLUME 4 NUMBER 9 SEPTEMBER 2003 NATURE IMMUNOLOGY

Figure 1 Cutaneous bombardment of ROSA26R mice with pCMV-Creplasmid DNA marks CD11c+ DCs that migrate to draining lymph nodes. (a) The ROSA26R transgene comprises the ubiquitous ROSA promoterfollowed by loxP (triangles), flanked by a transcriptional stop fragment andlacZ16. The stop fragment contains the phosphoglycerate kinase (pgk)promoter–driven neomycin phosphotransferase (neo) expression cassette andthe four tandem polyadenylation (pA4) sequences, which blocks transcrip-tion of the downstream lacZ. Transient Cre recombinase expression leads to irreversible deletion of the loxP-flanked neo cassette and permanentexpression of β-gal. (b–d) ROSA26R mice (five mice per group) wereimmunized with gold bullets coated with plasmid DNA encoding HA (b) orCre recombinase (c,d). Then, 60 h later, the lymph nodes were pooled, andCD11c+ DCs were enriched using magnetic sorting and analyzed by flowcytometry for expression of β-gal (CD11c– fraction, c; CD11c+ fraction, d).Percentages indicate the frequency of β-gal+ cells. Data are representative of >10 independent experiments.

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Figure 2 Phenotype of β-gal+ DCs. (a–g) Lymphnode cells from ROSA26R mice (n = 5)immunized 3 d earlier with CMV-Cre plasmid werepooled and stained for β-gal and the cell surfacemarkers CD11c, CD11b, DEC205, CD80, CD86,CD4, CD8 and MHC class II (filled histogram) orfluorochrome-matched isotype control antibodies(open histogram). The phenotypes of β-gal+CD11c+ DCs are representative of threeindependent experiments. (h) Expression ofmRNA for mouse langerin and β actin,determined by RT-PCR. Lane 1, CD11c+β-gal+DEC205+ DCs; lane 2, CD11c+β-gal–DEC205– DCs; lane 3, no template (negativecontrol). The amount of input RNA wasstandardized using real-time PCR for β-actin(data not shown).

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β-gal. Thus, delivery of the Cre-containing plasmid to the skin inducedexpression of β-gal in draining lymph nodes that was restricted toCD11c+ DCs.

Phenotype of β-gal+ DCs in draining lymph nodesTwo types of DCs inhabit the skin: Langerhans cells that reside in thesuprabasal regions of the epidermis, and dermal DCs that reside in theperivascular areas of the superficial plexus in the dermis19. To deter-mine whether the gene gun targets Langerhans cells and/or dermalDCs, we analyzed the phenotype of the β-gal+ antigen-bearing DCsthat migrate from the skin to the draining lymph nodes (Fig. 2). All ofthe β-gal+ DCs had very high expression of MHC class II and the acti-vation markers CD80 and CD86 (Fig. 2a–c), which is analogous to thephenotype of activated, skin-derived DCs described before20. A smallfraction of the CD11c+β-gal+ DCs expressed CD4 (9.8%) or CD8α(3%; Fig. 2f,g). More than 80% of the CD11c+β-gal+ cells expressedDEC205 and CD11b (Fig. 2d,e), which is typical of Langerhanscell–derived DCs21. The definitive marker for Langerhans cells is thetype II lectin langerin, or CD207 (refs. 22,23). To test whetherDEC205+β-gal+ DCs were Langerhans cells, we used β-gal+CD11c+DEC205+ and β-gal–CD11c+DEC205– DCs fromROSA26R mice that had been immunized 3 d earlier with CMV-Creplasmid DNA. We prepared RNA from these subsets and assayedexpression of mouse langerin by RT-PCR using langerin-specificprimers24. We found langerin mRNA expression in the β-gal+DEC205+

but not the β-gal–DEC205– fraction (Fig. 2h). Thus, cutaneous bom-bardment with a gene gun targets mainly DEC205+ Langerhans cellsbut also targets a small fraction of dermal DCs, and both types of cellsmigrate to draining lymph nodes.

