With Distinct Functional Properties Dendritic Cells

CX3CR1+c-kit+ Bone Marrow Cells Give Rise to CD103 + and CD103− Dendritic Cells with Distinct Functional Properties

This information is current as Maria-Luisa del Rio, Jose-Ignacio Rodriguez-Barbosa, of September 25, 2021. Jasmin Bölter, Matthias Ballmaier, Oliver Dittrich-Breiholz, Michael Kracht, Steffen Jung and Reinhold Förster J Immunol 2008; 181:6178-6188; ; doi: 10.4049/jimmunol.181.9.6178

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

؉ ؉ ؉ CX3CR1 c-kit Bone Marrow Cells Give Rise to CD103 and CD103؊ Dendritic Cells with Distinct Functional Properties1

Maria-Luisa del Rio,* Jose-Ignacio Rodriguez-Barbosa,† Jasmin Bo¨lter,* Matthias Ballmaier,‡ Oliver Dittrich-Breiholz,§ Michael Kracht,¶ Steffen Jung,ʈ and Reinhold Fo¨rster2*

Dendritic cells (DC) represent a rather heterogeneous cell population with regard to morphology, phenotype, and function and, ␣ ؉ like most cells of the immune system, are subjected to a continuous renewal process. CD103 (integrin E) DC have been identified as a major mucosal DC subset involved in the induction of tissue-specific homing molecules on T cells, but little is known about ؊ ؉ ؉ ؉ ؉ progenitors able to replenish this DC subset. Herein we report that lineage (lin) CX3CR1 c-kit (GFP c-kit ) bone marrow cells can differentiate to either CD11c؉CD103؊ or CD11c؉CD103؉ DC in vitro and in vivo. Gene expression as well as functional assays reveal distinct phenotypical and functional properties of both subsets generated in vitro. CD103؊ DC exhibit enhanced ؉ phagocytosis and respond to LPS stimulation by secreting proinflammatory cytokines, whereas CD103 DC express high levels Downloaded from of costimulatory molecules and efficiently induce allogeneic T cell proliferation. Following adoptive transfer of GFP؉c-kit؉ bone marrow cells to irradiated recipients undergoing allergic lung inflammation, we identified donor-derived CD103؉ DC in lung and the lung-draining bronchial lymph node. Collectively, these data indicate that GFP؉c-kit؉ cells contribute to the replenishment .of CD103؉ DC in lymphoid and nonlymphoid organs. The Journal of Immunology, 2008, 181: 6178–6188

PCs are an essential component of the immune system that lung-draining bronchial LN (brLN) have evolved to acquire op- http://www.jimmunol.org/ connects the innate and the adaptive immune responses. posing functions in presenting innocuous inhaled Ag. Thus, under They are involved in maintenance of tissue homeostasis, tolerogenic conditions, the CD103Ϫ DC present innocuous Ag to A ϩ ϩ inflammation, and the organization of the immune response (1). CD4 T cells, while the CD103 DC, which do not express Dendritic cells (DC)3 reside in peripheral tissues, such as the CD8␣, are specialized in presenting Ag exclusively to CD8ϩ T lung, and migrate under inflammatory as well as steady-state con- cells (9). Considering that in the lung, CD8␣ DC are virtually ditions to the regional lymph node, reflecting the continuous turn- missing and that most DC colonizing this tissue express CD11b, over process of these cells (2). Blood monocytes have been iden- we hypothesized that under homeostatic as well as inflammatory tified as a cell population that continuously replenishes those DC conditions bone marrow progenitors might contribute to the re- high high ϩ Ϫ by guest on September 25, 2021 that left peripheral organs during turnover. CCR2 Ly6C CX3 plenishment of CD103 and CD103 DC in the lung. low low low high CR1 and CCR2 Ly6C CX3CR1 cells are the two main To test this idea, we took advantage of a mutant mouse, in which populations of blood monocytes in mice (3). Recent findings enhanced GFP (eGFP) gene was knocked into the fractalkine re- high high low low low demonstrated that CCR2 Ly6C CX3CR1 and CCR2 Ly6C ceptor gene locus (CX3CR1). CX3CR1 is primarily expressed by high CX3CR1 cells are able to preferentially give rise to pulmonary subpopulations of NK cells, monocytes, macrophages, and DC but CD103ϩ and CD103Ϫ DC, respectively (4, 5). is also found on a subpopulation of bone marrow progenitor cells ϩ ϩ/gfp CD103 DC are also found in the intestinal lamina propria, (10). Using bone marrow from CX3CR1 mice, we sorted lin- Ϫ ϩ ϩ ϩ Ϫ ϩ ϩ mesenteric lymph nodes (LN), and skin-draining LN and have eage (lin) CX3CR1 c-kit CD11b /CD11b (GFP c-kit )as Ϫ ϩ Ϫ ϩ Ϫ been described as the major DC subset of the lung (6–8). We have well as lin CX3CR1 c-kit (GFP c-kit ) cells that were used as previously reported that CD103ϩ and CD103Ϫ DC residing in the a source of myeloid progenitors for the in vitro and in vivo differentiation of DC. We demonstrate that CD103Ϫ as well as ϩ ϩ ϩ ϩ † CD103 DC can be generated from both GFP c-kit and GFP c- *Institute of Immunology, Hannover Medical School, Hannover, Germany; Institute Ϫ Ϫ of Biomedicine, Immunology Section, University of Leoń, Leoń, Spain; ‡Clinic for kit cells cultured in vitro in the presence of GM-CSF. CD103 Pediatric Hematology and Oncology and §Institute of Biochemistry, Hannover Med- DC displayed up-regulated transcripts for genes involved in innate ical School, Hannover, Germany; ¶Rudolf-Buchheim Institute of Pharmacology, Uni- ʈ immunity, whereas those involved in costimulation were down- versity of Giessen, Giessen, Germany; and Department of Immunology, The Weiz- ϩ mann Institute of Science, Rehovot, Israel regulated when compared with CD103 DC, suggesting distinct Received for publication June 10, 2008. Accepted for publication September 3, 2008. functional activities of these two DC subsets. In vivo adoptive ϩ ϩ The costs of publication of this article were defrayed in part by the payment of page transfer of GFP c-kit cells to irradiated recipients undergoing an charges. This article must therefore be hereby marked advertisement in accordance allergic immune response allowed the induction of donor-derived with 18 U.S.C. Section 1734 solely to indicate this fact. CD103ϩ DC in the lung and the lung-draining LN. These results 1 This work has been supported by Deutsche Forschungsgemeinschaft Grant indicate that these progenitors can be recruited to sites of inflam- SFB587-B3 to R.F. mation where they further differentiate in situ to DC, which then 2 Address correspondence and reprint requests to Dr. Reinhold Fo¨rster, Institute of Immunology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, migrate to draining LN to induce adaptive immunity. Germany. E-mail address: [email protected] 3 Abbreviations used in this paper: DC, dendritic cell; BALF, bronchoalveolar lavage fluid; brLN, bronchial lymph node; eGFP, enhanced GFP; lin, lineage; LN, lymph Materials and Methods node; MDP, macrophage and DC progenitor; PI, propidium iodide; PRR, pattern Mice recognition receptor; SR, scavenger receptor. ϩ/gfp Mice expressing eGFP instead of the CX3CR1 gene (CX3CR1 )onthe Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 CD45.2 C57BL/6 (B6) background have been described elsewhere (10). www.jimmunol.org The Journal of Immunology 6179

