of February 16, 2018.This information is current as

Activated MonocytesImpacts Presentation by Dendritic Cells and Route of Antigen Uptake Differentially

and Michel C. NussenzweigAlice O. Kamphorst, Pierre Guermonprez, Diana Dudziak

ol.1001205http://www.jimmunol.org/content/early/2010/08/20/jimmun

published online 20 August 2010J Immunol 

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The Journal of Immunology

Route of Antigen Uptake Differentially Impacts Presentationby Dendritic Cells and Activated Monocytes

Alice O. Kamphorst,* Pierre Guermonprez,* Diana Dudziak,*,1 and Michel C. Nussenzweig*,†

Dendritic cells (DCs), which maintain tolerance and orchestrate T cell immune responses, comprise a heterogeneous group of cells.

For example, in the steady state, murine spleen contains pre-DC–derived CD8+ and CD82 conventional DCs. During inflammation,

monocytes become activated and acquire some DC-like features, such as expression of CD11c and MHC class II. Although each of

these cell types can present Ag, the relative efficiency of processing and presentation after Ag capture by different routes has not

yet been systematically compared. To this end, we administered OVA to various conventional DCs and activated monocytes by

receptor-mediated endocytosis, pinocytosis, or phagocytosis and measured internalization and presentation to MHC class I- and

MHC class II-restricted T cells. We find that CD82 DCs are more efficient than any other type of APC tested in terms of

presenting Ag to MHC class II-restricted T cells, irrespective of the route of Ag capture. In contrast, both subsets of splenic

DCs are highly effective in cross-presenting Ags to CD8+ T cells. DCs and activated monocytes cross-presented Ags delivered by

DEC205-mediated endocytosis and pinocytosis. However, DCs differ from activated monocytes in that the latter are several orders

of magnitude less efficient in presenting Ags captured by phagocytosis to CD8+ or CD4+ T cells. We conclude that DCs derived

from pre-DCs differ from monocyte-derived cells in that DCs process and present Ags efficiently irrespective of the route of Ag

capture. Our observations have significant implications for understanding initiation of immune responses and vaccination strat-

egies targeting DCs and activated monocytes. The Journal of Immunology, 2010, 185: 000–000.

Dendritic cells (DCs) were first recognized by their uniquemorphology (1) and later shown to be potent stimulatorsof theMLR (2) and effector T cell responses (3, 4). Unlike

macrophages or B cells, which are able to present Ag but are spe-cialized for phagocytosis and Ab production, respectively, DCs areprofessional APCs (5–7).DCs are closely related to monocytes and macrophages; how-

ever, they develop from committed progenitors that diverge frommonocytes and macrophages in the bone marrow (8) during thetransition between macrophage-and-DC progenitor (9, 10) andcommon-DC progenitor (11, 12). Common-DC progenitors give riseto pre-DCs, which have lost the potential to produce plasmacytoidDCs (8). Pre-DCs circulate in the blood and seed lymphoid (8) andnonlymphoid organs (13–15), where they divide (16, 17) and differ-entiate into subpopulations of conventional DCs. In the lymphoidorgans, DCs become incorporated into networks of cells and presentAg to migrating T cells (8, 18).

In humans as well as in mice, DCs are a heterogeneous groupof cells, composed of several subsets, all of which can express highlevels of CD11c and MHC class II (MHC-II) (19). In the mousespleen, resident DCs can be divided into two main subsets based onthe expression of CD8a, DEC205, and DCIR2: CD8+DEC205+ andCD82DCIR2+ DCs. In the steady state, both spleen DC subsets arederived from the same common progenitor, the pre-DC (8). However,during inflammation or infection the distribution of DCs in lymphoidorgans changes, and monocytes can also acquire DC-like features,such as expression of CD11c, MHC-II, and costimulatory molecules(20–22). For example, during Listeria infection, monocytes differ-entiate in the spleen into TNF/inducible NO synthase-producingCD11c+ cells (tip-DCs) (23). Similarly, cells with many features ofDCs can be derived frommonocytes cultured with cytokines, such asGM-CSF (GM) and IL-4 (24, 25). However, the precise function ofthese activated monocytes in vivo remains to be defined.Although both subsets of conventional DCs found in the spleen

excel in Ag processing and presentation, CD82DCIR2+ DCs aremore efficient thanCD8+DEC205+DCs in processingAgs forMHC-II presentation whenAgs are captured by endocytic receptors (26). Incontrast, CD8+DEC205+ DCs are superior in cross-presentingAgs toMHC class I (MHC-I) after ingestion of dead cells or during viralinfection (27–29). However, the two subsets have not yet beencompared systematically, either to each other or to other cells forpresentation of Ags captured by different mechanisms.In this paper,we compareMHC-I andMHC-IIAg presentation by

resting or activated spleenDCs, activatedmonocytes, GMand FLT3ligand (FL) bonemarrow culture-derivedDCs, and activated B cellsafter Ag capture by either receptor-mediated endocytosis, pinocy-tosis, or phagocytosis. We find that all the different cell types testedcan present Ags to MHC-II– or MHC-I–restricted T cells after Agcapture by DEC205-mediated endocytosis or pinocytosis, albeitwith different efficiencies. However, Ags captured by phagocytosisare presented effectively only by conventional DCs. CD82DCIR2+

DCs are the most efficient APCs for the MHC-II pathway, irre-spective of activation status or mode of Ag acquisition. Finally,

*Laboratory of Molecular Immunology, The Rockefeller University, New York, NY10065; and †Howard Hughes Medical Institute, Chevy Chase, MD 20815

1Current address: Laboratory of Dendritic Cell Biology, Department of Dermatology,University Hospital of Erlangen, Erlangen, Germany.

Received for publication April 15, 2010. Accepted for publication July 12, 2010.

This work was supported in part by National Institutes of Health Grant AI051573.M.C.N. is a Howard Hughes Medical Institute investigator. D.D. is supported byGerman Research Foundation Grants DU548/1-1 and DU548/2-1. A.O.K. was sup-ported by a Cancer Research Institute fellowship.