Transfection, not transfer, of Cre induces β-gal expressionCutaneous delivery of the pCMV-Cre plasmid by gene gun inducedexpression of β-gal in almost all epidermal cells. This raised the possi-bility that some of the CD11c+ cells in the lymph nodes might expressβ-gal by passive transfer of Cre protein or β-gal from other cells in theskin rather than by direct transfection. To test this, we targeted Creexpression to keratinocytes using a human keratinocyte 14 promoter(K14-Cre plasmid)25. Few, if any, CD11c+ DCs (or any other cell type)in the draining lymph nodes from ROSA26R mice immunized with theK14-Cre plasmid were β-gal+ on days 2, 8 or 12 (Fig. 3a). Nevertheless,we identified β-gal+ cells (keratinocytes) at the site of immunization by

immunohistochemical staining of frozen sections of abdominal skin ofmice immunized with K14-Cre (data not shown). Thus, the expressionof β-gal by DCs was solely due to direct transfection of plasmid DNAand not to passive uptake of the Cre recombinase gene, the Cre proteinor β-gal from other epidermal cells.

To rule out the possibility of a ‘trivial explanation’ for the higher fre-quency of β-gal+ cells seen in our system than found before8, we neededto determine whether Cre recombinase or β-gal could be transferredfrom dying immigrant DCs to resident DCs in the lymph nodes. Thus,we induced apoptosis in β-gal+ DCs by immunizing mice with a plas-mid that expressed both Cre recombinase (for β-gal marking) and cas-pase 2 (for induction of apoptosis). At 2.5 d after immunization, wefound only a few β-gal-marked DCs in the draining lymph nodes ofmice immunized with plasmids encoding Cre and caspase 2 comparedwith DCs from mice that received the pCMV-Cre plasmid (Fig. 3b).Our failure to detect substantial numbers of β-gal+ DCs in the lymphnodes of mice receiving Cre plus caspase 2 indicated that the trans-fected epidermal DCs may have died before reaching the lymph nodes.To allow caspase 2–expressing DCs to reach the lymph nodes in sub-stantial numbers and then undergo apoptosis, we reduced the caspase 2activity by using a plasmid that expressed Cre and a mutated caspase 2(ref. 26), which rendered the apoptotic activity 10% that of the wild-type caspase 2. If β-gal expression were due to direct transfection, thenDCs would be expected to undergo apoptosis due to caspase expres-sion, and hence fewer β-gal+ DCs would be found in the lymph nodes.However, if the number of β-gal+ DCs were equivalent to the numberfound with pCMV-Cre plasmid, that would indicate that some of the β-gal+ DCs passively acquired the marker. At 2.5 d, the frequency of β-gal+ DCs in mice immunized with the plasmid expressing Cre andmutant caspase 2 was similar to that of mice immunized with the plas-mid encoding Cre recombinase only (Fig. 3b). The number of β-gal+

DCs was reduced by day 7 and was undetectable by day 10 in recipientsof the mutant caspase 2 plasmid. Thus, apoptosis of β-gal+ DCs led todisappearance of marked DCs and not to spurious marking of neigh-boring resident DCs. Consequently, we conclude that the increased fre-quency of β-gal+ DCs in lymph nodes of mice receiving the pCMV-Creplasmid resulted from the improved efficiency of this system.

