Langerin+CD8α+ Dendritic Cells Are Critical for Cross-Priming and IL-12 Production in Response to Systemic Antigens

Kathryn J. Farrand, Nina Dickgreber, Patrizia Stoitzner, Franca Ronchese, Troels R. Petersen and Ian F. Hermans
J Immunol December 15, 2009, 183 (12) 7732-7742; DOI: https://doi.org/10.4049/jimmunol.0902707

Abstract

Distinct dendritic cell (DC) subsets differ with respect to pathways of Ag uptake and intracellular routing to MHC class I or MHC class II molecules. Murine studies suggest a specialized role for CD8α+ DC in cross-presentation, where exogenous Ags are presented on MHC class I molecules to CD8+ T cells, while CD8α− DC are more likely to present extracellular Ags on MHC class II molecules to CD4+ T cells. As a proportion of CD8α+ DC have been shown to express langerin (CD207), we investigated the role of langerin+CD8α+ DC in presenting Ag and priming T cell responses to soluble Ags. When splenic DC populations were sorted from animals administered protein i.v., the ability to cross-present Ag was restricted to the langerin+ compartment of the CD8α+ DC population. The langerin+CD8α+ DC population was also susceptible to depletion following administration of cytochrome c, which is known to trigger apoptosis if diverted to the cytosol. Cross-priming of CTL in the presence of the adjuvant activity of the TLR2 ligand N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-Cys-[S]-Serl-[S]-Lys4-trihydrochloride or the invariant NKT cell ligand α-galactosylceramide was severely impaired in animals selectively depleted of langerin+ cells in vivo. The production of IL-12p40 in response to these systemic activation stimuli was restricted to langerin+CD8α+ DC, and the release of IL-12p70 into the serum following invariant NKT cell activation was ablated in the absence of langerin+ cells. These data suggest a critical role for the langerin+ compartment of the CD8α+ DC population in cross-priming and IL-12 production.

Processing of exogenous Ags takes place in phagolysosomal compartments of the APC and is typically followed by presentation of Ag-derived peptide fragments by MHC class II molecules on the cell surface to drive activation of CD4+ T cells (1, 2, 3). Some APC have a heightened capacity to divert captured Ags into the MHC class I presentation pathway to stimulate CD8+ T cells, a process referred to as cross-presentation (4, 5). The stimulation of Ag-specific CD8+ T cell precursors and their subsequent differentiation into CTL with capacity to eradicate tumors or control intracellular pathogens that do not directly infect APC is likely to be dependent upon the cross-presentation process.

Accumulating evidence suggests that distinct dendritic cell (DC)4 subsets differ with respect to pathways of Ag uptake and intracellular routing to either MHC class I or MHC class II molecules (6). In murine models, Ags specifically targeted to molecules highly expressed on splenic CD8α+ DC, such as CD205 (DEC205), are efficiently cross-presented, whereas Ags targeted to molecules highly expressed on CD8α− DC, such as DCIR2, are preferentially presented on MHC class II molecules (7). Even under conditions in which the different DC subsets acquired equivalent amounts of Ag, CD8α+ DC were shown to have superior cross-presenting ability (8), which has been ascribed to higher levels of molecules involved in MHC class I presentation relative to other DC (7). Recent evidence points to the ability of the small GTPase Rac2 to maintain a higher pH in the phagolysomes of CD8α+ DC, thereby changing the proteolytic environment to one that favors cross-presentation (9).

There is now some evidence of heterogeneity within the CD8α+ DC population with respect to cross-presentation. One recent study exploited the requirement for cross-presented Ags to be diverted into the cytosol to deplete cells with a heightened propensity for the cross-presentation process. This was accomplished by injecting mice with horse cytochrome c (cyt c), which induces “suicide” in cells that acquire and divert this proapoptotic protein to the cytosol (10). Although the CD8α+ DC population was significantly depleted by this treatment, a proportion of CD8α+ DC was not affected by cyt c administration, suggesting that these remaining cells were incapable of cross-presentation. In a more recent study, Qiu et al. (11) showed that among splenic CD8α+ DC, the CD103+ langerin (CD207)+ subset was responsible for phagocytosis of apoptotic cells and tolerance of cell-associated Ags. These langerin+ DC may be particularly effective at screening the blood for self-Ags by virtue of their location in the marginal zone of the spleen (12, 13). Under appropriate conditions, these same cells may be responsible for stimulating immunity, as suggested by a study in which Ag was targeted to langerin+ cells with an anti-langerin Ab (14).

In this study, we investigated the role of the langerin+ subset of CD8α+ DC in cross-priming responses to soluble Ags in a transgenic model in which langerin+ cells could be specifically depleted in vivo. We show that only langerin+ CD8α+ DC are responsible for cross-presentation, with capacity to drive potent Ag-specific CTL responses if combined with appropriate activation stimuli such as TLR stimulation or concomitant activation of invariant NKT (iNKT) cells to provide “licensing” signals. The production of IL-12 in response to systemic activation stimuli is also dependent on langerin+CD8α+ DC, with the ratio of IL-12p40 to bioactive IL-12p70 manufactured determined by the activation stimulus used. The langerin+ subset of CD8α+ DC is therefore crucially involved in priming and differentiation of responses to cross-presented Ag.

Materials and Methods

Mice

Breeding pairs of the inbred strains C57BL/6 (CD45.2+) and B6.SJL- Ptprca Pepcb/BoyJ (CD45.1+) were obtained from The Jackson Laboratories and from the Animal Resource Centre. Also used were lang-DTREGFP and lang-EGFP knock-in mice, which express the human diphtheria toxin (DT) receptor and/or enhanced GFP (EGFP) under the control of the langerin promoter (15), CD1d−/− mice (16), which are devoid of Vα14 iNKT cells, TLR2−/− mice (17), OT-I mice, which are transgenic for a TCR recognizing a H-2Kb-restricted epitope from chicken OVA (OVA257–264) (18), and OT-II mice, with a TCR recognizing the I-Ab-restricted epitope OVA323–339 (19). For some adoptive transfer experiments, OT-I or OT-II animals were crossed with B6.SJL-Ptprca Pepcb/BoyJ animals, so that the congenic marker CD45.1 could be used to discriminate the transferred cells. All mice were maintained in the Biomedical Research Unit of the Malaghan Institute of Medical Research. Experiments were approved by a national animal ethics committee and performed according to established national guidelines. Data presented are representative of experiments performed two to four times.

In vitro culture media and reagents

Cell lines were maintained in complete medium consisting of IMDM supplemented with 2 mM glutamine, 1% penicillin-streptomycin, 5 × 10−5 M 2-ME (all Invitrogen), and 5% FBS (Sigma-Aldrich). Endotoxin-free chicken OVA, provided by T. Moran (Department of Microbiology, Mount Sinai School of Medicine, New York, NY), was isolated endotoxin free as previously described (20). Alternatively, Endograde OVA (Profos) was used. The iNKT cell ligand α-galactosylceramide (α-GalCer) was prepared as described previously (21) and was solubilized in 150 mM NaCl/0.5% Tween 20, hereafter referred to as vehicle. The TLR2 ligand N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-Cys-[S]-Serl-[S]-Lys4-trihydrochloride (Pam3Cys) (22) was from EMC Microcollections. Horse cyt c and DT were from Sigma-Aldrich.

Administration of Ags and adjuvants

All injected substances were diluted in PBS for i.v. administration. The doses used were (unless otherwise stated): 200 μg of OVA protein, 200 ng of α-GalCer, or 20 μg of Pam3Cys.

Flow cytometry

All Ab staining steps were performed on ice. Nonspecific FcR-mediated Ab staining was blocked by incubation for 10 min with anti-CD16/32 Ab (24G2, prepared in-house from hybridoma supernatant). Flow cytometry was performed on a BD Biosciences FACSCalibur or BD Biosciences LSRII SORP with data analysis using FlowJo software (Tree Star).