Address correspondence and reprint requests to Dr. Michel C. Nussenzweig, 1230 YorkAvenue, Box 220, New York, NY 10065. E-mail address: nussen@rockefeller.edu

The online version of this article contains supplemental material.

Abbreviations used in this paper: DC, dendritic cell; DCIR2-OVA, DCIR2-OVA mAb;aDCIR2-OVA, anti–DCIR2-OVAmAb; FL, FLT3 ligand; GM, GM-CSF; hDEC-OVA,human-DEC205-OVA mAb; ahDEC-OVA, anti–human-DEC205-OVA mAb; mBSA,methylatedBSA;mDEC-OVA,mouse-DEC205-OVAmAb;amDEC-OVA,anti–mouse-DEC205-OVA mAb; MHC-I, MHC class I; MHC-II, MHC class II; NP, 4-hydroxy-3-nitrophenil; tip-DC, TNF/inducible NO synthase-producing DC; WT, wild-type.

Copyright� 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001205

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both major spleen DC subsets (CD82 and CD8+) are similar incross-presentation, regardless of the route of Ag uptake.

Materials and MethodsMice

C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor,ME). Transgenic OT-II, OT-I, CD11c-hDEC (26), B1-8hi (30) mice, as wellas DEC-205–deficient and GM receptor-b–deficient (31) mice, were bredat the Rockefeller University; 6- to 10-wk-old mice were used in experi-ments. All experiments were performed in accordance with NationalInstitutes of Health guidelines and approved by the Rockefeller UniversityAnimal Care and Use Committee.

Production of chimeric Abs and other Ags

Chimeric anti–mouse-DEC205-OVA mAb (mDEC-OVA), anti–DCIR2-OVA mAb (DCIR2-OVA), and anti–human-DEC205-OVA mAb (hDEC-OVA) were expressed by transient transfection of 293T cells and purifiedwith Protein G (GE Healthcare, Piscataway, NJ) as described (32). OVA(Grade VA5503; Sigma-Aldrich, St. Louis, MO) was decontaminated fromLPS by multiple rounds of extraction with Triton-X 114 (Sigma-Aldrich)(33) and dialyzed extensively. 4-Hydroxy-3-nitrophenil (NP; Bio-search Technologies, Novato, CA) and biotin (Invitrogen, Carlsbad, CA)conjugation to OVA was performed according to the manufacturers’ in-structions. All Ags were tested for LPS contamination (Fisher-Cambrex/Fisher Scientific, Pittsburgh, PA) and decontaminated by Triton-X 114 ex-tractionwhennecessary.OVAwas adsorbed to 2-mMredbeads (Polysciences,Warrington, PA), according to the manufacturer’s protocol. Briefly, 0.5 ml2.5% suspension of beads was resuspended in 0.1M borate buffer pH 8.5 andincubated overnight with 500 mg OVA at 4˚C. Alternatively, to obtain 25%OVA-beads, beads were incubated overnight with 125 mg OVA and 375 mgkeyhole limpet hemocyanin (Sigma-Aldrich). Unbound protein was washedaway extensively, and OVA-adsorbed beads were resuspended in 1 ml PBS.Coupling was confirmed by flow cytometry with rabbit anti-OVA (Cappel)Abs, followed by anti–rabbit-Cy5 staining (Jackson ImmunoResearch Lab-oratories, West Grove, PA).

Isolation and sorting strategy for DCs and activated monocytes

DCs were isolated from mice that had been injected 9–12 d before eutha-nasia with 1–23 106 B16-FL melanoma cells (34). All experiments (exceptthose in Fig. 5) were also performed with DCs not expanded by FL isolatedfrom uninjected control mice, with identical results. For in vivo Ag targetingexperiments, mice were injected i.p. with 10 mg chimeric Abs 8–12 h beforeeuthanasia. Activated DCs and monocytes were isolated from mice thatwere primed and boosted with methylated BSA (mBSA) in CFA (Difco/BDDiagnostics, Franklin Lakes, NJ) and then injected i.p. 24 h before analysiswith 100 mg mBSA (Sigma-Aldrich), as described (22, 35). For DC andmonocyte isolation, spleens were removed, injected with 0.4 U/ml colla-genase D type II (Roche Biomedical Laboratories, Burlington, NC) inHBSS with 2% FCS (Life Technologies, Rockville, MD), cut into smallfragments and incubated at 37˚C for 30 min. Digestion was stopped, with 5mM EDTA for 5 min, before collection of cell suspensions. RBCs wereremoved by ACK lysis (Life Technologies). All subsequent steps wereperformed in PBS 2% FCS, and during incubations, cells were kept at 4˚C.Nonspecific binding was blocked with purified Fc block, and DCs wereenriched with anti-CD11c beads (Miltenyi Biotec, Auburn, CA), accordingto the manufacturer’s instructions. Enriched naive CD11c+ cells werestained and sorted as follows: CD8+ DCs (B2202, NK1.12, CD11chi, CD8+)and CD82 DCs (B2202, NK1.12, CD11chi, CD82, CD4+). EnrichedCD11c+ cells from immunized mice were stained and sorted as follows:iMono (CD82, CD11bhi, CD11cint, and Ly6C+), iCD82 DCs (CD82,Ly6C2, CD11bint, and CD11chi), and iCD8+ DCs (CD8+, CD11chi and,Ly6C2). In addition, cells were stained with B220, NK1.1, CD3, Ter119,and Ly6G to gate out non-DCs/non-monocytes. In experiments withCD11c-hDEC mice, cells were also stained with human CD205 to sortpositive cells. Sorted populations were collected in complete RPMI (RPMI1640 supplemented with 10% FCS, 10 mM HEPES, 2 mM L-glutamine,antibiotic/antimycotic, 1 mM sodium pyruvate, and 53 mM 2-ME; LifeTechnologies), and live cells were typically .95% pure.

Culture and sorting strategy for cultured DCs

Bone marrow cells were obtained by flushing femora and tibiae with RPMI1640 supplemented with 5% FCS. RBCs were removed by ACK lysis (LifeTechnologies) and washed with complete RPMI 1640.