Kinetics of β-gal+ DCs in draining lymph nodesThe pCMV-Cre plasmid system showed that the frequency of immi-grant DCs was about 100-fold higher than that reported before8,

NATURE IMMUNOLOGY VOLUME 4 NUMBER 9 SEPTEMBER 2003 909

Figure 3 β-gal marking of DCs results from direct transfection of DCs with Cre recombinase–encoding plasmid DNA, not passive uptake of Cre recombinaseor β-gal protein. (a) ROSA26R mice were immunized by gene gun with CMV-Cre or keratinocyte-specific promoter-driven Cre recombinase (K14-Cre) plasmid.Draining lymph nodes from six mice per group were pooled (times, above dot plots) and analyzed by flow cytometry for % β-gal+CD11c+DCs. Data arerepresentative of two experiments. (b) ROSA26 mice (five mice per group) were immunized by gene gun with DNA vaccines encoding Cre only (black bars),Cre and caspase 2 (gray bars) or Cre and mutant caspase 2 (open bars; apoptotic activity is 10% that of wild-type caspase 2). Data represent the frequency(± s.e.m. of two independent experiments) of β-gal+ DCs; times, horizontal axis.

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which raised the possibility that the kinetics might also be differentfrom those reported before. Thus, we determined the kinetics of theappearance of β-gal+CD11c+ DCs in draining lymph nodes. Wedetected β-gal+CD11c+ DCs by 6–12 h after immunization (Fig. 4a).We found that 12% of the CD11c+ DCs, or approximately 5 × 103 DCsper inguinal lymph node, were β-gal+ at the peak of the response onday 2.5. β-gal+ DCs were detectable through day 14 (Fig. 4a) but not atlater time points (days 15, 20 and 30; data not shown)

We designed the next experiment to determine whether the persis-tence of β-gal+ DCs was because of continued influx of DCs from theskin or extended survival of marked DCs in the lymph nodes. At dif-ferent time points, we used punch biopsy to remove abdominal skin atthe sites where the pCMV-Cre plasmid DNA was injected, and closedthe wounds with wound clips. We determined the presence of β gal+

DCs in the draining lymph nodes 7 d after immunization, regardless ofthe time of immunization site ablation. Ablation of the immunizationsite at 6 h and 12 h nearly abolished the appearance of marked DCs inthe draining lymph nodes (Fig. 4b). The effect of removal of theabdominal skin gradually abated until we found no difference in thefrequency of β gal+ DCs on day 5. These data showed that migration ofDCs from the skin to the draining lymph nodes occurs within the first3–4 d after immunization. Thus, the persistence of β gal+ DCs in thelymph nodes is not due to continuous migration but is due toextended survival.

β-gal+ DCs that persist in the lymph nodes are functional APCsTo determine whether the persisting DCs were functional APCs, weimmunized mice with a DNA plasmid that expressed both the Crerecombinase and ovalbumin (OVA) genes. Because 1 × 104 β gal+ DCswould be required for each well of a 96-well plate, one experimentwould have required >50 ROSA26R mice, which was not feasible.Instead, we isolated CD11c+ DCs from the draining lymph nodes ofC57BL/6 mice 5 or 10 d after immunization with plasmids encodingCre and OVA or Cre only, or with uncoated gold bullets devoid of DNA.We tested CD11c+ DCs for their ability to activate production of inter-feron-γ (IFN-γ) or proliferation by OVA-specific CD4+ T cells fromOT-II TCR-transgenic mice, which recognize the OVA peptide ofamino acids 323–338 (OVA(323–339) peptide) presented by I-Ab mol-ecules27. We added exogenous OVA peptide to some cultures as a positive control. In contrast, DCs from immunized mice presented arepertoire of peptides from the full-length OVA encoded by the DNAvaccine. CD11c+ DCs isolated from mice at each time point inducedIFN-γ production (Fig. 5a) and proliferation (Fig. 5b) directly ex vivo,although cells from day 5 were more effective than DCs from day 10.This correlates with the higher frequency of β gal+ DCs found on day 5(9%) than on day 10 (3%; Fig. 4a). These responses were dependent onthe presence of the gene encoding OVA in the plasmid, as plasmids con-taining Cre only did not stimulate cytokine production of proliferation.These data show that the DCs that persist are capable of activating