Phenotyping of APC

The maturation status of APC in response to adjuvants was assessed in lang-EGFP mice 5 h after administration. Spleens were removed and digested with Liberase and DNase I (both Roche) and RBC were lysed using RBC lysis buffer (Qiagen). The resulting single-cell suspension was stained with Abs against CD11c (clone Hl3; BD Pharmingen), CD8α (clone 53-6.7; eBioscience), CD24 (clone M1/69; BD Pharmingen), CD80 (clone 16-10A1; BD Pharmingen), CD86 (clone GL1; eBioscience), CD40 (clone 1C10; eBioscience), CD103 (clone M290; BD Pharmingen), and CD205 (clone NLDC145, prepared in-house). Dead cells were excluded by 4′,6-diamidino-2-phenylindole staining (BD Pharmingen). For intracellular expression of IL-12p40, mice were injected with adjuvants as above followed by i.v. injection of 250 μg of brefeldin A (Sigma-Aldrich) 2 h later. Spleens were removed 6 h after adjuvant administration and single-cell suspensions were generated as above and stained with Abs against CD11c and CD8α. Cells were fixed and stained for intracellular IL-12p40 (clone eB149/10H5 eBioscience) or isotype control Ab using a Fix & Perm cell permeabilization kit (Invitrogen) according to the manufacturer’s instructions.

Depletion of langerin+ cells in vivo

lang-DTREGFP mice were treated two times, 48 h apart, with the indicated doses (mg) of DT by i.p. administration to deplete langerin+ cells.

Depletion of cyt c-sensitive APC in vivo

C57BL/6 mice were treated two times, 24 h apart, with horse cyt c (Sigma-Aldrich) at the indicated concentrations by i.v. administration to deplete cells capable of diverting acquired Ag to the cytosol (10).

Isolation and FACS sorting of splenic DC

Groups of five mice were injected i.v. with 2 mg of OVA and then spleens were removed 14–16 h later, digested with Liberase and DNase I (both Roche) to provide a single-cell suspension, and enriched for DC using a Nycodenz gradient as previously described (23). Cells were further enriched with magnetic sorting (CD11c-MACS MicroBeads, clone N418; Miltenyi Biotec) and sorted using a FACSVantage-SE cell sorter (BD Biosciences) and the following Abs: CD8α-FITC (clone 53-6.7; eBioscience), CD4-PE (clone L3T4; eBioscience), and CD11c-allophycocyanin (clone HL3; BD Pharmingen). The three different splenic DC subsets (double-negative CD4−CD8α− DC, single-positive CD4+ DC and CD8α+ DC) were sorted to a purity above 97% for use in in vitro proliferation assays.

Isolation of T cells and in vitro proliferation assays

Spleens and lymph nodes from OT-I or OT-II mice were teased through a cell strainer, RBC were lysed with RBC lysis buffer, and then CD8+ or CD4+ cells were positively selected (from OT-I and OT-II, respectively) using magnetic beads (Miltenyi Biotec). The T cell suspensions (2 × 106 cells/ml) were then cultured in triplicate with the indicated numbers of sorted DC for 64 h, with proliferation measured by incorporation of [3H]thymidine (1 mCi/ml; Amersham Biosciences) during the last 16 h. All experiments presented were repeated two to three times with similar results.

Assessment of cross-presentation in vivo

T cells were isolated from an F1 cross between OT-I and B6.SJL-Ptprca Pepcb/BoyJ as described above and labeled with 1 μM CFSE (Molecular Probes/Invitrogen). Groups of C57BL/6 mice (n = 3) were injected i.v. with 2 × 106 CFSE-labeled transgenic cells and then 1 day later injected i.v. with OVA in the presence or absence of Pam3Cys. T cell proliferation was assessed in blood 3 days later by monitoring the decrease in CFSE fluorescence intensity as described elsewhere (24). Injected T cells were identified with anti-CD8-PerCP and anti-CD45.1-PE (clones 53-6.7 and A20 respectively; eBioscience).

Monitoring cross-priming in vivo

Groups of C57BL/6 mice (n = 5) were donated 2 × 104 OT-I × B6.SJL-Ptprca Pepcb/BoyJ CD8+ T cells by i.v. injection and then 1 day later, recipient mice were injected with OVA in combination with the adjuvant compounds described in the text. At different times after immunization, mice were bled from the lateral tail vein and PBL were stained directly ex vivo with Abs for TCR Vα2 (clone B20.1; eBioscience), CD8, and CD45.1.

Intracellular cytokine analysis of iNKT cells

Splenocytes were collected 2.5 h after administration of α-GalCer into recipient mice (n = 3–5), stained with α-GalCer-loaded mouse CD1d tetramer (ProImmune) and anti-CD3 Ab (clone 145-2C11; eBioscience), fixed, and permeabilized using BD Biosciences Cytofix/Cytoperm and then stained with Abs to murine IFN-γ (allophycocyanin, clone XMG1.2; BD Pharmingen) and Alexa Fluor 488-labeled IL-4 (clone 11B11, eBioscience) for analysis by flow cytometry.

Analysis of cytokine release into serum

Blood was collected from the lateral tail vein at different time intervals after glycolipid administration. Serum was collected after blood had clotted, and levels of cytokines IL-12p70, IL-4, and IFN-γ were assessed by bioplex cytokine bead arrays (Bio-Rad) according to the manufacturer’s instructions.

Results

Depletion of langerin+ spleen cells in lang-DTREGFP-transgenic mice

The endocytic receptor langerin (CD207) is a C-type lectin typically associated with Langerhans cells (LC), but is also known to be expressed in some DC in spleen and lymph nodes (13, 25, 26, 27). Within the spleen, expression of langerin can be visualized in lang-EGFP mice in which DNA encoding EGFP has been inserted into the endogenous mouse langerin locus (15). Typical expression of EGFP from the langerin locus in splenic DC (defined as CD11c+ cells) in these mice is shown in Fig. 1⇓A. As previously reported (13, 15, 26, 27), langerin expression is predominantly observed in the CD8α+ DC subset. The langerin+ DC also express CD24 and CD205 and exhibit higher levels of CD103 relative to langerin-negative DC and CD8α-negative DC (Fig. 1⇓B). We were interested in determining whether splenic langerin+CD8α+ DC were responsible for cross-presentation that has previously been ascribed to the CD8α+ DC subset as a whole. To undertake these studies, we used lang-DTREGFP knock-in mice in which DNA encoding the human DT receptor (DTR) has been fused with an EGFP sequence and inserted under the langerin promoter (15). Treatment with DT can therefore be used to selectively deplete langerin+ cells to establish their role in different physiological processes, a strategy that has been successfully used to acutely deplete LC (15). We used flow cytometry to quantify the DC populations in the spleen of lang-DTREGFP mice after DT treatment (Note that EGFP expression is lower in this strain than lang-EGFP mice and could not be used to discriminate langerin+ cells by flow cytometry). A range of doses of DT were used, with the treatment given twice, 2 days apart, and analysis performed 24 h after the last injection (Fig. 1⇓C). When compared with untreated animals, DT treatment with doses ranging from 100 to 1000 ng lead to a significant reduction in the percentage of CD8α+ DC in the spleen. After two doses of 350 ng of DT, depletion of CD8α+ DC was evident for a period of 3 days after the last DT treatment (Fig. 1⇓D). A trend showing a corresponding increase in CD4+ DC and double-negative DC was observed after DT treatment. Depletion was strictly dependent upon expression of the langerin-dtregfp transgene, as no reduction in CD8α+ DC was seen when C57BL/6 mice were treated with DT (data not shown).

FIGURE 1.
FIGURE 1.

DT treatment of lang-DTREGFP mice results in depletion of CD8α+ DC from the spleen. A, The gating strategy is shown for visualizing langerin+ cells within the CD8α+ subset of CD11c+ cells in lang-EGFP mice. B, Expression of the indicated markers on subsets of CD11c+ cells in lang-EGFP mice is shown relative to fluorescence minus one (fmo) unstained controls. C, Groups of lang-DTREGFP mice (n = 2) were treated twice with DT at the indicated doses, with injection 3 days and 1 day before analysis of spleen by flow cytometry. Subpopulations of DC within the CD11c+ population were discriminated on the basis of CD4 and CD8α expression. Graphs show mean percentages of cells within each DC population ± SEM. D, Groups of lang-DTREGFP mice (n = 2) were treated with 350 ng of DT (except where otherwise indicated) on the indicated days and the percentages and absolute numbers of cells within each DC population determined as in C. FSC, Forward scatter; DN, double negative.