ForGM-DCs differentiation (24), bonemarrow cells were plated at 13 106

cells/ml in complete RPMI 1640 with 3% vol/vol supernatant of J558L cellstransduced with murine GM (provided by A. Lanzavecchia, Institute forBiomedical Research, Bellinzona, Switzerland). Cells were cultured at 37˚Cand 5% CO2. Media were changed every 2 d, removing loosely adherent anddead cells. GM-DCswere collected at day 7. Nonspecific bindingwas blockedwith Fc block, and cells were stained with CD11c. CD11chi cells were sorted.

For FL-DC differentiation (36), bone marrow cells were plated at 1.5 3106 cells/ml in complete RPMI 1640with 100 ng/ml recombinant murine FL.FL was obtained by anti-FLAG purification of supernatant from Chinesehamster ovary (CHO) cells expressing recombinant murine FL-FLAG(kindly provided by C.G. Park, The Rockefeller University). Cells werecultured at 37˚C and 5% CO2, and FL-DCs were collected at day 10. Non-specific binding was blocked with purified Fc block, and cells were stainedand sorted as follows: FL-CD82 DCs (B2202, CD11chi, SIRPahi, andCD24lo) and FL-CD8+ DCs (B2202, CD11chi, SIRPalo, and CD24hi) (37).

In vitro activation of DCs was by addition of 1 mg/ml LPS (Sigma-Aldrich) for the last 18 h of culture. In experiments with CD11c-hDECmice, cells were also stained with human CD205 to sort positive cells.Sorted populations were collected in complete RPMI 1640, and live cellswere typically .95% pure.

B cell culture

Naive B cells were isolated from wild-type (WT) C57BL/6 mice or B1-8hi

mice, as indicated. For WT mice, single-cell suspensions from spleen wereincubated with anti-CD43 beads (Miltenyi Biotec) and enriched in LSColumns for CD432 naive B cells. For B1-8hi mice, single-cell suspen-sions from spleen and skin-draining lymph nodes were incubated withanti–Igk-PE (187.1; BD Biosciences, San Jose, CA), followed by anti-PE(Miltenyi Biotec) beads and anti-CD43 beads (38). Cells were enriched inLS Columns (Miltenyi Biotec) for CD432Igk2 naive l+ B cells (NP-specific). Enriched B cells were plated at 0.7 3 106 cells/ml in completeRPMI 1640 supplemented with 25 mg/ml LPS (Sigma-Aldrich) and 5 ng/ml IL-4 (Sigma-Aldrich) and cultured at 37˚C and 5% CO2 for 2–3 d.

In vitro phagocytosis

Enriched CD11c+ cells from spleen (naive control or immunized mice) orGM-DCswere incubatedwithOVA-adsorbedbeads at 108 cells/ml in completeRPMI a640, for 30 min at 37˚C and 5% CO2. Cells were washed 3 times withcold PBS (900 rpm, 5 min) and stained for cell sorting, as described above.

In vitro Ag delivery

Sorted APCs were counted, and 15,000 live cells/well were plated incomplete RPMI 1640 in 96-well round-bottom plates. For B cell blasts,30,000 live cells/well were plated. Ag was added at the indicated con-centrations. For receptor-mediated endocytosis (DEC-205 targeting andBCR targeting), all media were kept cold and Ag was pulsed for 20 min at4˚C. For pinocytosis, warm media with Ag (OVA-biotin) were added, andplates were incubated at 37˚C with 5% CO2 for 2 h. As controls in allexperiments, APCs were also incubated with peptides for 20 min at 4˚C:For OTI coculture we used OVA peptide EQLESIINFEKLTEW, and forOTII coculture we used OVA peptide LSQAVHAAHAEINEAGR, syn-thesized by the Proteomics Resource Center, The Rockefeller University.After Ag or peptide incubation, cells were washed 3 times with completeRPMI 1640 to remove excess free Ag, before assessment of Ag uptake orbefore incubation at 37˚C and 5% CO2, with OVA-specific T cells. Allincubations were done in duplicate wells.

Ag presentation assay

CD8+ and CD4+ OVA-specific T cells were isolated from OTI and OTIImice, respectively. CD8+ and CD4+ T cells were enriched with a CD8+

T cell isolation kit or a CD4+ T cells isolation kit, according to themanufacturer’s instructions (Miltenyi Biotec) with the addition of anti–CD11c-biotin to the Ab mixture. Enriched T cells were labeled with 1 mMCFSE (Molecular Probes/Invitrogen) in PBS 0.1% BSA for 10 min at37˚C. The reaction was quenched with FCS, and cells were washed 2–3times with complete RPMI 1640. 100,000 to 125,000 T cells were addedto each well containing APCs. Activation and division of OVA-specificT cells were determined by flow cytometry after culture at 37˚C and 5%CO2 for 60–65 h (OTI cells) or 80–85 h (OTII cells). For analysis, cellswere stained with Va2 PE, CD69 APC, CD8 or CD4 PerCP, and 0.5 mg/mlDAPI before acquisition on LSR II (BD Biosciences).

Quantification of Ag uptake

After removal of free Ag, APCs were resuspended in cytofix/cytoperm (BDBiosciences) for 15min at room temperature andwashed in Perm/Wash (BD

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Biosciences) and then in PBS 1% BSA 0.1% saponin. aDEC-OVA andNP-OVA captured by receptor-mediated endocytosis were detected withrabbit polyclonal anti-OVA (Cappel) followed by F(ab)92 donkey anti-Rabbit IgG-HRP (Jackson ImmunoResearch Laboratories). OVA-biotinwas detected with streptavidin-HRP (Jackson ImmunoResearch Labora-tories). HRP content was assessed by a fluorometric assay with amplex red(Invitrogen). Plates were excited at 530 nm, and emission was collected at590 nm on Cytofluor II (PerSeptive Biosystems, Ramsey, MN).