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Figure 5 Antigen-bearing DCs in the draining lymph nodes present vaccine-encoded OVA DNA to OVA-specific CD4 T cells ex vivo. (a) C57BL/6 mice (15mice per group) were immunized by gene gun with uncoated gold bullets or bullets coated with DNA plasmids encoding Cre and OVA, or Cre only. CD11c+

DCs were isolated by magnetic sorting (>95% purity) from draining lymph nodes 5 or 10 d after immunization. DCs (1 × 104) were cultured in duplicatewells with 1 × 104 OVA-specific CD4 T cells from a line established from OT-II TCR-transgenic mice, and 1 × 105 irradiated syngeneic filler spleen cells.After 24 h of incubation, the supernatants were assayed in duplicate for IFN-γ by enzyme-linked immunosorbent assay. Data (IFN-γ, in pg/ml ± s.d.) arerepresentative of two experiments. (b) DCs (1 × 104) were cultured for 60 h in triplicate with 1 × 104 magnetically sorted (>98% purity) CD4 T cells from OT-II mice and 1 × 105 irradiated syngeneic, splenic filler cells. [3H]thymidine was added during the last 12 h. Data represent [3H]thymidine incorporation (±s.d.). Results are representative of two experiments. Negative control, T cells and irradiated syngeneic, splenic filler cells incubated with the irrelevantOVA(257–264) peptide; positive control, OVA(323–339)

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Figure 4 Kinetics and persistence of β-galmarked DCs in draining lymph nodes afterimmunization by gene gun. (a) ROSA26R micewere immunized by gene gun with pCMV-Creplasmid DNA–coated gold bullets, and thefrequency of β-gal+CD11c+ DCs (± s.e.m.) wasdetermined. At each time point (horizontal axis),10–20 ROSA26R mice were analyzed. (b) OtherROSA26R mice were immunized by gene gunwith CMV-Cre plasmid in the abdominal skin,through an opening 6 mm in diameter cut in aparafilm template. Each mouse received threenonoverlapping shots (0.5 µg/shot), and theimmunization sites were then excised (times, horizontal axis). Draining lymph nodes of all mice were analyzed 7 d after immunization by flow cytometry forβ-gal+ DCs. The frequency of β-gal+ DCs in the draining lymph nodes for mice whose immunization site was ablated is plotted as a percent of control (micewhose skin was left intact). Data are representative of one of two experiments.

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T cells directly ex vivo, although the possibility of cross-presentation byresident DCs cannot be ruled out. We verified the specificity of T cellresponses by the observation that the relevant peptide OVA(323–339),but not the irrelevant peptide OVA(257–264), stimulated the indicatorT cells in the presence of irradiated filler cells.

DISCUSSIONIn this study, we used Cre/loxP recombination to mark DCs perma-nently in the skin and to track their migration to the draining lymphnodes. After immunization of abdominal skin by a gene gun, eachdraining inguinal lymph node contained approximately 5 × 103 anti-gen-bearing DCs, and these cells persisted for longer periods of timethan previously appreciated.

Our results differ from observations made previously, which esti-mated the frequency of antigen-bearing DCs in draining inguinallymph nodes to be 50–100 DCs per inguinal lymph nodes8 after immu-nization by gene gun with a β-gal-encoding plasmid (pCMV β-gal) tomark the DCs. This number was most likely underestimated because oflow or transient β-gal expression from the CMV promoter in the plas-mid. In contrast, the CMV promoter–driven Cre recombinase used inour study only needs to be expressed in low amounts or transiently toinduce permanent recombination in the genome and sustained highexpression of β-gal from the endogenous ROSA promoter16,17.Moreover, the FDG-mediated flow cytometry assay we used for detect-ing β-gal-expressing cells is much more sensitive than the histochemi-cal X-gal method used before8; a few molecules of β-gal are sufficientfor cleaving the FDG reagent used for flow cytometry18,28.