CD45.1 recipients, OT-I Thy1.1 and OT-II Ly5.1, mice that carry a trans- were harvested, minced under sterile conditions, and red cells were lysed genic TCR for the H2-Kb-restricted SIINFEKL peptide derived from in ACK lysing buffer for 2 min. Cells were then washed and suspended in b ϩ Ϫ ϩ OVA257–264 or for the H2-A -restricted ISQAVHAAHAEINEAGR pep- complete medium. CD103 and CD103 DC were sorted from GFP c- ϩ tide derived from OVA323–339, respectively, were also used in this study kit bone marrow cells that were differentiated in vitro in the presence of (11, 12). Mice were bred and maintained under specific pathogen-free con- GM-CSF for 5 days, seeded in U-bottom 96-well plates (5000 DC/well), ditions at the Central Animal Facility of Hannover Medical School or were and cocultured with 1.5 ϫ 105 allogeneic BALB/c splenocytes or 1.5 ϫ 105 purchased from Charles River Laboratories. All animal experiments were OT-I or OT-II T cells for 3 days. DC cultured with OT-I or OT-II cells conducted in accordance with local and institutional guidelines. were loaded with OVA prior culture. Proliferation of syngeneic, allogeneic, ϩ ϩ OT-I, and OT-II T cells in triplicate wells was then measured by pulsing In vitro DC differentiation from c-kit CX3CR1 bone marrow the cells with 1 ␮Ci/well methyl-[3H]thymidine for 16 h. Plates were har- progenitors vested and thymidine uptake was quantified in a beta counter (MicroBeta ϩ/gfp TriLux, PerkinElmer). Bone marrow cells from CX3CR1 mice were harvested by flushing femurs and tibias. Applying a cocktail of lineage-specific Abs (CD3, Microarray analysis of gene expression of CD103Ϫ and NK1.1, Ly6G, CD19, Ter-119), anti-CD11c, anti-c-kit mAb (CD117), and CD103ϩ DC eGFP as a marker for CX3CR1 expression, four populations of bone marrow cells were separated by flow sorting (see Fig. 1). The purity of sorted Total RNA was extracted from CD103Ϫ and CD103ϩ DC differentiated for Ͼ Ϫ ϩ ϩ populations was always 96%. After cell sorting, the four populations 5 days from lin CX CR1 c-kit bone marrow cells using RNeasy Mini ϫ 5 3 were subsequently cultured in 24-well plates at 1–5 10 cells/well in kit (Qiagen), transcribed into Cy3- or Cy5-labeled cRNA, respectively, and complete medium (RPMI 1640, 10% FCS, 2 mM L-glutamine, 1 mM so- cohybridized onto the same microarray. Samples derived from two inde- ␮ ϫ Ϫ5 dium pyruvate, 10 mM HEPES, 50 g/ml gentamicin, and 5 10 M pendent experiments were analyzed separately on two arrays, including one 2-ME) supplemented with 30 ng/ml GM-CSF (PeproTech/Tebu). Every dye-swap experiment. Whole Mouse Genome (4 ϫ 44,000) Oligo Microar- two to 3 days, the culture medium was collected, centrifuged to recover the ray kits (Agilent Technologies) were used in this study. Downloaded from cells in suspension, and then replated in fresh medium. Phenotypic analysis Sample processing, hybridization, and data extraction were performed was performed by flow cytometry to monitor the course of DC differenti- according to standard protocols (Agilent Technologies). Detailed informa- ation. GFPϩc-kitϩ bone marrow cells cultured for 5 days under GM-CSF ϩ ϩ ϩ Ϫ tion concerning experimental procedures as well as all microarray data treatment were sorted into CD11c CD103 and CD11c CD103 DC (pu- discussed in this publication have been deposited in the Gene National Ͼ rity 99%). Cytospins were prepared, air-dried, fixed in 3% paraformal- Center for Biotechnology Information Expression Omnibus (GEO, www. dehyde, and stained with H&E. ncbi.nlm.nih.gov/geo/) and are accessible through GEO series accession Ϫ ϩ ϩ number GSE10882, or directly via the following link: www.ncbi.nlm.