Flow cytometry analysis

The following Abs, purchased from either BE Biosciences or eBioscience(San Diego, CA), were used: CD3 (145-2C11), CD4 (L3T4), CD8 (53-6.7),CD11b (M1/70), CD11c (N418, HL3), CD16 and CD32/Fc block (2.4G2),CD19 (MB19-1 or eBio1D3), CD24/HSA (M1/69), CD45R/B220 (RA3-6B2), CD45.1 (A20), CD45.2 (104), CD69 ([1H].2F3), CD86 (GL1),CD115/CSF-1R (AFS98), CD135/Flk-2/Flt3 (A2F10), CD172a/SIRPa(P84), mouse CD205/mDEC-205 (205yefta), human CD205/hDEC-205(Mg38), DCIR2 (33D1), Fas/CD95 (Jo2), GL7, H2Kb (AF6-88.5), Ly6C(AL-21 or HK1.4), Ly6G (1A8), MHC-II (AF6-120.1 or M5/114.15.2),NK1.1 (PK136), Ter-119, and Va2 (B20.1). Streptavidin-PE and –PE-Cy5.5 were from eBioscience. Streptavidin APC was from BD Bio-sciences. Streptavidin Pacific Blue and DAPI were from Invitrogen. Datawere acquired on LSR-II (BD Biosciences). Analysis was performed usingDiva (BD Biosciences) or FlowJo (TreeStar, Ashland, OR).

Data analysis

Graphs were compiled on Prism software (GraphPad Software, San Diego,CA). Ag pulse curves representing Ag uptake or T cell proliferation wereadjusted to exponential one-phase association curves. Statistical analysiswas performed using Prism software, as described in each figure.

ResultsAg acquisition by DEC-205 or BCR-mediated endocytosis

CD8+ DCs excel in cross-presentation during infection with HSVand West Nile virus or after ingestion of apoptotic cells (27–29,39, 40). However, both CD8+ and CD82 DCs can cross-presentwhen they are incubated with high concentrations of soluble OVA(41), OVA immune complexes (42), or bacteria expressing OVA(40, 43). Whether these differences are related to the amount ofAg acquired, the mechanism of uptake, or some other cell intrinsicdifference between the two DC subsets is not known.To compare the Ag-presenting activity of the different DC sub-

sets after receptor-mediated endocytosis, we made use of mice thatexpress human DEC-205 under the control of the CD11c promoter(CD11c-hDEC) because all DC subsets in these mice expresshDEC-205 (26). OVAwas targeted to DCs in vivo by injecting chi-meric anti–human-DEC-205 mAbs that carry intact OVA (ahDEC-OVA).As controlswe used anti–mDEC-OVAand anti–DCIR2-OVA,which target either CD8+ or CD82 DCs in the spleen, respectively(26, 32). Following Ab injection, hDEC expressing CD82 and CD8+

splenic DCs were purified by cell sorting and evaluated for MHC-I orMHC-II presentation by coculturewith CFSE-labeledOVA-specificOTI and OTII transgenic T cells. As previously documented, CD82

DCs targeted in vivo with anti–DCIR2-OVA efficiently inducedOTII proliferation but little OTI proliferation, whereas CD8+ DCstargeted in vivo with anti–mDEC-OVA induced robust OTI andmore modest OTII proliferation (Fig. 1) (26). Consistent with thelow but detectable levels of mDEC-205 expressed by CD82 DCs(Supplemental Fig. 1A) (44), we found small amounts of cross-presentation by CD82 DCs upon anti–mDEC-OVA targeting (Fig.1A). When both DCs were targeted with anti–hDEC-OVA, CD82

DCsweremore effective at stimulatingOTII proliferation thanwereCD8+ DCs from the samemice (Fig. 1) (26). In contrast, both CD82

and CD8+ DCs targeted in vivo with anti–hDEC-OVA elicitedsimilarly robust levels of OTI proliferation (Fig. 1). These resultsindicate that both DC subsets have the same intrinsic potential tocross-present Ags when the Ag is delivered by DEC-205.

Although both subsets of DCs express similar levels of hDEC-205in the CD11c-hDEC transgenic mice (26), differences in Ag pre-sentation may nevertheless be the result of differences in theamounts of Ag captured. To explore this possibility, we isolatedspleen CD82 and CD8+ DCs from CD11c-hDEC mice, pulsedthem at 4˚C with varying amounts of anti–hDEC-OVA, and mea-sured the amount of cell-associated OVA (Fig. 2A, left). Replicaplates of the pulsed DCs were then cocultured with CFSE-labeledOTI or OTII transgenic T cells at 37˚C for 3–4 d to measure theirability to present OVA (Fig. 2B, left). As a further control, DCswere pulsed with peptides to measure their Ag-presenting activityindependent of Ag capture and processing (Fig. 2C, left).

FIGURE 1. hDEC205-mediated endocytosis in vivo promotes cross-pre-

sentation by both CD82 and CD8+ DC subsets. A, Representative histograms

showproliferation asmeasured byCFSEdyedilutionofOTI (upperpanels) and

OTII (lower panels) T cells upon culture with 5 3 103 CD82 or CD8+ DCs

isolated from CD11c-hDEC transgenic mice injected with anti–hDEC-OVA

(ahDEC-OVA), anti–mDEC-OVA (amDEC-OVA), or anti–DCIR2-OVA

(aDCIR2-OVA), as indicated.B, Summaryof three independent experiments as

in A. Panels show the percentage of divided, CFSE low OTI (upper panel) and

OTII (lower panel) after incubation with 53 103 (s, N,4) or 203 103 (d, n,

:) CD82 or CD8+DCs that were targetedwithOVA, as indicated. Each symbol

indicates independent experiments and represents the average of duplicate mea-

surements. Spleens of 3–10 injected mice were pooled in each experiment. Data

were analyzed by repeatedmeasures with ANOVA, and Tukey’s test was used to

compare groups. pp, 0.05; ppp, 0.01; pppp, 0.001.