Mature DCs are powerful APCs capable of initiating an immuneresponse by activating rare, naive antigen-specific T cells. This abilityof DCs depends on their longevity29,30 and abundance at the site of Tcell priming. In the lymph nodes, the interactions between DCs andCD4 or CD8 T cells are very efficient. These interactions have beenvisualized elegantly using two-photon laser scanning microscopy31–34.With this technique it was shown that even in the absence of antigen,DCs contact 500 different T cells in an hour31. In addition, DCs arehighly efficient in recruiting specific T cells in the presence of antigen;approximately 44% of adoptively transferred TCR-transgenic CD8 Tcells specific for lymphocytic choriomeningitis virus glycoproteininteracted with DCs pulsed with this glycoprotein in vivo. Moreover,each antigen-bearing DC could interact with ten or more differentantigen-specific T cells simultaneously31. Thus, the substantial fre-quency of migrating antigen-bearing DCs and their longevity reportedhere, combined with the high efficiency with which these cells seek outand activate naive T cells in situ, ensures that ample antigen presenta-tion and stimulation is available for the rare (1 in 105–106) antigen-specific T cells in the draining lymph nodes. In addition, the DCs thatpersist are potent APCs fully capable of activating T cells, directly exvivo, even 10 d after immunization.

Given the results of our immunization-site ablation studies, themigration of DCs from the immunization site to the draining lymphnodes occurs during the first 3–4 d after immunization by gene gun.Ablation at later days did not decrease the frequency of marked DCs inthe lymph nodes, demonstrating that the persistence of β-gal-markedDCs in the lymph nodes was not due to continued influx but rather topersistence of the DCs. Our data agree with previous studies showingthat skin resection at the immunization site before 3 d abrogated theimmune response, whereas removal at later time points did not, indi-cating that cells from the skin migrate to the lymph nodes over aperiod of 3 d (ref. 35). In summary, our study provides evidence for agreater frequency and longer persistence of antigen-bearing DCs thatmigrate from the skin to the draining lymph nodes than previously

appreciated. Our observations are pertinent to skin-derived DCs;whether they hold true for myeloid and lymphoid DCs warrants fur-ther investigation.

METHODSAnimals and immunization. ROSA26R mice were bred and maintained withthe approval of the Institutional Animal Care and Use Committee at the EmoryVaccine Center vivarium. Mice were housed in micro isolator units, fed ad libi-tum and cared for under United States Department of Agriculture guidelinesfor laboratory animals. Cohorts of ROSA26R mice 6–8 weeks of age weretreated with a single immunization by gene gun with gold particles coated withDNA vaccine plasmids. Immunization using the gene gun was done on shavedabdominal skin using a hand-held Accell gene delivery system as described35,36.Mice were immunized with doses containing 0.5 µg endotoxin-free plasmidDNA per 0.5 µg gold beads 1 µm in diameter (DeGussa-Huls) at a helium pres-sure setting of 400 pounds per square inch. Each mouse was immunized withfour doses given in nonoverlapping areas for a total of 2 µg DNA.

DC enrichment and flow cytometry. Isolation of DCs by collagenase D digestionof lymph nodes was done as described20 except that in all steps after collagenasetreatment, 2 mM EDTA containing buffer was used to prevent reassociation of Tcells with DCs. For magnetic enrichment of DCs, lymph node cells were incu-bated for 30 min on ice with Fc block (BD Pharmingen) followed by antibody toCD11c (N418)–coupled magnetic beads for 15 min, and were positively selectedusing AutoMACS (Miltenyi Corp). Flow cytometric detection of β-gal activity inviable cells was done using FDG (Molecular Probes) as described18,28. For surfacestaining, the DCs were stained with phycoerythrin-, peridinin chlorophyll pro-tein– or allophycocyanin-conjugated antibodies to CD11c, CD4, CD8α andCD11b or biotinylated antibodies to DEC205, B7.1 (CD80) and B7.2 (CD86),followed by allophycocyanin-conjugated streptavidin. Stained cells were analyzedon a Becton Dickinson FACS caliber flow cytometer, and flow cytometry data wasanalyzed with Flowjo software (Tree Star).