Adoptive transfer of lin CX CR1 c-kit bone marrow http://www.jimmunol.org/ 3 ϭ ϭ progenitors to recipients undergoing allergic lung inflammation nih.gov/geo/query/acc.cgi?token dbmhvccekomusjy&acc GSE10882. Criteria for visualization of microarray data for Figs. 3–6 are as follows: 1) CD45.1 recipient mice were immunized i.p. with 150 ␮g of OVA (grade All family members of a gene family of interest were selected as described VI; Sigma-Aldrich) in 200 ␮l aluminum hydroxide gel adjuvant (2.0% in the legends of Figs. 3–6. 2) Data from family members that do not show ϫ 5 Ϫ significant expression were excluded. Significant mRNA expression was Alhydrogel, Brenntag Biosector). Ten to 15 days later, 3 10 lin CX3 CR1ϩc-kitϩ bone marrow cells were adoptively transferred to nonirradi- defined by algorithms implemented within the data extraction software: ated or irradiated recipients (6 Gy) that were treated in an aerosol chamber feature extraction V.9.1.3.1 (FEV9.1.3.1). Data were used for visualization with OVA aerosol (1% in water) using a PariBoy vaporizer over 7 days as when at least in one channel of each of the two replicate microarray ex- described previously (13). At day 8, mice were sacrificed and lung, spleen, periments significant signal values were reached (corresponding to a “pos- and peripheral LN were analyzed by flow cytometry to monitor for the itive entry of 1” in the column labeled “IsWellAboveBackground” of the presence of CD45.2 donor-derived cells. FEV9.1.3.1 results file). 3) When multiple probes directed against the same by guest on September 25, 2021 mRNA species were present on the microarray, data were visualized for Isolation of lung, spleen, brLN, and bronchoalveolar lavage that particular probe that showed highest intensity levels across all four fluid (BALF) infiltrating cells channels of both microarrays (calculated by the arithmetic mean value of the “ProcessedSignal” values of all four subdatasets). 4) Highly significant Twenty-four hours after the last aerosol treatment, mice were anesthetized differential mRNA expression was defined as follows: “PValue” as calcu- and exsanguinated by perfusing the hearts with cold PBS until the lung lated by FEV9.1.3.1 for the corresponding probe was below 0.0001. lobes were free of blood. Lungs were perfused intratracheally three times ϩ ϩ with 1 ml each of Ca2 - and Mg2 -free PBS supplemented with 0.1 mM In vitro phagocytosis assays EDTA (Invitrogen) and the BALF was collected. RBC were lysed using ϩ ϩ ammonium chloride lysis buffer (ACK, BioWhittaker). Lung tissue was DC (2 ϫ 104) derived from GFP c-kit bone marrow cells collected at day then cut into small pieces and digested with collagenase A (Roche) plus 25 5 of culture were incubated with 2.5 ␮g/ml OVA-Cy5 at 37°C or 4°C for ␮g/ml DNase I (Roche) for 90 min at 37°C in a shaking platform (150 10 min, washed extensively in PBS, and analyzed by flow cytometry. rpm). The digested mixture was passed through a nylon mesh to remove Gram-negative Escherichia coli TOP10 (Invitrogen) and zymosan A undigested fragments and then subjected to a Percoll gradient. Spleen or (Sigma-Aldrich) were labeled with the fluorescent dye Cy5 (Amersham). A brLN donor-derived cells were obtained by enzymatic digestion using bacterial cell suspension of OD equivalent to 1 measured at 600 nm was complete medium supplemented with DNase I and collagenase A or col- prepared, whereas zymosan A particles were resuspended at 1 mg/ml. lagenase D (Roche), respectively, as previously described (9). Then, 100 and 300 ␮l of Cy5 were added to the bacterial and zymosan A suspensions, respectively. After1hofincubation in the dark, the reaction Flow cytometry was stopped by adding cold PBS. The bacterial and zymosan A suspen- The following mAbs were used in this study: CD45.1 (A20), CD45.2 sions were centrifuged at 10,000 rpm for 5 min and washed several times (104), CD3 (17A2), CD11b (M1/70.15), CD11c (HL3), CD19 (1D3), to remove the unbound fluorochrome. Cy5-labeled bacteria and zymosan A CD80 (16-10A1), CD86 (RMMP-1), CD40 (3/23), rat isotype control were incubated with opsonizing polyclonal Ab (Molecular Probes) for 15 (R35-95), NK1.1 (PK136), Ter-119 (Ly-76), MHC class II (IAb) (AF6- min and then washed and used in the phagocytosis assay. Phagocytosis assays were performed by incubating the nonopsonized or opsonized Cy5- 120.1), Ly6G (1A8), CD117 (104D2), and CD103 (M290) were purchased ϩ ϩ from BD Biosciences; PDL1 (MIH5), PDL2 (122), B7-H4 (188), 4-1BBL labeled bacteria or Cy5-labeled zymosan A with GFP c-kit -derived DC (TSK-1), and CD83 (Michel-17) were obtained from e-Bioscience. Fc re- (cultured for 5 days) and left for 20 min at 37°C. Cells were washed in cold ceptors were blocked by preincubating the cell suspensions with 2 ␮g/ml PBS containing 0.02% Na2-EDTA and samples were analyzed by flow of blocking anti-FcR mAb (2.4G2) before adding the specified mAb to cytometry. reduce nonspecific binding (14). Dead cells and debris were excluded from acquisition by staining with DAPI (4Ј,6Ј-diamido-2-phenylindole hydro- Cytokine production chloride) or propidium iodide (PI) staining. Flow cytometry acquisition CD103ϩ and CD103Ϫ DC were sorted at day 5 from in vitro cultures of was conducted on a LSR II cytometer (BD Biosciences) and data analysis GFPϩc-kitϩ cells and incubated overnight in complete medium supple- was performed using WinList version 5 (Verity Software House). mented with either 1 ␮g/ml R848 (InvivoGen), 16 ␮g/ml CpG (Amer- MLR and T cell proliferation assays sham), 1 ␮g/ml LPS (Sigma-Aldrich), or were left untreated. Supernatants were analyzed for the presence of IL-6, IL-10, MCP-1, IFN-␥, TNF, and Murine syngeneic B6, allogeneic BALB/c, OT-I Thy1.1, or OT-II Ly5.1 IL-12 (p70 subunit) using a cytometric bead array following the manufac- splenocytes were prepared and used as responder cells. To this end, spleens turer’s instructions (BD Biosciences). ϩ ϩ ϩ Ϫ 6180 CX3CR1 c-kit BM-DERIVED CD103 AND CD103 DC

CD11cϩCD103Ϫ DC cells differentiated from GM-CSF-treated GFPϩc-kitϩ progenitors cultured for 5 days exhibited a DC-like morphology with a kidney-shaped nuclei and long cytoplas- matic extensions, as shown in Fig. 2C. CD103Ϫ DC do not transform into CD103ϩ DC or vice versa We next compared the capacity of GFPϩc-kitϩ and GFPϩc-kitϪ cells to proliferate and expand in vitro in the presence of GM-CSF. c-kitϪ bone marrow cells (GFPϩc-kitϪ) survived without further ϩ ϩ FIGURE 1. Cell sorting strategy for the isolation of CX3CR1 c-kit expansion for 5 days but were progressively diminished at later ϩ Ϫ ϩ ϩ ϩ and CX3CR1 c-kit bone marrow cells. Bone marrow cells were har- time points. In contrast, c-kit cells (GFP c-kit ) expanded 50- ϩ/gfp vested from CX3CR1 mice and labeled with anti-c-kit, anti-CD11c, fold during the first 5 days of culture. Cell counts then stayed and a cocktail of lineage-specific Abs (CD3, NK1.1, Ly6G, CD19, and rather constant until day 10 and slowly decayed afterward (Fig. ϩ Ϫ Ter-119 mAbs). Based on the differential expression of CX3CR1, two ϩ Ϫ Ϫ int 2D). To test whether CD103 and CD103 DC keep their phe- CD11c subsets (R2 and R3) were identified. Lin CD11c CX3CR1 ϩ Ϫ ϩ ϩ Ϫ notyperegardingCD103expression,CD11c CD103 andCD11c cells (R4) were further sorted into c-kit and c-kit cells and used in ϩ ϩ ϩ further studies. CD103 DC were sorted from day 5 cultures of GFP c-kit cells and were further cultured for 2 days in the absence or presence of LPS (1 ␮g/ml). Interestingly, under both culture conditions, DC Results did not change their CD103 expression status while, as expected, Downloaded from LPS induced DC maturation as shown by increased expression of CX CR1 and c-kit expression distinguish two populations of 3 MHC class II molecules (Fig. 2E and data not shown). precursors within the bone marrow ϩ/gfp Enhanced expression of costimulatory molecules in resting and Bone marrow cells of CX3CR1 heterozygous B6 mice were ϩ stained with a cocktail of lineage-specific mAb (CD3, NK1.1, LPS-treated CD103 DC ϩ Ϫ