The langerin+ cells are required for cross-presentation, but not for presentation to CD4+ T cells

To establish whether splenic langerin+ cells contribute to the presentation of Ag to T cells in vivo, lang-EGFP mice were injected with OVA protein and then DC were sorted from the spleen on the basis of CD11c, CD8α, and langerin-EGFP expression and assessed for their capacity to stimulate proliferation of OVA-specific TCR-transgenic T cells from OT-I and OT-II mice (CD8+ and CD4+ T cells, respectively; Fig. 2⇓A). Although all sorted DC were capable of stimulating some proliferation of CD4+ T cells, only DC expressing both CD8α and langerin-EGFP were capable of stimulating CD8+ T cells. We then isolated DC from lang-DTREGFP mice injected with OVA protein as above and either treated with DT or left untreated. Although the limited CD4+ T cell proliferation stimulated by all DC populations was unaffected by prior DT-mediated depletion of langerin+ cells, cross-presentation to CD8+ T cells by CD8α+ DC was severely reduced (Fig. 2⇓B). Thus, the langerin−CD8α+ DC that remain after DT treatment do not contribute to cross-presentation; only the langerin+ subset of CD8α+ DC has this capacity.

FIGURE 2.
FIGURE 2.

Cross-presentation is limited to langerin+CD8α+ DC in the spleen. A, lang-EGFP mice were injected i.v. with 2 mg of OVA protein and then 16 h later the indicated DC subtypes were FACS sorted from the spleen and incubated with OVA-specific CD4+ or CD8+ T cells in vitro. Proliferation of Ag-specific cells was assayed by measuring incorporation of [3H]thymidine over the last 16 h of a 64-h culture period. DC from untreated animals did not induce Ag-specific T cell proliferation (data not shown). Results are the average of triplicate wells ± SEM. B, Lang-DTREGFP mice were treated twice with 350 ng of DT, 48 h apart, or were left untreated, and then all animals were injected i.v. with 2 mg of OVA protein 1 day after the last DT injection. The indicated DC subtypes were sorted from the spleen 16 h later and used to stimulate OVA-specific T cells as in A.

We next investigated the role of langerin+ cells in presentation of Ag to CD4+ and CD8+ T cells in vivo. Naive CFSE-labeled OT-I and OT-II cells were transferred into lang-DTREGFP mice that had either been treated with DT or left untreated, and then animals were immunized by i.v. injection of OVA, with the TLR2-ligand Pam3Cys serving as an adjuvant (22). Assessment of proliferation of the CFSE-labeled cells 72 h later showed a significant reduction in the Ag-specific CD8+ T cell response in animals depleted of langerin+ cells (Fig. 3⇓). Proliferation of CD8-negative cells of the transferred cohort, which were CD4+ T cells, were largely unaffected by the absence of langerin+ cells. We conclude that langerin+ cells are critically involved in cross-presentation of systemic soluble Ags, but their involvement in presentation of Ag via MHC class II molecules to CD4+ T cells is limited.

FIGURE 3.
FIGURE 3.

Cross-presentation in vivo is dependent on langerin+ cells. Lang-DTREGFP animals were treated twice with 350 ng of DT, 48 h apart, or left untreated and then donated cohorts of CFSE-labeled transgenic T cells from OT-I or OT-II animals that had been crossed with B6.SJL-Ptprca Pepcb/BoyJ animals, thereby enabling use of the congenic marker CD45.1 to monitor expansion of the transferred CD4+ and CD8+ populations after i.v. injection with 200 μg of OVA protein and 20 μg of Pam3Cys. A, Representative FACS profiles of the transferred cells in the blood 66 h after Ag administration are presented. B, Percentage (±SEM) of transferred cells in each cell division for DT-treated and untreated animals (n = 2).

The langerin+ cells are efficient at shuttling acquired soluble Ag to the cytosol

Intravenous administration of horse cyt c was previously shown to specifically deplete cells capable of shuttling Ag through the cytosol, thereby removing cells capable of cross-presentation (10). We investigated whether there is an overlap between cells that are sensitive to horse cyt c and the langerin+ cells we have identified as being involved in cross-presentation. We first confirmed that CD11c+ cells selected from cyt c-treated animals were indeed incapable of cross-presentation, whereas in untreated animals cross-presentation was observed in the CD8α+ subset (Fig. 4⇓A). We then treated lang-EGFP mice with horse cyt c and assessed whether langerin+ cells were depleted in vivo. Analyses of spleen tissue 24 h after the last administration of different doses of horse cyt c showed a dose-dependent depletion of a significant proportion of CD8α+ DC (Fig. 4⇓, B and C). Interestingly, we also noted a significant reduction of the CD4+ DC population, with a corresponding increase in the double-negative DC population, which was more dramatic than previously reported in the study of Lin et al (10). The authors of this previous study also noted some reduction in only the CD4+ DC population when yeast-derived cyt c was used. Because cyt c from lower eukaryotes has a much lower affinity for mammalian Apaf-1 and thus cannot initiate cell death, the reduction in the CD4+ DC population remains an as yet unexplained off-target effect. Significantly, in our experiments, a dramatic dose-dependent reduction in langerin+ CD8α+ DC was observed in response to horse cyt c, whereas langerin−CD8α+ DC were unaffected by the treatment. Thus, we can confirm that there is heterogeneity within the CD8α+ DC population with respect to sensitivity to cyt c and that this sensitivity to cyt c is positively correlated with the expression of langerin.

FIGURE 4.
FIGURE 4.

The langerin+ cells capable of cross-presentation are depleted by administration of cyt c. A, Proliferation of OVA-specific cells by DC subtypes sorted from the spleens of C57BL/6 animals treated with 5 mg of cyt c 12 and 36 h before Ag administration or left untreated and then injected i.v. with 2 mg of OVA protein. B, Representative FACS profiles of splenic CD11c+ cells from individual lang-DTREGFP mice treated with cyt c or left untreated. C, Analysis of splenic DC subsets in groups of lang-DTREGFP mice 24 h after the last cyt c treatment at the indicated doses. Graphs show percentage and absolute number of cells within each DC population for individual mice in each treatment group.

The langerin+ splenic DC respond rapidly to systemic activation stimuli

DC function can be enhanced through the stimulation of pathogen recognition receptors such as TLRs (28) or through interaction with cells of the innate immune system, such as iNKT cells (29, 30, 31). We therefore investigated what impact these activities can have on the phenotype of langerin+ DC. Intravenous injection of the TLR2 ligand Pam3Cys or the potent iNKT cell ligand α-GalCer into lang-EGFP mice resulted in enhanced expression of the costimulatory molecules CD80 and CD86 on CD8α+ and CD8α− DC in the spleen, indicating maturation of all splenic DC subsets (Fig. 5⇓). Interestingly, the basal level of CD86 expression in naive mice was higher for langerin+CD8α+ DC than for the other DC subsets. When CD40 was examined, expression in response to the injected stimuli was more pronounced on the langerin+CD8α+ subset at the 5-h time point examined compared with both the langerin−CD8α+ and CD8α− subsets. Thus, the langerin+CD8α+ DC subset has a heightened propensity to up-regulate molecules involved in T cell stimulation in response to systemic stimuli.

FIGURE 5.
FIGURE 5.

The langerin+CD8α+ DC have a heightened propensity to up-regulate CD40 in response to systemic stimuli. lang-EGFP mice were injected with Pam3Cys or α-GalCer and then 5 h later the expression of the indicated maturation markers on CD11c+ DC subtypes was assessed and compared with expression in untreated animals. FACS profiles are representative of results from groups of three animals.