DNA vaccine plasmids. Cre recombinase cDNA with a nuclear localizationsequence from SV40 was cloned downstream of the CMV promoter andupstream of a bovine growth hormone poly adenylation (pA) signal sequence(CMV-Cre). This Cre recombinase–encoding DNA vaccine plasmid was usedfor immunization by gene gun. Dual expression vectors were constructed bycloning CMV–caspase 2–pA or CMV–mutant caspase 2–pA cassettes26 down-stream of CMV–Cre–pA. Similarly, a dual expression vector expressing Cre andovalbumin was constructed by cloning CMV–OVA–pA expressing full-lengthOVA cDNA downstream of pCMV-Cre. The keratinocyte-specific Cre expres-sion construct K14-Cre was generated by cloning the gene encoding Crerecombinase downstream of the K14 promoter25. All constructs were tested byrestriction enzyme digestion, sequence analysis and transient transfectionexperiments in vitro. Large-scale preparations of these plasmids were madeusing endotoxin-free Qiagen kit.

RT-PCR for mouse langerin. ROSA26R mice (n = 15) were immunized by genegun with CMV-Cre plasmid and 3 d later, CD11c+ DCs were first purified usingmagnetic sorting and then subjected to high-speed cell sorting on a MoFlowcytometer. Two populations, β-gal+CD11c+DEC205+ DCs and β-gal–CD11c+DEC205– DCs, were isolated. DNA-free RNA was prepared fromthe two sorted cell populations using an automated Magna Pure LC instrument(Roche Applied Science). The RNA was reverse-transcribed using oligo dTprimers and the First Strand cDNA synthesis kit from Invitrogen. The reverse-transcription reactions were subjected to PCR with β-actin primers (25 cycles)or mouse langerin–specific primers (35 cycles) as described24. The PCR cyclingparameters were 94 °C for 1 min, 58 °C for 2 min and 72 °C for 3 min.

Immunization-site ablation. Cohorts of 6- to 8-week-old ROSA26R mice wereimmunized with three nonoverlapping doses of gold bullets coated with CMV-Cre plasmid DNA. A parafilm template with an opening 6 mm in diame-ter was placed on the shaved abdomen of each mouse, and gold bullets weredelivered through the 6-mm opening as described35. Each mouse receivedthree nonoverlapping shots (0.5 µg DNA/shot). The immunization sites weremarked. At various time points after immunization, the marked immunization

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sites were excised using a punch biopsy 8 mm in diameter. After the immuniza-tion-site skin was removed (time of removal, Fig. 5), the wound was closed withsutures or wound clips. The draining lymph nodes of all mice were analyzed forβ-gal+ DCs at day 7 after immunization.

T cell activation. The ability of DCs from mice immunized by gene gun to acti-vate T cells was tested in vitro. OVA-specific T cell lines were produced by cul-ture of naive CD4+ transgenic cells from OT-II mice with OVA and irradiatedfiller cells for 3–6 weeks. CD11c+ DCs were isolated from draining lymph nodesof mice immunized with control uncoated gold bullets or gold bullets coatedwith DNA vaccines encoding Cre and OVA, or Cre only. CD11c+ cells (1 × 104)were plated with 1 × 104 OVA-specific T cells in presence of 1 × 105 irradiatedsplenocytes from C57BL/6 mice as a source of filler cells. After being incubatedfor 24 h at 37 °C, culture supernatants were collected and assayed for IFN-γusing a cytokine detection kit (R&D systems). To measure the proliferativeresponses, 1 × 104 naive CD4+ OVA-specific TCR-transgenic cells from OT-IImice were magnetically sorted (>95% purity) and incubated with 1 × 104

CD11c+ DCs and 1 × 105 irradiated splenocytes for 48 h and then pulsed with1.0 µCi [3H]thymidine. After 12 h, cells were collected and assayed for[3H]thymidine incorporation to measure proliferation.