Ly6G, CD19, Ter-119) excluding CD11b, together with anti-c-kit Based on our previous studies showing that CD103 and CD103 http://www.jimmunol.org/ and anti-CD11c. Together with eGFP signals identifying brLN DC differ with regard to their capacity to present and cross- ϩ Ϫ CX3CR1 cells, several populations of lin cells could be iden- present Ag (9), and based on the stable expression of CD103 ob- ϩ tified (Fig. 1). Two CD11c subsets, one being GFPlow (R2) one served in the above-described experiments, we reasoned that Ϫ being GFPhigh (R3), as well as a GFPintCD11c population (R4) CD103 might also be a suitable marker that allows the differenti- were readily detectable. Further analysis of the latter subset re- ation of functionally distinct DC subsets generated in vitro. To test ϩ Ϫ vealed two populations of progenitor cells: GFP c-kit (R5) and this hypothesis, we analyzed gene expression of CD11cϩCD103ϩ ϩ ϩ GFP c-kit (R6). The latter population represents ϳ20% of the and CD11cϩCD103Ϫ DC sorted from day 5 applying high-density ϩ gfp/ϩ ϩ GFP cells in bone marrow of CX3CR1 mice. These GFP oligonucleotide arrays. Data obtained from DC differentiated from ϩ c-kit cells are similar to the recently described macrophage and GFPϩc-kitϩ cells cultures in the absence of maturation stimuli by guest on September 25, 2021 Ϫ ϩ ϩ Ϫ Ϫ DC progenitor (MDP) progenitors (lin CX3CR1 c-kit CD11b ) indicated that CD103 DC express lower levels of several co- (15), although note that we did not include anti-CD11b mAb in the stimulatory molecules of the TNF and Ig superfamily, as well as linage cocktail for reasons discussed in the Discussion. MHC class II molecules (Fig. 3A). Interestingly, CD103Ϫ DC expressed higher levels of B7-H3 (CD276) (Fig. 3A). Flow cytom- GFPϩc-kitϩ bone marrow cells differentiate in vitro to CD103ϩ Ϫ etry on resting DC, as well as on DC activated with LPS for 24 h, and CD103 DC in the presence of GM-CSF confirmed the tendency that CD103Ϫ DC show reduced levels for Further analysis of the two CD11cϩ subsets revealed that these some costimulatory molecules such as CD83, CD86, and PDL2, cells represent immature stages of committed DC that do not fur- while others such as CD40, CD80, and PDL1 were expressed to ther differentiate in vitro (data not shown). Thus, these populations similar amounts on CD103Ϫ and CD103ϩ DC (Fig. 3B). were not further analyzed in the present study. To evaluate the To test whether CD103ϩ and CD103Ϫ DC differ with regard to potential of GFPϩc-kitϩ and of GFPϩc-kitϪ to give rise to Ag presentation, both populations were used in allogeneic T cell CD11cϩ DC following in vitro culture, both populations were proliferation assays. While both DC populations were equally po- sorted and incubated for 5 days in the presence of GM-CSF. Since tent to induce syngeneic T cell proliferation, CD103ϩ DC were c-kit is a surface marker of hematopoietic progenitor cells that is ϳ3-fold more efficient in stimulating allogeneic T cells compared progressively down-regulated during cell differentiation (16), we with CD103Ϫ DC (Fig. 3C). However, analysis of T cells activated analyzed c-kit expression of GFPϩc-kitϩ during a period of 7 days with specific Ag gave different results. Neither immature CD103ϩ of in vitro culture. Starting at day 1, c-kit was continuously down- nor immature CD103Ϫ DC, loaded with OVA, were able to acti- regulated, while as early as day 3, CD11cϩ cells could be identi- vate OVA-specific MHCI-restricted OT-I or MHCII-restricted fied. At day 7, c-kitϩ cells were virtually absent, while ϳ70% of OT-II cells (Fig. 3D). Applying LPS-matured DC in this assay, we all cells expressed CD11c, suggesting that these cells differentiated observed that CD103Ϫ and CD103ϩ DC activated OT-I cells to a to DC (Fig. 2A). Next, we tested whether cells of GFPϩc-kitϩ or similar degree, while CD103Ϫ DC were seemingly more efficient GFPϩc-kitϪ could be differentiated in vitro to CD103ϩ DC. As in activating OT-II cells (Fig. 3D). ϩ ϩ shown in Fig. 2B, considerable amounts of CD11c CD103 and Ϫ CD11cϩCD103Ϫ cells could be identified at day 5 in cultures of Increased phagocytic activity in CD103 DC both GFPϩc-kitϩ as well as GFPϩc-kitϪ cells. A subset of cells Macrophages and DC express a large repertoire of pattern recog- expressing a low amount of CD103 was already present at day 3 in nition receptors (PRR) (17). Among the PRRs, TLRs, which reg- GFPϩc-kitϪ cell cultures (Fig. 2B). Data obtained from these in ulate gene expression, are usually distinguished from those vitro experiments indicate that GFPϩc-kitϩ as well as GFPϩc-kitϪ involved in effector immune functions such as phagocytosis (18– cells possess the capacity to differentiate to CD11cϩCD103ϩ and 20). Within the scavenger receptor (SR)-A family, Msr1 and CD11cϩCD103Ϫ DC. Cytospins of sorted CD11cϩCD103ϩ and MARCO genes were 6.4- and 12.5-fold increased in CD103Ϫ The Journal of Immunology 6181

compared with CD103ϩ DC. Similarly, within the SR-B family CD36, Scarb1 and Scarb2 gene expression were increased 5.4-, 4.1-, and 2.2-fold, respectively. Genes from SR-D family (CD68) and SR-E (LOX-1) were up-regulated 3.8- and 7.9-fold, whereas SR-PSOX (CXCL16) was down-regulated 2.2-fold in CD103Ϫ DC. There was also a signiﬁcantly increased expression of other scavenger-like receptor genes in CD103Ϫ compared with CD103ϩ DC such as Fpr-rs2 (formyl peptide receptor, 25.1-fold), CD93 (C1qr1, 17.9-fold), CD163 (hemoglobin scavenger receptor, 12.2- fold), or CD14 (2.6-fold), whereas the mRNA level for CD6 was 2.6-fold decreased (Fig. 4A). Three major groups of proteins within the C-type lectin family are known to play a role in the process of phagocytosis: group VI (mannose receptor family), group II (CD209, Langerin and Clec molecules), and group V (NK cell receptor-like molecules) (18). Remarkably, the mRNA level for these phagocytic receptors was highly up-regulated in CD103Ϫ compared with CD103ϩ DC. Within group VI, the mannose receptor (Mrc1) was strongly up-