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CD82 and CD8+ splenic DCs captured similar amounts of OVAwhen pulsed with anti–hDEC-OVA (Fig. 2A, left) and showedsimilar intrinsic Ag-presenting activity when pulsed with peptides(Fig. 2C, left). When Ag was titrated under conditions when DCswere present in excess, the results of in vitro targeting mirroredthose obtained in vivo in that the two subsets were equivalent forMHC-I cross-presentation and also that CD82 DCs were in-trinsically more efficient than CD8+ DCs in processing and pre-

senting Ags on MHC-II (Fig. 2B, left; Supplemental Fig. 2A). Onlysmall differences in presentation were found when the number ofAPCs were titrated: CD8+ DCs were ∼3-fold more effective thanCD82 DCs for cross-presentation when,10,000 DCs were present(Supplemental Fig. 2B, upper panels). In contrast, CD82 DCs wereseveral orders of magnitude more efficient than CD8+ DCs forMHC-II presentation, irrespective of the number of DCs assayed(Supplemental Fig. 2B, lower panels). We conclude that CD82 and

FIGURE 2. Ag presentation after DEC205-mediated endocytosis in vitro. A–C, APCs from CD11c-hDEC mice were isolated from naive (solid lines) or

mBSA-CFA–immunized (dashed lines) mice (left panels), or cultured from bone marrow (right panels). A, The y-axis shows relative cell-associated OVA,

as measured by rabbit anti-OVA and developed with anti–rabbit-HRP, after targeting with the indicated concentrations of ahDEC-OVA on the x-axis. B,

Activation and proliferation of OTI (upper panels) and OTII (lower panels) T cells in response to OVA containing APCs. The y-axis shows the percentage

of divided T cells. C, Graphs show OTI (upper panels) and OTII (lower panels) T cell proliferation in response to peptide pulsed APCs. D–F, APCs were

isolated or cultured from spleen or bone marrow of WT mice. D and E, As in A and B, but targeting was with amDEC-OVA. F, As in C. A–F, Graphs

represent pooled data from three to seven independent experiments.

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CD8+ splenic DCs are similar in terms of their ability to cross-present Ags on MHC-I when the Ag is captured by DEC-205.During some infections or inflammation, monocytes become ac-

tivated and can differentiate into GM-dependent tip-DCs (22, 23,35, 45) (Supplemental Fig. 3). Conversely, conventional DCs areFL dependent, GM independent, and derived from pre-DCs (16). Tocompare activated monocytes with conventional DCs, we immu-nized hDEC-205 transgenic micewith CFA (35) to induce monocyteactivation. Activated monocytes and conventional DCs were puri-fied by cell sorting and compared after targeting with anti–hDEC-OVA. Activated monocytes had similar levels of CD86, 2-fold higherlevels of MHC-I, but lower levels of MHC-II when compared withconventional DCs from naive control mice (Supplemental Figs. 3C,4). Conventional DCs isolated from the immunized mice (iCD8+

and iCD82 DCs) had higher levels of CD86, MHC-I and MHC-IIwhen compared with control DCs (Supplemental Figs. 3C, 4).All cell types isolated from the immunized mice (iCD8+ and

iCD82 DCs, and iMono) were similar to control DCs in Ag uptakeafter anti–hDEC-OVA targeting (Fig. 2A, left) (46). However, DCsfrom immunized mice were more efficient than their naive coun-terparts with regard to the presentation of pulsed peptides. In con-trast, activated monocytes were less efficient than DCs, especiallyfor MHC-II presentation, when pulsed with peptides (Fig. 2C, left).Consistent with the peptide presentation experiments, activatedmonocytes performed poorly in MHC-II presentation, but showedlevels of cross-presentation that approached those of conventionalDCs (Fig. 2B, left). We conclude that activated monocytes resembleconventional DCs in their ability to cross-present Ags captured byDEC205-mediated endocytosis. However, under the same conditions,activated monocytes are far inferior to naive or activated conventionalspleen DCs with regard to MHC-II presentation.

Cultured DCs and B cells

DCs obtained by culturing murine bone marrow cells with GM arewidely used as APCs (24). A second method for producing DCsin vitro involves murine bone marrow culture in the presence ofFL (FL-DCs) (36). FL-DCs are heterogeneous, and contain cellsthat resemble CD8+ and CD82 splenic DCs (FL-CD8+ and FL-CD82 DCs, respectively), as well as plasmacytoid DCs in termsof cell surface marker and transcription factor expression (37, 47).Both FL- and GM-DCs can be activated with TLR agonists, suchas LPS, to increase the expression of MHC and costimulatorymolecules (36, 48, 49), as well as Ag processing (50, 51) and thestability of MHC-peptide complexes on the cell surface (6, 52).Moreover, endogenous mouse DEC-205 expression is induced oncultured DCs upon TLR ligation (36, 53).To compare the ability of tissue culture-derived DCs and con-

ventional DCs to present Ags captured by receptor-mediated en-docytosis, we targeted GM- and FL-DCs obtained from hDECtransgenic mice with anti–hDEC-OVA. CD86, MHC-I, andMHC-IIexpression by GM- and FL-DCs was equal to or higher than that inspleen DCs (Supplemental Fig. 4). However, GM-DCs accumu-lated more OVA than did spleen DCs or FL-DCs after anti–hDEC-OVA targeting (Fig. 2A, right). Nevertheless, GM-DCs were lesseffective than spleen DCs in cross-presentation (Fig. 2B, right;Supplemental Fig. 2A). Consistent with their high levels of MHC-IIexpression, GM-DCs were efficient in presenting pulsed peptidesto MHC-II–restricted T cells (Fig. 2C, right). However, the samecells were intermediate, between CD8+ and CD82 DCs, for MHC-II presentation after Ag capture by DEC205-mediated endocytosis(Fig. 2B, right; Supplemental Fig. 2A).FL-DCs were separated into CD82 (Sirpahi) and CD8+ (CD24hi)

subsets. Both FL-DC subsets induced less OTI proliferation than didsplenic DCs (Fig. 2B, right). However, FL-CD8+ DCs were almost