ACKNOWLEDGMENTSThe authors thank R. Germain for advice; B. Pulendran and members of the Jacoblaboratory for discussions; and I. Williams and P. Lambeth for human keratinocytetransgenic construct. J.A.K. was supported by grants from the Foundation FightingBlindness, the National Eye Institute (National Institutes of Health; EY13459 andP30 EYO06360), and a gift from M. and M. Powell. J.W. was supported by aNational Research Service Award grant (F32 EY06985) from the National EyeInstitute. J.J. was supported by National Institutes of Health grant AI RO1524751.

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Received 18 April; accepted 10 July 2003Published online at http://www.nature.com/natureimmunology

1. Banchereau, J. & Steinman, R.M. Dendritic cells and the control of immunity. Nature392, 245–252 (1998).

2. Banchereau, J. et al. Immunobiology of dendritic cells. Annu. Rev. Immunol. 18,767–811 (2000).

3. Lanzavecchia, A. & Sallusto, F. Regulation of T cell immunity by dendritic cells. Cell106, 263-266 (2001).

4. Mellman, I. & Steinman, R.M. Dendritic cells: specialized and regulated antigen pro-cessing machines. Cell 106, 255–258 (2001).

5. Pulendran, B., Palucka, K. & Banchereau, J. Sensing pathogens and tuning immuneresponses. Science 293, 253–256 (2001).

6. Liu, Y.J. Dendritic cell subsets and lineages, and their functions in innate and adap-tive immunity. Cell 106, 259–262 (2001).

7. Lipscomb, M.F. & Masten, B.J. Dendritic cells: immune regulators in health and dis-ease. Physiol. Rev. 82, 97–130 (2002).

8. Porgador, A. et al. Predominant role for directly transfected dendritic cells in antigenpresentation to CD8+ T cells after gene gun immunization. J. Exp. Med. 188,1075–1082 (1998).

9. Condon, C., Watkins, S.C., Celluzzi, C.M., Thompson, K. & Falo, L.D. DNA-based immu-nization by in vivo transfection of dendritic cells. Nat. Med. 2, 1122–1128 (1996).

10. Bouloc, A., Walker, P., Grivel, J.C., Vogel, J.C. & Katz, S.I. Immunization through der-mal delivery of protein-encoding DNA: a role for migratory dendritic cells. Eur. J.Immunol. 29, 446–454 (1999).

11. Macatonia, S.E., Knight, S.C., Edwards, A.J., Griffiths, S. & Fryer, P. Localization ofantigen on lymph node dendritic cells after exposure to the contact sensitizer fluores-

cein isothiocyanate. Functional and morphological studies. J. Exp. Med. 166,1654–1667 (1987).

12. Ruedl, C., Koebel, P., Bachmann, M., Hess, M. & Karjalainen, K. Anatomical origin ofdendritic cells determines their life span in peripheral lymph nodes. J. Immunol.165, 4910–4916 (2000).

13. Ruedl, C., Koebel, P. & Karjalainen, K. In vivo-matured Langerhans cells continue totake up and process native proteins unlike in vitro-matured counterparts. J. Immunol.166, 7178-7182 (2001).

14. Kamath, A.T., Henri, S., Battye, F., Tough, D.F. & Shortman, K. Developmentalkinetics and lifespan of dendritic cells in mouse lymphoid organs. Blood 100,1734–1741 (2002).

15. Sauer, B. Manipulation of transgenes by site-specific recombination: use of Crerecombinase. Meth. Enzymol. 225, 890–900 (1993).

16. Soriano, P. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat.Genet. 21, 70–71 (1999).

17. Zambrowicz, B.P. et al. Disruption of overlapping transcripts in the ROSA β geo 26gene trap strain leads to widespread expression of β-galactosidase in mouse embryosand hematopoietic cells. Proc. Natl. Acad. Sci. USA 94, 3789–3794 (1997).