regulated (73.1-fold), while the expression of DEC-205 was Downloaded from slightly diminished (Ϫ1.9-fold; Fig. 4A). The high mRNA level for the mannose receptor correlated with an enhanced ability of the CD103Ϫ DC to engulf Cy5-labeled OVA (Fig. 4B), a process known to be mediated via this receptor (21). Within the C-type lectin group II, mRNA expression levels of

all CD209 members (CD209a–e) were moderately increased (be- http://www.jimmunol.org/ tween 1.6- and 3.4-fold), while Langerin was 4.9-fold increased in CD103ϩ DC. In contrast, members of the Clec family were increased 2- to 18-fold in the CD103Ϫ DC (Fig. 4A). Within the NK cell receptor-like molecules of C-type lectin group V, Clec7a (Dectin-1, ␤-glucan receptor) and Clec1b genes were 4.3- and 8.6- fold increased, while another set of genes within this group, such as CD94, CD72, and CD69, were 6.6-, 3.2-, and 4.8-fold decreased in CD103ϩ cells (Fig. 4A). Further groups of phagocytosis receptors studied were ficollins by guest on September 25, 2021 (Fcna and Fcnb) and chinitases (Chi3l3, Chi3l4, and Chi3l1). All mRNAs for these molecules were remarkably up-regulated in CD103Ϫ DC, with Chi3l3 (52.2-fold) showing the strongest difference in expression compared with CD103ϩ DC (Fig. 4A). The overall up-regulation of phagocytosis-associated genes in the CD103Ϫ DC correlated with their capacity to engulf fluorochrome-labeled E. coli and yeast particles (zymosan) from S. cer- evisiae (Fig. 4C). Optimal phagocytosis of some microorganisms depends on the deposition of serum opsonins on the surface of the microbe that coat the target cells and facilitate the uptake and subsequent de- struction upon interaction with Fc␥ and complement receptors. Most genes encoding for the Fc receptors were either below de- tection threshold (CD89, poly-IgR) or expressed at comparable levels (CD64, Fcer1a, and FcRN) in CD103Ϫ and CD103ϩ DC (data not shown). Only Fcgr3a (CD16), Fcgr2b (CD32), and Fcer1g (high-affinity IgE receptor) were significantly higher expressed in CD103Ϫ DC (Fig. 4A). Regarding the genes encoding for the complement receptors CR2 (CD21), CR3 (CD11b/CD18), Ϫ ϩ FIGURE 2. GM-CSFdrivesthedifferentiationofmurinelin CX3CR1 c- CR4 (CD11c/CD18), and C3ar1, comparable levels of mRNA ex- ϩ Ϫ ϩ Ϫ ϩ Ϫ kit and lin CX3CR1 c-kit bone marrow cells to CD103 and CD103 pression were detected in both DC subsets (data not shown). Sig- ϩ ϩ ϩ Ϫ DC. After sorting, GFP c-kit (1 ϫ 105 cells/well) and GFP c-kit (5 ϫ nificantly higher expression levels of CD93 (C1qr1, 17.9-fold) and 105 cells/well) were cultured in 24-well plates in the presence of GM-CSF and the course of DC differentiation was monitored for the next 7 days. A, c-kit down-regulation and CD11c up-regulation on GFPϩc-kitϩ during differentiation are shown. B, GFPϩc-kitϩ and GFPϩc-kitϪ bone marrow cells cultured in vitro give rise to CD103ϩ DC to different extents. C, Cytospins kitϪ (5 ϫ 105 cells/well). E, CD11cϩCD103ϩ and CD11cϩCD103Ϫ DC of sorted CD11cϩCD103ϩ and CD11cϩCD103Ϫ DC differentiated from were sorted from day 5 cultures of GFPϩc-kitϩ bone marrow cells. Both day 5 cultures of GFPϩc-kitϩ progenitors were fixed in paraformaldehyde populations were further cultured for 2 days in the absence or presence of and stained with H&E. D, GFPϩc-kitϩ (1 ϫ 105 cells/well) exhibited an LPS (1 ␮g/ml) and analyzed for the expression of CD103 and IAb. Rep- increased potential to expand and survive in vitro compared with GFPϩc- resentative data from three independent experiments are shown. ϩ ϩ ϩ Ϫ 6182 CX3CR1 c-kit BM-DERIVED CD103 AND CD103 DC Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 3. CD103ϩ DC derived from GFPϩc-kitϩ display higher levels of costimulatory molecules compared with CD103Ϫ DC. A, CD11cϩCD103ϩ and CD11cϩCD103Ϫ DC were sorted at day 5 from cultures of nonstimulated GFPϩc-kitϩ bone marrow cells and subjected to microarray analysis. The x-axis shows relative expression levels of costimulatory molecules belonging to the TNF/TNFR and Ig superfamily. Mean ratio values were calculated from two independent experiments and are shown as bars. The error bar ends directly, representin individual ratio values from the two experiments performed. Ratio data are shown in ϩ log2 scale, whereby x-axis labels are reconverted to non-log values for clarity. Filled bars indicate transcripts that are present at higher levels in CD103 DC, while open bars show the transcript that is more abundant in CD103Ϫ DC. GenBank accession numbers are provided as identifiers next to gene names. Asterisks indicate genes that show highly significant differences in mRNA expression in one (*) or both (**) microarray experiments, respectively (for details see Materials and Methods). B, At day 5 of in vitro culture of bone marrow GFPϩc-kitϩ cells, cultures were either left untreated (Resting) or activated with 1 ␮g/ml LPS (Mature). After 24 h, cells were analyzed for the expression of CD103 and costimulatory molecules. Red solid lines indicate PIϪCD11cϩCD103ϩ; black solid lines, PIϪCD11cϩCD103Ϫ; dotted lines, respective isotype controls. CD103ϩ and CD103Ϫ DC were flow-sorted from GFPϩc-kitϩ, cultured in duplicates (5000/well), and incubated for 3 days with (C) 1.5 ϫ 105 isogenic (B6) or allogenic (BALB/c) splenocytes or (D) with 1.5 ϫ 105 OVA-specific transgenic T cells and pulsed with [3H]thymidine for 16 h. Data shown are means Ϯ SD from three independent experiments. The Journal of Immunology 6183 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 4. Increased phagocytic activity of CD103Ϫ DC compared with CD103ϩ DC. A, Same experiment as described in Fig. 3A. Gene expression proﬁles of phagocytosis receptors are shown. B, GFPϩc-kitϩ-derived DC differentiated in vitro for 5 days were incubated at 4°C or 37°C for 10 min with OVA-Cy5 (2.5 ␮g/ml) and then washed extensively in cold PBS. Cells were gated on PIϪCD11cϩCD103ϩ (red line) or PIϪCD11cϩCD103Ϫ (black line) and analyzed for the uptake of OVA-Cy5. C, DC were generated as described for B and incubated for 20 min at 37°C with nonopsonized or opsonized Cy5-labeled E. coli or with nonopsonized or opsonized Cy5-labeled zymosan A. PIϪCD11cϩ cells were gated and analyzed for the expression of CD103 and the uptake of Cy5-labeled particles. ϩ ϩ ϩ Ϫ 6184 CX3CR1 c-kit BM-DERIVED CD103 AND CD103 DC