The langerin+ splenic DC are the major source of IL-12 in response to systemic activation stimuli

It is known that activation of iNKT cells with a potent stimulus such as α-GalCer is followed by iNKT cell proliferation and a cascade of intercellular interactions leading to the release of large quantities of cytokines into the serum (32, 33). The iNKT cells themselves are responsible for the release of a spectrum of cytokines including the archetypal Th1 cytokine IFN-γ and the archetypal Th2 cytokine IL-4. Activated iNKT cells also directly stimulate APC to release IL-12 (34, 35), and this cytokine, in turn, drives NK cells to release more IFN-γ (36). In this context, presentation of glycolipid by splenic CD8α+ DC has been shown to be required for iNKT cell-mediated release of IL-12p70 following administration of α-GalCer (37). We therefore investigated whether initiation of iNKT cell activation and/or stimulation of release of IL-12p70 could be attributed to the langerin+ subset of CD8α+ DC. The profile of cytokines released by NKT cells and their accumulation in the serum was therefore examined when α-GalCer was administered to lang-DTREGFP mice that had been treated with DT or left untreated. Interestingly, depletion of langerin+ cells before α-GalCer administration had little effect on accumulation of IL-4 in the serum, indicating that the iNKT cells can be activated by APC other than langerin+ cells (Fig. 6⇓A). This is supported by intracellular cytokine staining of the activated iNKT cells themselves, which showed IL-4 and IFN-γ expression was not affected by depletion of langerin+ cells (Fig. 6⇓B). However, accumulation of IL-12p70 and IFN-γ in the serum was almost entirely ablated in the absence of langerin+ cells (Fig. 6⇓A). Thus, langerin+ cells are not required for the initial activation of iNKT cells, but are required for downstream release of large quantities of IL-12p70 into the serum. Failure to elicit IL-12p70 in sufficient quantity is, in turn, likely to abrogate the transactivation of NK cells, which are the major contributors of IFN-γ into the serum.

FIGURE 6.
FIGURE 6.

The langerin+ cells are dispensable for activation of iNKT cells, but are required for iNKT cell-triggered release of IL-12p70. A, Levels of the indicated cytokines were measured in the serum of groups of lang-DTREGFP mice (n = 5) that had been treated with DT or left untreated and then administered α-GalCer i.v. 1 day later. Mean serum concentrations ± SEM for each group are presented. B, Intracellular cytokine staining was used to assess cytokine production by splenic iNKT cells 2.5 h after α-GalCer administration into groups of lang-DTREGFP mice (n = 3) treated as in A. The splenic iNKT cells were identified by flow cytometry with α-GalCer-loaded CD1d tetramers. Mean percentages of cytokine-producing cells ± SEM for each group are presented.

To examine IL-12 production directly in langerin+ cells, intracellular cytokine staining was used to examine expression of the IL-12p40 subunit in splenic DC subsets in lang-EGFP mice after i.v. injection of α-GalCer or Pam3Cys. This analysis revealed a clear distinction in function between the DC subsets, with langerin+CD8α+ DC being the prime producers of IL-12p40 in response to either systemic stimulus (Fig. 7⇓, A and B). Interestingly, administration of α-GalCer resulted in significantly higher concentrations of IL-12p70 in the serum than administration of Pam3Cys, while the levels of intracellular IL-12p40 induced in langerin+CD8α+ DC by either stimulus were similar (Fig. 7, B and C). Activated iNKT cells may therefore be potent drivers of the IL-12p35 subunit that is required to combine with IL-12p40 to form the bioactive IL-12p70 heterodimer, which may reflect the strong provision of CD40 signaling by these cells.

FIGURE 7.
FIGURE 7.

The langerin+CD8α+ DC are the prime producers of IL-12p40 in response to systemic activation stimuli. A, Production of the IL-12p40 subunit of IL-12 was assessed in splenic DC by intracellular cytokine staining 6 h after administration of the indicated stimuli into groups of lang-EGFP mice (n = 3). Representative FACS profiles with gating on CD11c+ cells are shown. B, Graph showing mean percentages of cells (±SEM) of each DC subtype producing IL-12p40 for each group of animals treated as in A. C, Graph of mean serum levels of IL-12p70 (±SEM) in groups of C57BL/6 animals (n = 5) 6 h after treatment with the indicated stimuli.

Enhanced cross-priming of CTL responses in the presence of TLR2 stimulation or activation of iNKT cells is dependent on langerin+ DC

Having established a role for langerin+ DC in cross-presentation of protein Ag and IL-12 production, we next investigated whether these cells are ultimately responsible for cross-priming effector CTL in vivo. Cohorts of OVA-specific TCR-transgenic CD8+ T cells (1 × 104 cells) were transferred into lang-DTREGFP mice that had been treated with DT or left untreated, and then OVA protein was administered i.v. in the presence of Pam3Cys or α-GalCer or a combination of both. Expansion of the OVA-specific transgenic T cell population was then monitored in the blood by flow cytometry. When assessed on day 7 after administration of OVA, prior depletion of langerin+ cells with DT ablated responses initiated with Pam3Cys alone and significantly reduced CD8+ T cell responses elicited in the presence α-GalCer alone or in combination with α-GalCer, relative to untreated controls (Fig. 8⇓A). Interestingly, at later time points (14 days and beyond), CD8+ T cell responses elicited in the presence α-GalCer showed some recovery, with differences between DT-treated and untreated groups no longer statistically significant. Thus, the impact of depleting langerin+ cells was to reduce the early proliferative burst of CD8+ T cells elicited in the presence of iNKT cell stimulation. In a separate experiment, depletion of cross-presenting APC was achieved by administration of horse cyt c in C57BL/6 hosts (Fig. 8⇓B). Again, the impact of depletion of cross-presenting APC was to severely reduce responses initiated in the presence of Pam3Cys and significantly reduce the early proliferative burst of the CD8+ T cell responses induced in the presence of iNKT cell stimulation.

FIGURE 8.
FIGURE 8.

Depletion of langerin+ cells attenuates cross-priming of CD8+ T cell responses to soluble Ag. A, Groups of lang-DTREGFP animals were treated with DT to deplete langerin+ cells or left untreated and then donated cohorts of CD45.1+ OVA-specific transgenic T cells (from the cross of OT-I × B6.SJL-Ptprca Pepcb/BoyJ). One day later, animals were administered 200 μg of OVA protein alone or in the presence of Pam3Cys, α-GalCer, or both. Blood was collected at designated time points and analyzed for the presence of transgenic T cells using TCR-specific Abs and anti-CD45.1. Mean percentage (±SEM) of transferred Vα2+CD45.1+ cells as a proportion of CD8+ T cells in blood are shown for each treatment group (n = 5). B, As in A, except cyt c was used to deplete cells capable of cross-presentation.

Discussion

The langerin expression has generally been regarded as a hallmark of LC distributed within the epidermis and skin-derived LC found in skin-draining lymph nodes. However, it was recently shown that langerin is also expressed by a CD8α+ DC subtype in skin-draining and non-skin-draining, lymphoid organs (13, 26, 27). The same observation was made with the lang-EGFP mouse strain used here, in which EGFP is expressed under the control of the langerin promoter (15). The availability of both the lang-EGFP and lang-DTREGFP mouse strains permitted us to specifically examine the role of langerin+CD8α+ DC in responses to systemic Ags.

We report that animals depleted of the langerin+ fraction of the CD8α+ DC population are compromised in their ability to cross-present blood-borne Ags and cross-prime Ag-specific CTL in the presence of an adjuvant. Depletion of langerin+ cells also had a negative impact on the release of bioactive IL-12 in response to systemic activation stimuli. Because langerin+CD8α+ DC were susceptible to apoptosis following i.v. administration of cyt c, it is likely that the process of cross-presentation utilized by these cells involves diverting acquired Ags into the cytoplasm for proteasomal degradation before presentation on MHC class I molecules. The potent capacity to cross-prime CTL is also likely to reflect an enhanced capacity for langerin+CD8α+ DC to up-regulate CD40 in response to systemic stimuli, which was seen here in response to the TLR2 ligand Pam3Cys and the iNKT cell ligand α-GalCer. Our data therefore suggest functional heterogeneity within the CD8α+ DC population, with a primary role for initial priming and shaping of CD8+ T cells responses to blood-borne Ags resting with the langerin+ subfraction.