18. Nolan, G.P., Fiering, S., Nicolas, J.F. & Herzenberg, L.A. Fluorescence-activated cellanalysis and sorting of viable mammalian cells based on β-D-galactosidase activityafter transduction of Escherichia coli lacZ. Proc. Natl. Acad. Sci. USA 85,2603–2607 (1988).

19. Stingl, G. Dendritic cells of the skin. Dermatol. Clin. 8, 673-679 (1990).20. Henri, S. et al. The dendritic cell populations of mouse lymph nodes. J. Immunol.

167, 741–748 (2001).21. Pulendran, B., Banchereau, J., Maraskovsky, E. & Maliszewski, C. Modulating the

immune response with dendritic cells and their growth factors. Trends Immunol. 22,41–47 (2001).

22. Valladeau, J. et al. Langerin, a novel C-type lectin specific to Langerhans cells, is anendocytic receptor that induces the formation of Birbeck granules. Immunity 12,71–81 (2000).

23. Stoitzner, P. et al. Visualization and characterization of migratory Langerhans cells inmurine skin and lymph nodes by antibodies against Langerin/CD207. J. Invest.Dermatol. 120, 266–274 (2003).

24. Takahara, K. et al. Identification and expression of mouse Langerin (CD207) in den-dritic cells. Int. Immunol. 14, 433–444 (2002).

25. Williams, I.R. et al. IL-7 overexpression in transgenic mouse keratinocytes causes alymphoproliferative skin disease dominated by intermediate TCR cells: evidence for ahierarchy in IL-7 responsiveness among cutaneous T cells. J. Immunol. 159,3044–3056 (1997).

26. Sasaki, S., Amara, R.R., Oran, A.E., Smith, J.M. & Robinson, H.L. Apoptosis-medi-ated enhancement of DNA-raised immune responses by mutant caspases. Nat.Biotechnol. 19, 543–547 (2001).

27. Barnden, M.J., Allison, J., Heath, W.R. & Carbone, F.R. Defective TCR expression intransgenic mice constructed using cDNA-based α- and β-chain genes under the con-trol of heterologous regulatory elements. Immunol. Cell Biol. 76, 34–40 (1998).

28. Fiering, S.N. et al. Improved FACS-Gal: flow cytometric analysis and sorting of viableeukaryotic cells expressing reporter gene constructs. Cytometry 12, 291–301 (1991).

29. Josien, R. et al. TRANCE, a tumor necrosis factor family member, enhances thelongevity and adjuvant properties of dendritic cells in vivo. J. Exp. Med. 191,495–502 (2000).

30. Nopora, A. & Brocker, T. Bcl-2 controls dendritic cell longevity in vivo. J. Immunol.169, 3006-3014 (2002).

31. Bousso, P. & Robey, E. Dynamics of CD8+ T cell priming by dendritic cells in intactlymph nodes. Nat. Immunol. 4, 579–585 (2003).

32. Parker, I., Wei, S.H. & Miller, M.J. Two-photon imaging of lymphocyte motility andantigen response in intact lymph node. Nat. Rev. Immunol. 2, 872–880 (2002).

33. Rauer, H. et al. Two-photon imaging of lymphocyte motility and antigen response inintact lymph node. J. Biol. Chem. 276, 12249–12256 (2001).

34. Stoll, S. & Germain, R.N. Dynamic imaging of T cell-dendritic cell interactions inlymph nodes. Immunol. Rev. 189, 51–63 (2002).

35. Torres, C.A., Iwasaki, A., Barber, B.H. & Robinson, H.L. Differential dependence ontarget site tissue for gene gun and intramuscular DNA immunizations. J. Immunol.158, 4529–4532 (1997).

36. Ross, T.M., Xu, Y., Bright, R.A. & Robinson, H.L. C3d enhancement of antibodies tohemagglutinin accelerates protection against influenza virus challenge. Nat.Immunol. 1, 127–131 (2000).

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