10-fold less efficient than their splenic counterparts for MHC-Ipresentation after exogenous peptide loading (Fig. 2C, right). ForMHC-II presentation, FL-CD82 DCs were more efficient than FL-CD8+ DCs, the latter showing efficiency similar to that of theirsplenic counterpart CD8+ DCs (Fig. 2B, right). We conclude thatboth GM- and FL-DCs are capable of MHC-I and MHC-II pre-sentation when Ag is captured by DEC205-mediated endocytosis;however, these cells are less active than conventional spleen DCs.To determine how activation by TLR ligation alters DCs ability to

present Ags acquired through receptor-mediated endocytosis, westimulated GM- or FL-DCs with LPS andmeasured Ag presentationafter targeting with anti–mDEC-OVA.Naive CD8+DCs from spleenwere used as controls in all experiments. As expected, stimulationwith LPS enhanced expression of MHC-I and MHC-II and co-stimulatory molecules in all culture-derived DCs (SupplementalFig. 4) (6). GM-DCs activated with LPS expressed high levels ofDEC-205 and thus captured more Ag than any of the other DCstested (Fig. 2D). In addition, when loaded with exogenous peptide,LPS-GM-DCs performed better than control CD8+ DCs in activat-ing MHC-I– and MHC-II–dependent T cell responses (Fig. 2F).However, LPS-GM-DCs were far less effective than CD8+ DCs andno better than resting GM-DCs in cross-presentation of Ags ac-quired by DEC205-mediated endocytosis (Fig. 2E, SupplementalFig. 2C). Finally, LPS stimulation also decreased the relative effi-ciency of GM-DCs with regard to processing Ag to be presented onMHC-II (Fig. 2E).LPS-FL-DCs captured similar amounts of Ag as control splenic

CD8+ DCs (Fig. 2D), but showed increased activation of MHC-I– and MHC-II–restricted T cells after peptide loading (Fig. 2F).Surprisingly, LPS stimulation had a positive impact on Ag pre-sentation on FL-CD8+, but not on FL-CD82 DCs (Fig. 2E, Sup-plemental Fig. 2C). In conclusion, LPS failed to enhance MHC-Iand MHC-II presentation by all cultured DCs, with the exceptionof FL-CD8+ DCs.B cells express constitutive low levels of DEC-205 (53) but in-

crease DEC-205 expression upon activation: Germinal centerB cells have higher DEC-205 expression than naive B cells (Sup-plemental Fig. 1A), and B cells express high levels of DEC-205after culture with LPS and IL-4 or anti-CD40 and IL-4 (Supple-mental Fig. 1B). LPS/IL-4 activated B cells, which also expressedhigher levels of CD86, MHC-I, and and MHC-II than did splenicDCs (Supplemental Fig. 4), were compared with DCs for Ag pro-cessing and presentation after targeting with anti–mDEC-OVA.Activated B cells captured more Ag than did CD8+ DCs (Fig.2D), and were equivalent to CD8+ DCs in presenting exogenouslyloaded peptide to MHC-I– and MHC-II– restricted T cells (Fig.2F). Consistent with these observations, activated B cells were alsosimilar to CD8+ DCs in Ag presentation to MHC-II–restrictedT cells after Ag targeting to DEC-205 (Fig. 2E). However, B cellblasts induced far less OTI proliferation and were much less effi-cient than DCs in cross-presentation (Fig. 2E, Supplemental Fig.2C). When the relative amount of Ag uptake is taken into account,similar results were obtained for MHC-II presentation for Agstargeted to the B cell AgR instead of DEC-205 (Fig. 3A, 3B) onB cells from B1-8hi mice, specific for NP (30). However, Ag cap-tured by DEC-205 was approximately 3 times more efficient thanthe B cell AgR for cross-presentation (Fig. 3A, 3B). This differenceis not dependent on BCR cross-linking, because aDEC-OVA, to-gether with BSA-NP, was equivalent to aDEC-OVA alone.We conclude that, when Ag is captured by DEC205-mediated

endocytosis, FL-DCs (both CD8+ and CD82) resemble conven-tional spleen DCs with regard to efficiency of Ag presentation.Furthermore, Ag delivery by endocytosis through DEC-205 is notsufficient to ensure efficient cross-presentation, because B cells

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and LPS-GM-DCs were almost 10-fold less effective in cross-presentation than splenic DCs.

Ag presentation after pinocytosis

To determine whether the observed differences in Ag presentationwere cell intrinsic or dependent on the route of Ag capture, weloaded cells with Ag by pinocytosis, using high concentrations ofsolubleOVA (up to 100mg/ml). Although some preparations ofOVAdisplay mannose residues that serve as ligands for mannose recep-tors, we found no detectable binding at 4˚C of our OVA preparationto any of the cell types tested (Supplemental Fig. 5A, 5B).Both spleen DC subsets obtained from naive and immunized mice

showed similar levels of OVA endocytosis after incubation at 37˚C(Fig. 4A, left). Despite their similarities in Ag uptake and presentationof exogenous peptides, OVA captured by bulk pinocytosis was pre-sented more effectively by CD82 than CD8+ DCs to MHC-II–restricted T cells (Fig. 4B, 4C, left). In addition, DCs obtained fromimmunized mice were more efficient in MHC-II Ag presentation thanwere naive DCs, but the differences between the subsets did notchange qualitatively (Fig. 4B, left). Therefore, CD82 DCs are moreefficient than CD8+ DCs at presentation to MHC-II–restricted T cells,irrespective of the route of Ag uptake or activation status.Despite Ag uptake at levels similar to that in conventional DCs,

activated monocytes showed low levels of MHC-II presentation

of pinocytosed Ag, which correlated with low levels of presentationwith pulsed peptide (Fig. 4, left). However, activated monocyteswere similar to spleen DCs for cross-presentation (Fig. 4B, left).We conclude that activated monocytes can cross-present pinocy-tosed Ags as efficiently as spleen DCs; however, these cells are farless effective in MHC-II presentation when Ag is captured byeither DEC205-mediated or bulk phase endocytosis.Among cultured DCs, FL-DCs acquired amounts of OVA by pi-

nocytosis similar to what their spleen DC counterparts acquired(Fig. 4A, right). Whereas FL-CD82 DCs were equivalent to spleenDCs for cross-presentation and far more efficient with regard toMHC-II presentation of pinocytized Ag, FL-CD8+ DCs were lesseffective inboth tasks (Fig. 4B, right; Supplemental Fig. 5C).GM-DCsaccumulated similar amounts of OVA by bulk phase pinocytosisand performed similarly to CD8+ DCs for cross-presentation and in-termediate between the two DC subsets for MHC-II presentation(Fig. 4A, 4B, right).In contrast, activated B cells, which were active in endocytosis

and presentation of peptides, were nearly inactive in processing Agacquired by bulk phase pinocytosis for presentation on MHC-II orMHC-I (Fig. 4, right; Supplemental Fig. 5C). Therefore, weconclude that both the cell type and the route of endocytosis in-fluence the efficiency of Ag presentation.