Depletion of langerin+ cells had only a limited impact on the induction of CD4+ T cell responses to i.v. Ags in the presence of adjuvant. This observation fits with previous studies using Ab-targeted Ags, where stimulation of CD4+ T cells was favored when Ags were targeted directly to CD8α− DC rather than CD8α+ DC (38). Despite the heterogeneity in the CD8α+ DC population described here, Ags targeted to the CD8α+ population as a whole have been shown to be efficiently cross-presented to CD8+ T cells (7). This bias in DC function has been attributed to differences in Ag processing and presentation mechanisms, with molecules involved in MHC class I presentation shown to be enriched in the CD8α+ DC. It has recently been postulated that differences in phagosomal oxidation and pH may alter proteolytic activity in endocytic and phagocytic pathways in the different DC populations (9). Thus, with the exception of the protease cathepsin S, overall proteolytic activity in the phagosomes of CD8α+ DC was shown to be generally lower than in the phagosomes of CD8α− DC, which is likely to significantly alter the rate and quality of peptide generation. Since none of these studies have taken into account the heterogeneity in the CD8α+ DC population implied by our studies, it is likely that more stark differences exist between the langerin+CD8α+ DC population and those APC that favor stimulation of CD4+ T cells, or indeed the langerin− fraction of CD8α+ DC, which were unable to cross-present in our studies.

Depletion of langerin+ cells resulted in a severely impaired CD8+ T cell response in the presence of either Pam3Cys or α-GalCer. In repeated experiments, most notably when α-GalCer was used as adjuvant, the greatest negative impact was on the early kinetics of the CD8+ T cell response. At later time points (beyond 14 days after Ag administration), responses became statistically indistinguishable from levels seen in nondepleted animals (Fig. 8⇑). A similar delay in the kinetics of the CD8+ T cell response was observed when cyt c was used to deplete APC before Ag administration (Fig. 8⇑). These data perhaps suggest that depletion was incomplete or that some residual cross-presentation was performed by an undefined APC population that was not susceptible to either depletion strategy. However, if the depletion strategies were incomplete, the net impact would be to lower the overall dose of cross-presented Ag, which is thought to lower the magnitude of the peak of an induced CD8+ T cell response rather than induce a delay in the kinetics (39). It is also possible that the reduced cytokine release in response to α-GalCer in depleted animals has some bearing on the kinetics of the T cell response. However, it has been shown that stimulation in a low presence of inflammatory mediators such as IFN-γ and IL-12 also tends to lower the magnitude of the response, as well as prolong the contraction phase of a CD8+ T cell response, rather than delay the initial proliferative phase (40).

Timing of the peak of a CD8+ T cell response has been linked to the peak of functional Ag display by DC in vivo (41). It is therefore possible that efficient Ag display by langerin+CD8α+ DC may drive early T cell proliferation and that in the absence of these cells, Ag display is restricted to an APC population with a slower capacity for cross-presentation. In this regard, it has been shown that some DC can store Ag in a lysosome-like organelle depot for several days before it is cross-presented (42). Also, we have recently observed that Ag can be cross-presented in vivo for up to 4 days in the absence of adjuvant and as long as 10 days in the presence of adjuvant, as determined by transferring OT-I cells at different times after i.v. OVA administration (K. J. Farrand, N. Dickgreber, C. M. Hayman, G. F. Painter, V. Cerundolo, T. R. Petersen, and I. F. Hermans, manuscript in preparation). Resident langerin+CD8α+ DC are rapidly depleted from the spleen in response to microbial stimuli (11) or α-GalCer (discussed below); therefore, it is possible that the delayed cross-priming is performed by newly recruited langerin+CD8α+ DC that populate the spleen over the following days, although this needs further investigation. Intriguingly, different “waves” of cross-presented Ag display may contribute to the effector and memory components of an induced CD8 T cell response, with rapid display by resident langerin+CD8α+ DC being critical for the effector phase and the slower display driving memory.

Splenic CD8α+ DC have been shown to be the major producers of IL-12p40 and p70 in response to microbial or T cell stimuli compared with splenic CD4+ DC or CD4−CD8α− DC (43, 44, 45). However, in response to systemic stimuli, we have now identified the langerin+ fraction of CD8α+ DC as the primary source of IL-12, as indicated by intracellular cytokine staining for IL-12p40 in response to Pam3Cys or α-GalCer. Administration of α-GalCer typically induces a cascade of cellular interactions, including iNKT cell-mediated induction of IL-12 release by DC (34, 35). In the absence of langerin+ cells, iNKT cells are still stimulated to release IL-4 and IFN-γ, but there is no subsequent release of large quantities of IL-12p70 into the serum. The failure to produce IL-12 prevents transactivation of NK cells, and the release of large quantities of IFN-γ into the serum typically mediated by NK cells is abrogated. Given the role of CD40 signaling in enhancing IL-12p70 production (46, 47), the heightened propensity of langerin+CD8α+ DC to release of IL-12p70 in response to α-GalCer may reflect the ability of these cells to rapidly up-regulate CD40 in response to systemic stimuli (Fig. 5⇑). Activated iNKT cells, in turn, are a significant source of CD40L for this interaction. The cytokines IL-4 and IFN-γ released by iNKT cells may also play a role in this process by programming DC to limit p40 homodimer formation and promote p70 production (48, 49). Indeed, in IL-4-deficient animals, IL-12p70 release following iNKT cell stimulation is severely compromised (N. Dickgreber, unpublished observation).

Although initial studies with langerin-specific Abs showed that CD8α+ DC were localized to the red pulp and marginal zones surrounding the white pulp of the spleen, with a minority in the T cell area (13, 50), other studies using mAbs against CD205 (DEC205) and DCIR2 as markers for CD8α+ DC and CD8α− DC cells, respectively, had located the bulk of the CD8α+ DC population to the T cell-rich periarterial lymphoid sheaths (7, 51). More recently, newly developed langerin-specific mAbs showed that large numbers of langerin+CD8α+ DC were indeed distributed within the marginal zone (12). A further study showed that CD103+langerin+ DC within the marginal zone were capable of phagocytosis of apoptotic cells and could migrate into the T cell zone for cross-presentation of cell-associated Ags (11). It is therefore likely that the langerin+ cells are specifically positioned to screen the blood for circulating particulate Ags, including apoptotic cells. In the absence of activating stimuli, movement to the T cell zones induces peripheral tolerance to the apoptotic cargo of self-origin. However, it has also been reported that in the presence of a microbial stimulus, langerin+ cells move en masse from the marginal zone, with a subsequent increase in the T cell areas, and by 24–48 h the cells disappear entirely. These cells are therefore likely to be responsible for cross-priming responses to their cargo in the T cell zone. We have also observed disappearance of langerin+CD8α+ DC from the spleen over a similar time frame in response to α-GalCer administration, which was partly attributable to TNF-α, suggesting some form of activation-induced cell death that may serve to limit excessive adaptive responses (H. Simkins, K. J. Farrand, P. Stoitzner, I. F. Hermans, and F. Ronchese, manuscript in preparation).

In the steady state, the lymphoid organs contain DC distinct from plasmacytoid DC that can be categorized into resident or migratory phenotypes. The resident cells, which have primarily been discussed here, arrive in a precursor form from the bloodstream and carry out all their functions within the lymphoid tissue. Migratory DC, on the other hand, sample Ags in peripheral tissues and migrate into lymph nodes via the lymph for subsequent interaction with T cells. In skin-draining lymph nodes, this population includes migratory LC and a population of langerin+CD103+ dermal DC, which both lack expression of CD8α (52). The mediastinal lymph nodes draining the lung also contain migratory langerin+CD8α− cells (53). Because i.v. administered Ags are known to recirculate via the lung, we cannot rule out the possibility that these migratory DC contribute to the responses to i.v. OVA in our experiments, as these cells will also be susceptible to DT treatment. Studies are ongoing to address this issue.

The significance of langerin expression by some CD8α+ DC is not clear. In LC, expression of langerin induces the formation of Birbeck granules, a hallmark feature of this cell type (25, 54). No Birbeck granules have been detected in the cytoplasm of sorted CD8α+ DC nor can Birbeck granule expression be induced by stimulating CD8α+ DC with anti-langerin Abs, as has been observed in human epidermal LC (27). The langerin molecule itself is characterized by an extracellular recognition domain, which binds carbohydrate ligands in a Ca2+-dependent manner, and has been shown to have specificity for mannose, N-acetylglucosamine, and fucose (54, 55). This capacity for sugar recognition, along with endocytic properties (56), raises the possibility that langerin is directly involved in Ag presentation. However cross-presentation of OVA by CD8α+ splenic DC is not impaired in langerin-deficient mice (57), so the improved cross-presentation by langerin+CD8α+ DC described here cannot be explained by langerin-mediated uptake.