C5ar1 (4.6-fold) were found in CD103Ϫ DC (Fig. 4A and data not shown). TLR agonists stimulate CD103Ϫ DC to secrete proinflammatory cytokines Microarray analysis also revealed increased transcripts for the genes encoding most of the TLRs (TLR1, TLR2, TLR4, TLR6, TLR7, and TLR8) in CD103Ϫ DC, with TLR7 mRNA being in particular up-regulated (34-fold; Fig. 5A). This observation prompted us to assess whether this differential pattern of TLR receptor expression was associated with a differential capacity to secrete proinflammatory cytokines when stimulated with TLR ligands. To that end, supernatants of stimulated CD103ϩ and CD103Ϫ DC were quantified by cytometric bead arrays. Consis- tent with the higher levels of TLR4 and TLR7 expression on CD103Ϫ DC, the addition of LPS (TLR4 agonist) and R848 (TLR7 agonist) induced a stronger production of TNF-␣ and IL-6 in stimulated CD103Ϫ DC compared with CD103ϩ DC, while no significant changes of secretion of IL-10, MCP-1, IFN-␥, or IL-12 Downloaded from cytokines could be observed (Fig. 5B). The TLR9 agonist CpG induced equivalent amounts of TNF-␣ in both populations but slightly less IL-6 secretion in CD103Ϫ DC. This finding cannot be attributed to TLR9 mRNA expression levels, which were comparable in both DC cell populations (Fig. 5B). http://www.jimmunol.org/ CD103Ϫ DC express inflammatory chemokines With regard to chemokine and chemokine receptor expression we observed that most chemokines that were differentially expressed were up-regulated in CD103Ϫ DC, suggesting that these cells might recruit T cells (via CXCL10 and CXCL4), neutrophils (CXCL1, CXCL2), and monocytes to places of infection and inflammation. Chemokine receptors relevant for the recruitment of monocytes to sites of inflammation are CCR2 and CX3CR1, although CCR1 and CCR5 play also a substantial role in this process by guest on September 25, 2021 (22). The detailed analysis of the microarray experiments suggests that CD103Ϫ DC seemingly secrete higher amounts of chemokines than do the CD103ϩ DC, particularly those that would attract monocytes such as the ligands for CCR2 (CCL2, CCL6, CCL7, and CCL9). With regard to chemokine receptors, CCR6 and CCR7 were up-regulated in CD103ϩ DC, while CD103Ϫ DC expressed higher levels of CCR2 (Fig. 6A). Additional chemokine receptors, namely CCR1, CCR4, and CXCR4, were significantly detectable but do not show strong differences in mRNA abundance. Apart from those mRNAs differentially expressed in CD103Ϫ and CD103ϩ DC reported so far, many others were also differentially regulated between both subsets. Among them, signal regulatory protein ␤1 precursor (sirp␤1) and thrombospondin 1 (thbs1) were 103.9- and 39.4-fold up-regulated in CD103Ϫ DC, respectively. It is known that sirp␤1 plays a role in regulating phagocytosis of CD8␣Ϫ (but not CD8␣ϩ) splenic DC (23), whereas thbs1 is mainly involved in apoptotic cell engulfment (24) (Fig. 6B). ϩ CD103 DC are known to build tight junction strands with epi- FIGURE 5. Distinct TLR agonists trigger CD103Ϫ DC to secrete proinflam- thelial cells. Thus, it was no surprise to see that tjp1 (zonula oc- matory cytokines. A, Same experiment as described in Fig. 3A. Gene expression cludens 1 gene, ZO-1) and claudin-1 (cldn1) mRNAs were 10.4- profiles of TLRs are shown. B, CD103ϩ and CD103Ϫ DC were sorted from day ϩ and 3-fold increased, respectively, in CD103 DC (Fig. 6B and 5 cultures of GFPϩc-kitϩ cells and cultured for 16 h in the presence of 1 ␮g/ml data not shown). LPS, 1 ␮g/ml R848, or 16 ␮g/ml CpG or were left untreated (PBS). Cytokines present in the culture supernatants were analyzed applying cytokine bead arrays. GFPϩc-kitϩ bone marrow cells differentiate in vivo into CD103Ϫ and CD103ϩ DC competent to migrate to lymphoid organs and sites of inflammation had been induced by repetitive administration of OVA aerosol. We next examined whether GFPϩc-kitϩ cells might contribute Eight days after adoptive transfer of GFPϩc-kitϩ cells into sub- in vivo to CD103Ϫ and CD103ϩ DC. To that end, GFPϩc-kitϩ lethally irradiated CD45.1 recipients undergoing a local inflam- bone marrow cells were adoptively transferred into nonirradi- matory response in the lung, DC derived from the adoptively ated or irradiated recipients in which a local lung inflammation transferred donor cells could be identified in spleen, lung, brLN, The Journal of Immunology 6185 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 6. Differential gene expression of chemokine and chemokine receptors on CD103Ϫ DC compared with CD103ϩ DC. Same experiment as described in Fig. 3A. Gene expression proﬁles of chemokine and chemokine receptors (A) and gene expression proﬁles of the 40 top-ranking genes showing the strongest mean expression difference between CD103Ϫ and CD103ϩ DC (B) are shown. Only those genes of the highest ranking top 40 were selected for presentation that have not already been depicted in Figs. 3–6A. For those genes where multiple probes directed against the same mRNA were present within the 40 top-ranking ratios, the value of the highest ranking probe is shown. ϩ ϩ ϩ Ϫ 6186 CX3CR1 c-kit BM-DERIVED CD103 AND CD103 DC