Ag presentation after phagocytosis

To further analyze the effects of the route of Ag uptake on Agpresentation, we delivered OVA to APCs by phagocytosis of OVA-adsorbed polystyrene beads. Flow cytometrywas used to purify cellsthat had captured a single fluorescent bead to normalize the amountof Ag captured. Internalization was confirmed by analysis on ImageStreamcytometry (Supplemental Fig. 6).Naive and activated splenicDCs presented Ag acquired by phagocytosis in a manner that wassimilar to Ag captured by DEC205-mediated or bulk phase pino-cytosis (Fig. 5A). CD82 DCs remained more efficient than CD8+ inMHC-II presentation, and cross-presentation was similar in bothtypes of DCs (Fig. 5A). Decreasing the amount of OVA on the beadsdid not alter the results (Fig. 5B). However, neither activated mono-cytes nor GM-DCs, nor LPS-activated GM-DCs, presented phago-cytized Ags to MHC-I– or MHC-II–restricted T cells to any appre-ciable degree (Fig. 5A). Similar results were obtained when weassayed splenic monocytes from Listeria-infected mice (tip-DCs)(Supplemental Fig. 7). We conclude that activated monocytes andGM-DCs are far less effective in Ag presentation than are conven-tional DCs when the Ag is acquired by phagocytosis.

DiscussionWe have systematically compared conventional spleen DCs withtissue culture-derived DCs and with activated monocytes for pre-sentation of Ag acquired by DEC205-mediated endocytosis, pi-nocytosis, or phagocytosis. We find that conventional spleen DCsdiffer from all other cells tested in that they excel in Ag presentationirrespective of the route of Ag uptake and activation status. Incontrast, the mechanism of Ag capture has a profound influence onthe efficiency of Ag presentation by other APCs. For example,activated monocytes and GM-DCs (which are monocyte derived)are nearly as effective as conventional DCs in cross-presentation ofAgs captured by DEC205-mediated and bulk phase endocytosis,but they are nearly inactive when the same Ags are captured byphagocytosis. Similarly, activated B cells are as effective as CD8+

DCs for MHC-II Ag presentation when the Ag is delivered byreceptor-mediated endocytosis (DEC-205 or the B cell AgR), butthe same cells are ineffective when the Ag enters the cell by bulkphase endocytosis.

FIGURE 3. DEC205-mediated endocytosis promotes cross-presentation

in B cells. NP-specific B cells were isolated from B1-8hi transgenic mice

and stimulated with LPS and IL-4 for 50–60 h. A, The y-axis shows relative

cell-associated OVA, after targeting with the indicated concentrations of

amDEC-OVA, or amDEC-OVA and BSA-NP, or OVA-NP on the x-axis.

Maximum cell-associated OVAwas normalized to 100 in each experiment.

B, Activation and proliferation of OTI (upper panel) and OTII (lower

panel) T cells in response to OVA-containing B cells. The y-axis shows the

percentage of divided T cells. A and B represent pooled data from three

independent experiments.

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As has been previously documented, Ag uptake was at least 100-fold more efficient when delivered through specific receptors,compared with bulk phase pinocytosis (54). However, not allreceptors deliver Ag to intracellular compartments with equal effi-ciency. For example, DEC-205 is more efficient in Ag internal-ization than is DCIR2 (26). When equivalent amounts of capturedAg are compared, however, the efficiency of presentation by DCswas similar for bulk phase and DEC205-mediated endocytosis.B cells differed from DCs in that Ags taken up by bulk phasepinocytosis were presented inefficiently when compared withreceptor-mediated uptake. Unlike DCs, whose primary function isto initiate immunity, B cells present Ags to recruit specific cognateT cell help. The relative inefficiency at processing and presenta-tion of Ags acquired by bulk phase endocytosis guarantees that T cellhelp remains focused during an immune response. Only B cells that

capture Ag through specific AgRs will recruit T cell help. In contrast,the DCs’ ability to present a range of Ags acquired by pinocytosisserves to broaden the scope of T cell immunity.Several groups have suggested that cross-presentation is a spe-

cialized function of CD8+ DCs (27–29, 39), whereas others havecome to the opposite conclusion (43, 55–57). However, many ofthese experiments failed to consider the amount or route of Agcapture. For instance, CD8+ DCs are far better at taking updead cells than are CD82 DCs and therefore are favored in termsof presenting Ag contained in dead cells (41). Similarly, CD8+

DCs are the preferred Ag cross-presenting cells in viral (29, 58) orL. monocytogenes (27) infections; however, this, too, may be dueto differential access to the Ag. We have shown that CD8+ DCs areenriched in components of the MHC-I processing pathway, but wedid not measure the intrinsic MHC-I Ag presenting capacity of

FIGURE 4. Ag presentation after pinocytosis. APCs

were isolated from spleen or cultured from bone mar-

row or spleen of WT mice. A, The y-axis shows relative

cell-associated OVA, developed with streptavidin-HRP,

after incubation of APCs with the indicated concen-

trations of OVA-biotin on the x-axis. B, Activation and

proliferation of OTI (upper panels) and OTII (lower

panels) T cells in response to OVA-containing APCs.