Is there a human counterpart to langerin+CD8α+ DC? Gene expression studies of murine DC subsets showed preferential expression of the type II membrane protein Clec9A on CD8α+ DC, which lead to the identification of a human ortholog expressed by human BDCA-3+ DC (58, 59). Since murine Clec9A+ cells and langerin+CD8α+ DC have been shown to be related functionally in terms of capacity to cross-present cell-associated Ags, it will be interesting to determine whether a Clec9A+BDCA3+ DC population with similar function is located within the marginal zones of human spleen. The langerin molecule itself has recently been shown to be expressed in human spleen, although in the T cell zone rather than marginal zone (60). Langerin+ cells can also be detected in other human tissues such as lymph nodes, gut, lung epithelium (61), and kidney (62); whether any of these populations represents a non-LC langerin+ cell type with similar function to murine langerin+CD8α+ DC remains to be determined.

Considerable effort is now being directed at vaccination strategies that specifically target Ags to DC with known function. Studies in mice have shown a benefit to targeting vaccine-encoded Ags to lymphoid-resident CD8α+ DC to cross-prime CD8+ T cell responses to intracellular infections and tumors. The data presented here support a more refined strategy in which the langerin+CD8α+ DC subset of the spleen is specifically targeted. In fact, Ags conjugated to anti-langerin Abs have been shown to be efficiently presented on both MHC class I and II molecules (14), although this activity can potentially be attributed to other langerin+ APC in the host. Further definition of the splenic langerin+CD8α+ DC subset may uncover more effective targets for this form of immunotherapy.

Acknowledgments

We thank Adrien Kissenpfennig and Bernard Malissen for providing the lang-DTREGFP and lang-EGFP mice, the personnel of the Biomedical Research Unit of the Malaghan Institute of Medical Research for animal husbandry, and Kylie Price and Brigitta Mester for FACS support.

Disclosures

The authors have no financial conflict of interest.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 This work was supported by the New Zealand Health Research Council and the Cancer Society of New Zealand. I.F.H. was supported by a New Zealand Health Research Council Sir Charles Hercus Fellowship.

  • ↵2 Current address: Department of Dermatology, Innsbruck Medical University, A-6020 Innsbruck, Austria.

  • ↵3 Address correspondence and reprint requests to Dr. Ian F. Hermans, Malaghan Institute of Medical Research, PO Box 7060, Wellington 6242, New Zealand. E-mail address: ihermans{at}malaghan.org.nz

  • ↵4 Abbreviations used in this paper: DC, dendritic cell; α-GalCer, α-galactosylceramide; DT, diphtheria toxin; iNKT, invariant CD1d-dependent NK-like T cell; Pam3Cys, N-Palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-Cys-[S]-Serl-[S]-Lys4-trihydrochloride; Cyt c, cytochrome c; LC, Langerhans cell; EGFP, enhanced GFP; DTR, DT receptor.

  • Received August 18, 2009.
  • Accepted October 14, 2009.