these cells to epithelia via its binding to E-cadherin (25, 26). In- testinal CD11bϩCD103ϩ DC possess the ability to induce ϩ␣ ␤ ϩ ϩ ϩ CCR9 4 7 gut-tropic CD8 effector T cells (7). Most CD103 DC in the spleen also expresses CD8␣ (M.-L. del Rio and R. Fo¨rster, unpublished results), a DC subset known to be localized in the T cell area in this organ. CD8␣ϩ DC, however, are almost absent in the lung, where CD11cϩCD11bϩ and plasmacytoid DC (CD11clowGr1ϩB220ϩ120G8ϩ) are the predominant DC populations (27). We and others have previously shown that within the CD11cϩCD11bϩ DC, the expression of CD103 allows to distinguish two subpopulations: CD103ϩ (CD11chighCD11blow) DC, which represents the major DC subset in the lung, and a CD103Ϫ (CD11cint CD11bhigh) DC subset (8, 9). Two subsets of bone marrow progenitor cells giving rise to de- ϩ/gfp fined myeloid lineages have recently been identified in CX3CR1 Ϫ ϩ Ϫ mice: myeloblast progenitor (lin c-kit CX3CR1 ) and MDP Ϫ ϩ ϩ (lin c-kit CX3CR1 ) (15). MDP have been identified in partic- ϩ/gfp Ϫ ϩ ular in the bone marrow of CX3CR1 mice (lin CX3CR1 c- kitϩ) as the main progenitor intermediary for monocytes and mac- Downloaded from rophages, as well as DC (both CD11bϩ and CD11bϪ) (15), but not for plasmacytoid DCs. Plasmacytoid DCs develop from a distinct bone marrow precursor cell differentiated in vitro in the presence of Flt3L (28, 29). Fogg et al. have shown that MDP immediately convert in vitro into CD11cϩCD11bϩ DC cells upon exposure to GM-CSF, but not into macrophages, which were only derived after http://www.jimmunol.org/ culturing MDP with M-CSF in vitro (15). Our study made use of Ϫ ϩ ϩ ϩ Ϫ “MDP-like” progenitors (lin CX3CR1 c-kit CD11b /CD11b ), which are similar to the MDP progenitor described by Fogg et al. with the only difference that in our work, CD11b was not used in ϩ ϩ Ϫ the lineage cocktail to ensure that c-kit CX3CR1 CD11b ϩ ϩ ϩ (MDP) and their immediate progenies (c-kit CX3CR1 CD11b ) were represented in the progenitor cell population (15). This con- ϩ

sideration was based on the fact that within the lung, both CD103 by guest on September 25, 2021 and CD103Ϫ DC express CD11b. Thus, to assess whether GFPϩ c-kitϩ and GFPϩc-kitϪ bone marrow cells are able to generate CD103ϩ and CD103Ϫ DC in vitro, these progenitors were sorted and differentiated in vitro in the presence of GM-CSF. Although both populations were able to give rise to CD11cϩCD103ϩ and ϩ Ϫ ϩ ϩ FIGURE 7. ϩ ϩ ϩ CD11c CD103 DC, we focused on the GFP c-kit cells, which, GFP c-kit bone marrow cells give rise to CD103 DC in ϩ Ϫ vivo that are recruited to sites of inflammation. A, GFPϩc-kitϩ bone mar- compared with GFP c-kit cells, possess a high ability to prolif- row cells were sorted and adoptively transferred (3 ϫ 105) into sublethally erate and expand in vitro. irradiated CD45.1 OVA-sensitized mice that were further treated for 7 days Data obtained in the present study support a model in which ϩ with OVA aerosol (1%). At day 8, percentages of donor-derived CD11c CD103ϩ and CD103Ϫ DC generated from GFPϩc-kitϩ cells rep- Ϫ ϩ DC (left panel) or percentages of CD103 and CD103 DC (right panel) resent two distinct DC populations: no substantial conversion from in lung, brLN, spleen, and mesenteric LN were calculated. B, Absolute sorted CD103Ϫ DC into CD103ϩ DC (or vice versa) was observed numbers of donor-derived CD11cϩCD103ϩ and CD11cϩCD103Ϫ DC within the following 4 days of culture (Fig. 2E and data not were determined in different compartments as indicated 8 days after adoptive transfer. Data are means Ϯ SD from two independent experiments with shown), arguing against the idea that one of the two populations three mice each per group and experiment. serves as a progenitor for the other. Furthermore, since sorted CD103ϩ and CD103Ϫ DC kept their CD103 expression profile and mesenteric LN. Of interest, 50–60% of these DC express even in the presence of LPS, a TLR4 ligand known to induce DC CD103 (Fig. 7A). With regard to absolute numbers, donor-de- maturation, we also trust that expression of CD103 is not a mat- ϩ Ϫ rived DC were preferentially found in spleen and brLN (Fig. uration marker. Both nonmatured CD103 and CD103 DC dis- 7B). We failed to identify donor-derived cells expressing CD19, played low expression of MHCII as well as costimulatory mole- CD3, or NK1.1 cells, indicating that the adoptively transferred cules, but they showed significant functional differences with GFPϩc-kitϩ cells predominantly differentiate to DC under the regard to many aspects of DC biology, suggesting that these two experimental conditions chosen. Taken together, these in vivo DC subsets might have acquired marked specialization required to observations support the hypothesis that GFPϩc-kitϩ bone marrow face distinct requirements. cells can differentiate to CD103ϩ and CD103Ϫ DC capable of mi- CD103Ϫ DC showed a higher capacity than CD103ϩ DC to engulf grating to inflamed tissues and their draining LN. nonopsonized as well as opsonized microorganisms and to secrete proinflammatory cytokines upon ligation with TLR agonists. Similar Discussion to our findings, CD103Ϫ DC isolated from mesenteric LN have been ␣ ␣ ␣ ␤ ␣ CD103 ( E)isthe -chain of the E 7 integrin that is expressed on reported to secrete proinflammatory cytokines (mainly TNF- and human and mouse lymphocytes and facilitates the adhesion of IL-6) following stimulation with TLR agonists (30). The Journal of Immunology 6187