The y-axis shows the percentage of divided T cells. C,

Graphs show OTI (upper panels) and OTII (lower

panels) T cell proliferation in response to peptide

pulsed APCs. A–C represent pooled data from three to

five independent experiments.

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CD8+ versus CD82 cells directly (26). The data presented in thispaper show that when the amount of Ag captured and the route ofinternalization are taken into consideration, the two subsets ofconventional spleen DCs have similar abilities with regard tocross-presentation. In agreement with others, we found that therewere small differences in MHC-I cross-presentation by the twoDC subsets: CD8+ DCs were slightly more efficient than CD82

DCs after phagocytosis of OVA-adsorbed beads (40) and also afterDEC205-mediated endocytosis when limiting numbers of DCs arecompared. However, these differences were far less pronouncedthan the difference between either subset of DCs and APCs de-rived from monocytes.In contrast to cross-presentation, CD82 DCs are more efficient

than CD8+ DCs and most other APCs tested for processing andpresenting Ags in MHC-II, irrespective of the route of Ag ac-quisition or maturation status (26). Thus, in the steady state thissubset may be key to maintaining CD4+ T cell tolerance to self-Ags (32, 59–61). Nevertheless, activation improves MHC-II pre-sentation by CD8+ DCs and may enhance their ability to activateCD4+ T cells during infection.Activated monocytes, including GM-DCs and tip-DCs, share many

featuresof conventional spleenDCs, but arise fromdistinctprogenitors(8, 10, 22, 62). For example, whereas conventional DCs are normal inCCR22/2 mice, tip-DCs do not develop in the spleen of these miceafter Listeria infection because monocytes fail to emigrate from thebone marrow (63). In addition, activated monocytes do not expresscharacteristic DC lectins (DCIR2 or DEC-205), or high levels of Flk2(FL receptor), but instead express the M-CSF receptor, like theirmonocyte progenitors (Supplemental Figs. 3D, 7B). Finally, CD8+

DCs and activatedmonocytes rely ondifferentmechanisms to enhancecross-presentation (64). Although activated monocytes contribute toimmunity against influenza (65), Lesihmania (66), and Listeria (23),

they do not appear to be responsible for the initiation of adaptive im-mune responses. This is consistent with these cells’ lower levels ofMHC-II surface expression and poor performance in MHC-II pre-sentation, irrespective of the mechanism of Ag capture, includingpeptide pulsing. Notably, monocyte-derived DCs do not contributesignificantly to the steady-state pool of DCs in lymphoid (8) andmay only contribute to CD1032 populations of CD11c+ cells in non-lymphoid organs (13, 14), which have poor T cell stimulation capacity(14, 67).Although they appear to be most closely related to activated

monocytes and tip-DCs (62, 68), the physiologic counterparts ofbone marrow-derived GM-DCs remain to be defined. GM-DCscan present Ags captured by DEC205-mediated or bulk phaseendocytosis; these cells also transport MHC-II to the cell surfaceafter activation by TLR ligation (6, 48, 50, 51). However, prioractivation did not have a positive impact on their ability to presentAgs to T cells. In contrast, FL-DCs resembled conventional spleenDCs in this important respect (i.e., Ag presentation was enhancedby activation through TLR ligation). In addition, mirroring theirsplenic counterparts, FL-CD82 DCs were better than FL-CD8+

DCs in activating MHC-II–restricted T cells. FL-DCs originatefrom the same progenitor (pre-DCs), rely on the same cytokine(FL) for differentiation and expansion, and are more similar toconventional DCs with regard to Ag processing and presentation.Thus, GM-DCs may not be the best cell type to investigate the roleof conventional DCs in immune responses.In keeping with the idea that the primary function of activated

monocytes and GM-DCs might be as innate immune effector cellsthat destroy phagocytized Ag (23, 62), these cells were far lessefficient in presenting phagocytized Ag than were conventionalDCs. This observation is consistent with the finding that monocyte-derived mononuclear phagocytes tend to have more developed

FIGURE 5. Ag presentation after phagocytosis. A,

APCs were isolated from spleen of naive or mBSA-

CFA–immunized mice. GM-DCs were derived from

bone marrow. Enriched populations were incubated

with OVA-beads before sorting. Panels show activation

and proliferation of OTI (left panels) and OTII (right

panels) T cells after incubation with the indicated

number of APCs in the x-axis, that were sorted to

contain a single OVA-bead. The y-axis shows the per-

centage of divided T cells. In lower panels, each

symbol indicates independent experiments and repre-

sents the average of duplicate measurements where

2500 sorted APCs were incubated with T cells. Data

were analyzed by ANOVA, and Tukey’s test was used

to compare groups. pp , 0.05; pppp , 0.001. B, As in

A, but DCs from naive mice were incubated with beads

adsorbed with 25% OVA and 75% keyhole limpet he-

mocyanin protein. A and B represent pooled data from

two to seven independent experiments.

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lysosomes that may hamper the escape of processed peptides intoMHC loading compartments (7, 69).We have shown that cell intrinsic differences have a profound

impact onAghandling forMHC-II and cross-presentation.Althoughmost APCs studied have the ability to process Ag, in non-DCs theroute of Ag capture has a profound impact on its subsequent pro-cessing and presentation. Our observations have significant impli-cations for understanding the development of adaptive immuneresponses and targeted vaccination strategies.

AcknowledgmentsWe thank C.G. Park for cells producing rFL, K. Yao for production and pu-

rification of rFL, D. Bosque and T. Eisenreich for genotyping and caring for

mice, E. Pamer for L. monocytogenes, T. Schwickert for B1-8hi mice, C.G.

Park and C. Chong for CD11c-hDECmice, G. Begley (Amgen) for GMCSF-

Rb–deficient mice, K.Velizon and O. Uche for the multiple long sorts, C.

Bare from The Rockefeller University Flow Cytometry facility for operating

the Image Stream100 flow cytometer, members of the laboratory, and R.M.

Steinman for helpful discussions and critical reading of the manuscript.

DisclosuresThe authors have no financial conflicts of interest.

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