References

  1. ↵
    Trombetta, E. S., M. Ebersold, W. Garrett, M. Pypaert, I. Mellman. 2003. Activation of lysosomal function during dendritic cell maturation. Science 299: 1400-1403.
    OpenUrlAbstract/FREE Full Text
  2. ↵
    Trombetta, E. S., I. Mellman. 2005. Cell biology of antigen processing in vitro and in vivo. Annu. Rev. Immunol. 23: 975-1028.
    OpenUrlCrossRefPubMed
  3. ↵
    Wilson, N. S., J. A. Villadangos. 2005. Regulation of antigen presentation and cross-presentation in the dendritic cell network: facts, hypothesis, and immunological implications. Adv. Immunol. 86: 241-305.
    OpenUrlCrossRefPubMed
  4. ↵
    Bevan, M. J.. 1976. Cross-priming for a secondary cytotoxic response to minor H antigens with H-2 congenic cells which do not cross-react in the cytotoxic assay. J. Exp. Med. 143: 1283-1288.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    Heath, W. R., G. T. Belz, G. M. Behrens, C. M. Smith, S. P. Forehan, I. A. Parish, G. M. Davey, N. S. Wilson, F. R. Carbone, J. A. Villadangos. 2004. Cross-presentation, dendritic cell subsets, and the generation of immunity to cellular antigens. Immunol. Rev. 199: 9-26.
    OpenUrlCrossRefPubMed
  6. ↵
    Villadangos, J. A., P. Schnorrer. 2007. Intrinsic and cooperative antigen-presenting functions of dendritic-cell subsets in vivo. Nat. Rev. Immunol. 7: 543-555.
    OpenUrlCrossRefPubMed
  7. ↵
    Dudziak, D., A. O. Kamphorst, G. F. Heidkamp, V. R. Buchholz, C. Trumpfheller, S. Yamazaki, C. Cheong, K. Liu, H. W. Lee, C. G. Park, et al 2007. Differential antigen processing by dendritic cell subsets in vivo. Science 315: 107-111.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    Schnorrer, P., G. M. Behrens, N. S. Wilson, J. L. Pooley, C. M. Smith, D. El-Sukkari, G. Davey, F. Kupresanin, M. Li, E. Maraskovsky, et al 2006. The dominant role of CD8+ dendritic cells in cross-presentation is not dictated by antigen capture. Proc. Natl. Acad. Sci. USA 103: 10729-10734.
    OpenUrlAbstract/FREE Full Text
  9. ↵
    Savina, A., A. Peres, I. Cebrian, N. Carmo, C. Moita, N. Hacohen, L. F. Moita, S. Amigorena. 2009. The small GTPase Rac2 controls phagosomal alkalinization and antigen crosspresentation selectively in CD8+ dendritic cells. Immunity 30: 544-555.
    OpenUrlCrossRefPubMed
  10. ↵
    Lin, M. L., Y. Zhan, A. I. Proietto, S. Prato, L. Wu, W. R. Heath, J. A. Villadangos, A. M. Lew. 2008. Selective suicide of cross-presenting CD8+ dendritic cells by cytochrome c injection shows functional heterogeneity within this subset. Proc. Natl. Acad. Sci. USA 105: 3029-3034.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    Qiu, C. H., Y. Miyake, H. Kaise, H. Kitamura, O. Ohara, M. Tanaka. 2009. Novel subset of CD8α+ dendritic cells localized in the marginal zone is responsible for tolerance to cell-associated antigens. J. Immunol. 182: 4127-4136.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    Idoyaga, J., N. Suda, K. Suda, C. G. Park, R. M. Steinman. 2009. Antibody to Langerin/CD207 localizes large numbers of CD8α+ dendritic cells to the marginal zone of mouse spleen. Proc. Natl. Acad. Sci. USA 106: 1524-1529.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    McLellan, A. D., M. Kapp, A. Eggert, C. Linden, U. Bommhardt, E. B. Brocker, U. Kammerer, E. Kampgen. 2002. Anatomic location and T-cell stimulatory functions of mouse dendritic cell subsets defined by CD4 and CD8 expression. Blood 99: 2084-2093.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    Idoyaga, J., C. Cheong, K. Suda, N. Suda, J. Y. Kim, H. Lee, C. G. Park, R. M. Steinman. 2008. Cutting edge: langerin/CD207 receptor on dendritic cells mediates efficient antigen presentation on MHC I and II products in vivo. J. Immunol. 180: 3647-3650.
    OpenUrlAbstract/FREE Full Text
  15. ↵
    Kissenpfennig, A., S. Henri, B. Dubois, C. Laplace-Builhe, P. Perrin, N. Romani, C. H. Tripp, P. Douillard, L. Leserman, D. Kaiserlian, et al 2005. Dynamics and function of Langerhans cells in vivo: dermal dendritic cells colonize lymph node areas distinct from slower migrating Langerhans cells. Immunity 22: 643-654.
    OpenUrlCrossRefPubMed
  16. ↵
    Mendiratta, S. K., W. D. Martin, S. Hong, A. Boesteanu, S. Joyce, L. Van Kaer. 1997. CD1d1 mutant mice are deficient in natural T cells that promptly produce IL-4. Immunity 6: 469-477.
    OpenUrlCrossRefPubMed
  17. ↵
    Takeuchi, O., K. Hoshino, T. Kawai, H. Sanjo, H. Takada, T. Ogawa, K. Takeda, S. Akira. 1999. Differential roles of TLR2 and TLR4 in recognition of Gram-negative and Gram-positive bacterial cell wall components. Immunity 11: 443-451.
    OpenUrlCrossRefPubMed
  18. ↵
    Hogquist, K. A., S. C. Jameson, W. R. Heath, J. L. Howard, M. J. Bevan, F. R. Carbone. 1994. T cell receptor antagonist peptides induce positive selection. Cell 76: 17-27.
    OpenUrlCrossRefPubMed
  19. ↵
    Barnden, M. J., J. Allison, W. R. Heath, F. R. Carbone. 1998. Defective TCR expression in transgenic mice constructed using cDNA-based α- and β-chain genes under the control of heterologous regulatory elements. Immunol. Cell Biol. 76: 34-40.
    OpenUrlCrossRefPubMed
  20. ↵
    Brimnes, M. K., L. Bonifaz, R. M. Steinman, T. M. Moran. 2003. Influenza virus-induced dendritic cell maturation is associated with the induction of strong T cell immunity to a coadministered, normally nonimmunogenic protein. J. Exp. Med. 198: 133-144.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    Lee, A., K. J. Farrand, N. Dickgreber, C. M. Hayman, S. Jurs, I. F. Hermans, G. F. Painter. 2006. Novel synthesis of α-galactosyl-ceramides and confirmation of their powerful NKT cell agonist activity. Carbohydr. Res. 341: 2785-2798.
    OpenUrlCrossRefPubMed
  22. ↵
    Aliprantis, A. O., R. B. Yang, M. R. Mark, S. Suggett, B. Devaux, J. D. Radolf, G. R. Klimpel, P. Godowski, A. Zychlinsky. 1999. Cell activation and apoptosis by bacterial lipoproteins through Toll-like receptor-2. Science 285: 736-739.
    OpenUrlAbstract/FREE Full Text
  23. ↵
    McLellan, A. D., G. C. Starling, D. N. Hart. 1995. Isolation of human blood dendritic cells by discontinuous Nycodenz gradient centrifugation. J. Immunol. Methods 184: 81-89.
    OpenUrlCrossRefPubMed
  24. ↵
    Lyons, A. B.. 2000. Analysing cell division in vivo and in vitro using flow cytometric measurement of CFSE dye dilution. J. Immunol. Methods 243: 147-154.
    OpenUrlCrossRefPubMed
  25. ↵
    Valladeau, J., V. Clair-Moninot, C. Dezutter-Dambuyant, J. J. Pin, A. Kissenpfennig, M. G. Mattei, S. Ait-Yahia, E. E. Bates, B. Malissen, F. Koch, et al 2002. Identification of mouse langerin/CD207 in Langerhans cells and some dendritic cells of lymphoid tissues. J. Immunol. 168: 782-792.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    Flacher, V., P. Douillard, S. Aït-Yahia, P. Stoitzner, V. Clair-Moninot, N. Romani, S. Saeland. 2008. Expression of langerin/CD207 reveals dendritic cell heterogeneity between inbred mouse strains. Immunology 123: 339-347.
    OpenUrlCrossRefPubMed
  27. ↵
    Douillard, P., P. Stoitzner, C. Tripp, V. Clair-Moninot, S. Aït-Yahia, A. D. McLellan, A. Eggert, N. Romani, S. Saeland. 2005. Mouse lymphoid tissue contains distinct subsets of langerin/CD207 dendritic cells, only one of which represents epidermal-derived Langerhans cells. J. Invest. Dermatol. 125: 983-994.
    OpenUrlCrossRefPubMed
  28. ↵
    Takeda, K., T. Kaisho, S. Akira. 2003. Toll-like receptors. Annu. Rev. Immunol. 21: 335-376.
    OpenUrlCrossRefPubMed
  29. ↵
    Fujii, S., K. Shimizu, C. Smith, L. Bonifaz, R. M. Steinman. 2003. Activation of natural killer T cells by α-galactosylceramide rapidly induces the full maturation of dendritic cells in vivo and thereby acts as an adjuvant for combined CD4 and CD8 T cell immunity to a coadministered protein. J. Exp. Med. 198: 267-279.
    OpenUrlAbstract/FREE Full Text
  30. ↵
    Hermans, I. F., J. D. Silk, U. Gileadi, M. Salio, B. Mathew, G. Ritter, R. Schmidt, A. L. Harris, L. Old, V. Cerundolo. 2003. NKT cells enhance CD4+ and CD8+ T cell responses to soluble antigen in vivo through direct interaction with dendritic cells. J. Immunol. 171: 5140-5147.
    OpenUrlAbstract/FREE Full Text
  31. ↵
    Silk, J. D., I. F. Hermans, U. Gileadi, T. W. Chong, D. Shepherd, M. Salio, B. Mathew, R. R. Schmidt, S. J. Lunt, K. J. Williams, et al 2004. Utilizing the adjuvant properties of CD1d-dependent NK T cells in T cell-mediated immunotherapy. J. Clin. Invest. 114: 1800-1811.
    OpenUrlCrossRefPubMed
  32. ↵