The relationship between macrophages and DC is still a subject CD103Ϫ CD11bhigh lung DC, respectively (5). Additional studies of intense debate (31). It remains unclear which array of molecules revealed that Ly6Chigh cells can give rise to DC and Ly6Clow cells would allow discrimination between DC and macrophages (32). to macrophages in infectious peritonitis and the healing myocar- Indeed, CD103Ϫ DC display several markers that are also found dium, respectively (39, 40). Along this line, the adoptive transfer on macrophages and also possess some features that could be at- of GFPϩc-kitϩ progenitors described herein allows for an efficient tributed to macrophages (e.g., high phagocytic activity, secretion repopulation of CD11cϩCD103ϩ and CD11cϩCD103Ϫ DC in the of proinflammatory cytokines upon ligation with TLR, poor co- lung of irradiated recipients undergoing allergic lung inflamma- stimulatory activity of allogeneic T cells). Nevertheless, we pro- tion. Note that we cannot formally rule out that some of the donor- vide evidence that the CD103Ϫ cells that are derived from GFPϩ derived CD11cϩCD103Ϫ cells in the lung might be macrophages c-kitϩ bone marrow cells cultured in the continuous presence of since lung macrophages also express CD11c and might have up- GM-CSF are indeed bona fide DC. As described in the present regulated CD11b due to the allergic airway inflammation (4, 8). study for GFPϩc-kitϩ cells, MDP precursors grown in the pres- CD103ϩ DC are localized in the airway mucosa next to the ence of GM-CSF but in the absence of stromal cells give rise to basal surface of bronchial epithelial cells where E-cadherin is CD11cϩCD11bϩ DC but not to macrophages. Furthermore, these present, and they are therefore more readily exposed to airway- cells, as the cell described in the present study, display a DC-like carrying stimuli than CD103Ϫ DC, which are situated in the sub- morphology and phenotype (15). Finally, the CD103Ϫ cells of the epithelial regions (8). Based on their location, it seems possible present study express CD11c, a marker widely used for the iden- that the differential distribution of both subpopulations and the tification of DC in mice. refractory behavior of CD103ϩ DC to produce cytokines and che-

Chemokines are essential components of the innate as well as mokines may serve as a mechanism to prevent continuous leuko- Downloaded from the adaptive immune system (33, 34). Within the lung, CD103Ϫ cyte recruitment and their activation in response to slight pertur- DC produce higher levels of chemokines than CD103ϩ DC or even bations of the lung microenvironment. In this model, CD103ϩ DC macrophages, making them a potential target in asthma therapy would be the ﬁrst cell type of the innate immune system to sense (35, 36). In vitro-generated CD103Ϫ DC are also considerably the danger signal, while the CD103Ϫ would be the innate mech- more efﬁcient than CD103ϩ DC in secreting a large variety of anism responsible for the antigenic clearance that would activate

inflammatory chemokines. Thus, it is tempting to speculate that an inflammatory response facilitating the recruitment of neutro- http://www.jimmunol.org/ once a first wave of monocytes/progenitors reaches the inflamed phils and monocytes to the site of inflammation. tissue and differentiates to DC, CD103Ϫ DC derived from these In summary, we show that CD103ϩ and CD103Ϫ DC can be precursors would initiate the secretion of chemokines that then generated in vitro under GM-CSF treatment from GFPϩc-kitϩ and would not only attract neutrophils but also monocytes through GFPϩc-kitϪ bone marrow cells. Compared with CD103ϩ DC, CCR1, CCR2, and CCR5 (22). This mechanism would act as a CD103Ϫ DC exhibited enhanced phagocytosis activity and secre- positive feedback loop that ensures the maintenance of a contin- tion of inflammatory cytokines. Furthermore, adoptive transfer of uous flow of DC precursors indispensable for the maintenance of GFPϩc-kitϩ cells into aerosol-sensitized and subsequently irradi- the DC pool and for the persistence of an inflammatory immune ated mice is able to reconstitute CD103ϩ and CD103Ϫ DC pop- response. ulations in lymphoid and nonlymphoid compartments, which in- by guest on September 25, 2021 The CD103ϩ and CD103Ϫ DC subsets generated in vitro from dicates that these precursors contribute to the pool of CD103ϩ and GFPϩc-kitϩ bone marrow cells did not display the capacity to CD103Ϫ DC in the lung. differentially stimulate CD8 and CD4 Ag-specific T cells that we recently reported for lung-derived CD103ϩ and CD103Ϫ DC that Acknowledgments had taken up Ag in vivo in the lung (9). The reason for this dif- We thank Christina Reimer for cell sorting. ference is currently unclear. However, it seems likely that DC residing in peripheral organs such as the lung receive additional Disclosures signals that further drive their differentiation leading to distinct Ag The authors have no financial conflicts of interest. ϩ presentation and cross-presentation profile. Since CD103 DC re- References side between epithelial cells, it seems possible that these epithelial 1. Hume, D. A. 2006. The mononuclear phagocyte system. Curr. Opin. Immunol. cells provide defined differentiation factors that are not present in 18: 49–53. our in vitro cultures. 2. Holt, P. G., S. Haining, D. J. Nelson, and J. D. Sedgwick. 1994. Origin and steady-state turnover of class II MHC-bearing dendritic cells in the epithelium of The mechanisms that are involved in replenishing pulmonary the conducting airways. J. Immunol. 153: 256–261. DC are not well understood, but it has recently been suggested that 3. Geissmann, F., S. Jung, and D. R. Littman. 2003. Blood monocytes consist of two monocytes contribute to the pool of pulmonary DC under both principal subsets with distinct migratory properties. Immunity 19: 71–82. 4. Landsman, L., C. Varol, and S. Jung. 2007. Distinct differentiation potential of steady-state and inflammatory conditions (4). It is well established blood monocyte subsets in the lung. J. Immunol. 178: 2000–2007. that lung DC, probably like most DC residing in peripheral organs, 5. Jakubzick, C., F. Tacke, F. Ginhoux, A. J. Wagers, N. van Rooijen, M. Mack, M. Merad, and G. J. Randolph. 2008. Blood monocyte subsets differentially give are subjected to an active process of cell turnover (37). Lung DC ϩ Ϫ rise to CD103 and CD103 pulmonary dendritic cell populations. J. Immunol. numbers decay rapidly after irradiation treatment due to their con- 180: 3019–3027. stant migration to the lung draining LN even in the absence of 6. Kilshaw, P. J. 1993. 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Immunol. 176: 2161–2172. 9. Del Rio, M. L., J. I. Rodriguez-Barbosa, E. Kremmer, and R. Forster. 2007. high low tion, Ly6C cells still give rise to DC while Ly6C cells pri- CD103Ϫ and CD103ϩ bronchial lymph node dendritic cells are specialized in marily differentiate to macrophages and to a lesser extent to DC presenting and cross-presenting innocuous antigen to CD4ϩ and CD8ϩ T cells. J. Immunol. 178: 6861–6866. (38). Further studies done by Randolph and colleagues suggest that 10. Jung, S., J. Aliberti, P. Graemmel, M. J. Sunshine, G. W. Kreutzberg, A. Sher, high low ϩ Ly6C and Ly6C cells differentiate to CD103 DC and and D. R. Littman. 2000. Analysis of fractalkine receptor CX3CR1 function by ϩ ϩ ϩ Ϫ 6188 CX3CR1 c-kit BM-DERIVED CD103 AND CD103 DC

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