    Kawano, T., J. Cui, Y. Koezuka, I. Toura, Y. Kaneko, K. Motoki, H. Ueno, R. Nakagawa, H. Sato, E. Kondo, H. Koseki, M. Taniguchi. 1997. CD1d-restricted and TCR-mediated activation of vα14 NKT cells by glycosylceramides. Science 278: 1626-1629.
    OpenUrlAbstract/FREE Full Text
  33. ↵
    Bendelac, A., P. B. Savage, L. Teyton. 2007. The biology of NKT cells. Annu. Rev. Immunol. 25: 297-336.
    OpenUrlCrossRefPubMed
  34. ↵
    Tomura, M., W. G. Yu, H. J. Ahn, M. Yamashita, Y. F. Yang, S. Ono, T. Hamaoka, T. Kawano, M. Taniguchi, Y. Koezuka, H. Fujiwara. 1999. A novel function of Vα14+CD4+NKT cells: stimulation of IL-12 production by antigen-presenting cells in the innate immune system. J. Immunol. 163: 93-101.
    OpenUrlAbstract/FREE Full Text
  35. ↵
    Kitamura, H., K. Iwakabe, T. Yahata, S. Nishimura, A. Ohta, Y. Ohmi, M. Sato, K. Takeda, K. Okumura, L. Van Kaer, et al 1999. The natural killer T (NKT) cell ligand α-galactosylceramide demonstrates its immunopotentiating effect by inducing interleukin (IL)-12 production by dendritic cells and IL-12 receptor expression on NKT cells. J. Exp. Med. 189: 1121-1128.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    Carnaud, C., D. Lee, O. Donnars, S. H. Park, A. Beavis, Y. Koezuka, A. Bendelac. 1999. Cutting edge: cross-talk between cells of the innate immune system: NKT cells rapidly activate NK cells. J. Immunol. 163: 4647-4650.
    OpenUrlAbstract/FREE Full Text
  37. ↵
    Schmieg, J., G. Yang, R. W. Franck, N. Van Rooijen, M. Tsuji. 2005. Glycolipid presentation to natural killer T cells differs in an organ-dependent fashion. Proc. Natl. Acad. Sci. USA 102: 1127-1132.
    OpenUrlAbstract/FREE Full Text
  38. ↵
    Carter, R. W., C. Thompson, D. M. Reid, S. Y. Wong, D. F. Tough. 2006. Preferential induction of CD4+ T cell responses through in vivo targeting of antigen to dendritic cell-associated C-type lectin-1. J. Immunol. 177: 2276-2284.
    OpenUrlAbstract/FREE Full Text
  39. ↵
    Badovinac, V. P., B. B. Porter, J. T. Harty. 2002. Programmed contraction of CD8+ T cells after infection. Nat. Immunol. 3: 619-626.
    OpenUrlCrossRefPubMed
  40. ↵
    Badovinac, V. P., B. B. Porter, J. T. Harty. 2004. CD8+ T cell contraction is controlled by early inflammation. Nat. Immunol. 5: 809-817.
    OpenUrlCrossRefPubMed
  41. ↵
    Badovinac, V. P., J. T. Harty. 2006. Programming, demarcating, and manipulating CD8+ T-cell memory. Immunol. Rev. 211: 67-80.
    OpenUrlCrossRefPubMed
  42. ↵
    van Montfoort, N., M. G. Camps, S. Khan, D. V. Filippov, J. J. Weterings, J. M. Griffith, H. J. Geuze, T. van Hall, J. S. Verbeek, C. J. Melief, F. Ossendorp. 2009. Antigen storage compartments in mature dendritic cells facilitate prolonged cytotoxic T lymphocyte cross-priming capacity. Proc. Natl. Acad. Sci. USA 106: 6730-6735.
    OpenUrlAbstract/FREE Full Text
  43. ↵
    Hochrein, H., K. Shortman, D. Vremec, B. Scott, P. Hertzog, M. O'Keeffe. 2001. Differential production of IL-12, IFN-α, and IFN-γ by mouse dendritic cell subsets. J. Immunol. 166: 5448-5455.
    OpenUrlAbstract/FREE Full Text
  44. ↵
    Maldonado-Lopez, R., T. De Smedt, P. Michel, J. Godfroid, B. Pajak, C. Heirman, K. Thielemans, O. Leo, J. Urbain, M. Moser. 1999. CD8α+ and CD8α− subclasses of dendritic cells direct the development of distinct T helper cells in vivo. J. Exp. Med. 189: 587-592.
    OpenUrlAbstract/FREE Full Text
  45. ↵
    Reis e Sousa, C., S. Hieny, T. Scharton-Kersten, D. Jankovic, H. Charest, R. N. Germain, A. Sher. 1997. In vivo microbial stimulation induces rapid CD40 ligand-independent production of interleukin 12 by dendritic cells and their redistribution to T cell areas. J. Exp. Med. 186: 1819-1829.
    OpenUrlAbstract/FREE Full Text
  46. ↵
    Schulz, O., A. D. Edwards, M. Schito, J. Aliberti, S. Manickasingham, A. Sher, C. Reis e Sousa. 2000. CD40 triggering of heterodimeric IL-12p70 production by dendritic cells in vivo requires a microbial priming signal. Immunity 13: 453-462.
    OpenUrlCrossRefPubMed
  47. ↵
    Cella, M., D. Scheidegger, K. Palmer-Lehmann, P. Lane, A. Lanzavecchia, G. Alber. 1996. Ligation of CD40 on dendritic cells triggers production of high levels of interleukin-12 and enhances T cell stimulatory capacity: T-T help via APC activation. J. Exp. Med. 184: 747-752.
    OpenUrlAbstract/FREE Full Text
  48. ↵
    Kalinski, P., H. H. Smits, J. H. Schuitemaker, P. L. Vieira, M. van Eijk, E. C. de Jong, E. A. Wierenga, M. L. Kapsenberg. 2000. IL-4 is a mediator of IL-12p70 induction by human Th2 cells: reversal of polarized Th2 phenotype by dendritic cells. J. Immunol. 165: 1877-1881.
    OpenUrlAbstract/FREE Full Text
  49. ↵
    Hochrein, H., M. O'Keeffe, T. Luft, S. Vandenabeele, R. J. Grumont, E. Maraskovsky, K. Shortman. 2000. Interleukin (IL)-4 is a major regulatory cytokine governing bioactive IL-12 production by mouse and human dendritic cells. J. Exp. Med. 192: 823-833.
    OpenUrlAbstract/FREE Full Text
  50. ↵
    Neuenhahn, M., K. M. Kerksiek, M. Nauerth, M. H. Suhre, M. Schiemann, F. E. Gebhardt, C. Stemberger, K. Panthel, S. Schroder, T. Chakraborty, et al 2006. CD8α+ dendritic cells are required for efficient entry of Listeria monocytogenes into the spleen. Immunity 25: 619-630.
    OpenUrlCrossRefPubMed
  51. ↵
    Banchereau, J., F. Briere, C. Caux, J. Davoust, S. Lebecque, Y. J. Liu, B. Pulendran, K. Palucka. 2000. Immunobiology of dendritic cells. Annu. Rev. Immunol. 18: 767-811.
    OpenUrlCrossRefPubMed
  52. ↵
    Bursch, L. S., L. Wang, B. Igyarto, A. Kissenpfennig, B. Malissen, D. H. Kaplan, K. A. Hogquist. 2007. Identification of a novel population of Langerin+ dendritic cells. J. Exp. Med. 204: 3147-3156.
    OpenUrlAbstract/FREE Full Text
  53. ↵
    Sung, S. S., S. M. Fu, C. E. Rose, Jr, F. Gaskin, S. T. Ju, S. R. Beaty. 2006. A major lung CD103 (αE)-β7 integrin-positive epithelial dendritic cell population expressing Langerin and tight junction proteins. J. Immunol. 176: 2161-2172.
    OpenUrlAbstract/FREE Full Text
  54. ↵
    Valladeau, J., O. Ravel, C. Dezutter-Dambuyant, K. Moore, M. Kleijmeer, Y. Liu, V. Duvert-Frances, C. Vincent, D. Schmitt, J. Davoust, et al 2000. Langerin, a novel C-type lectin specific to Langerhans cells, is an endocytic receptor that induces the formation of Birbeck granules. Immunity 12: 71-81.
    OpenUrlCrossRefPubMed
  55. ↵
    Stambach, N. S., M. E. Taylor. 2003. Characterization of carbohydrate recognition by langerin, a C-type lectin of Langerhans cells. Glycobiology 13: 401-410.
    OpenUrlAbstract/FREE Full Text
  56. ↵
    Valladeau, J., V. Duvert-Frances, J. J. Pin, C. Dezutter-Dambuyant, C. Vincent, C. Massacrier, J. Vincent, K. Yoneda, J. Banchereau, C. Caux, et al 1999. The monoclonal antibody DCGM4 recognizes Langerin, a protein specific of Langerhans cells, and is rapidly internalized from the cell surface. Eur. J. Immunol. 29: 2695-2704.
    OpenUrlCrossRefPubMed
  57. ↵
    Kissenpfennig, A., S. Ait-Yahia, V. Clair-Moninot, H. Stossel, E. Badell, Y. Bordat, J. L. Pooley, T. Lang, E. Prina, I. Coste, et al 2005. Disruption of the langerin/CD207 gene abolishes Birbeck granules without a marked loss of Langerhans cell function. Mol. Cell. Biol. 25: 88-99.
    OpenUrlAbstract/FREE Full Text
  58. ↵
    Sancho, D., D. Mourao-Sa, O. P. Joffre, O. Schulz, N. C. Rogers, D. J. Pennington, J. R. Carlyle, C. Reis e Sousa. 2008. Tumor therapy in mice via antigen targeting to a novel, DC-restricted C-type lectin. J. Clin. Invest. 118: 2098-2110.
    OpenUrlCrossRefPubMed
  59. ↵
    Caminschi, I., A. I. Proietto, F. Ahmet, S. Kitsoulis, J. Shin Teh, J. C. Lo, A. Rizzitelli, L. Wu, D. Vremec, S. L. van Dommelen, et al 2008. The dendritic cell subtype-restricted C-type lectin Clec9A is a target for vaccine enhancement. Blood 112: 3264-3273.
    OpenUrlAbstract/FREE Full Text
  60. ↵
    Steiner, Q. G., L. A. Otten, M. J. Hicks, G. Kaya, F. Grosjean, E. Saeuberli, C. Lavanchy, F. Beermann, K. L. McClain, H. Acha-Orbea. 2008. In vivo transformation of mouse conventional CD8α+ dendritic cells leads to progressive multisystem histiocytosis. Blood 111: 2073-2082.
    OpenUrlAbstract/FREE Full Text
  61. ↵
    Chikwava, K., R. Jaffe. 2004. Langerin (CD207) staining in normal pediatric tissues, reactive lymph nodes, and childhood histiocytic disorders. Pediatr. Dev. Pathol. 7: 607-614.
    OpenUrlCrossRefPubMed
  62. ↵
    Segerer, S., F. Heller, M. T. Lindenmeyer, H. Schmid, C. D. Cohen, D. Draganovici, J. Mandelbaum, P. J. Nelson, H. J. Grone, E. F. Grone, et al 2008. Compartment specific expression of dendritic cell markers in human glomerulonephritis. Kidney Int. 74: 37-46.
    OpenUrlCrossRefPubMed
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