APCs, such as dendritic cells (DC), can present glycolipid Ags on CD1d molecules to NKT cells. This interaction activates DC and NKT cells, leading to release of cytokines and enhanced T cell responses. Thus, glycolipid Ags are currently being tested as adjuvants for immunotherapy. We were interested in the interaction of murine skin DC with NKT cells in skin-draining lymph nodes. We observed that all skin DC subsets expressed CD1d upon migration to the lymph nodes. Moreover, skin DC were able to present the synthetic glycolipid Ag α-galactosylceramide (α-GalCer) to the NKT cell hybridoma DN32.D3. Intradermally injected α-GalCer was presented by migratory skin DC and lymph node DC to NKT hybridoma cells in vitro. When we injected α-GalCer intradermally into the skin, the numbers of various leukocyte subsets in the draining lymph nodes did not change significantly. However, T and B cells as well as NKT cells up-regulated the activation marker CD69. Coapplication of α-GalCer with the tumor model Ag OVA induced strong cytolytic CD8+ T cell function that could inhibit the growth of B16 melanoma cells expressing OVA. However, mice that were devoid of migratory skin DC developed similar cytotoxic immune responses after intradermal immunization, indicating that skin DC are not required for the adjuvant properties of NKT cell activation and Ag presentation by this immunization route. In conclusion, migratory skin DC are able to interact with NKT cells; however, intradermally applied glycolipids are presented predominantly by lymph node DC to NKT cells.

Dendritic cells (DC)3 are mandatory for the initiation of T cell responses by virtue of their ability to activate naive T cells to develop effector function. Distributed all over the body, DC acquire and process Ag, which is then presented as peptides on MHC molecules to T cells. DC need to be activated for the induction of optimal T cell responses. There are various mechanisms by which DC sense danger signals and become activated, for example, through their TLR and/or cytokine receptors. An alternative activation route is through their interaction with members of the innate immune system, such as NKT cells (1).

NKT cells express a semi-invariant TCR α-chain that allows them to recognize a restricted repertoire of glycolipids presented on CD1d molecules (2). APCs, such as DC, can present glycolipid Ags to NKT cells. This interaction is dependent on CD40 ligation and results in the rapid release of large amounts of Th1 and Th2 cytokines and chemokines. CD40 ligation on DC induces up-regulation of costimulatory molecules, such as CD80 and CD86, and production of IL-12, which in turn further enhances NKT cell activation as characterized by induction of CD69. The activation of DC causes enhanced Ag presentation and improved stimulation of CD4+ and CD8+ T cell responses (3). NKT cells are present in most lymphatic tissues, with the highest density in liver (30%) and lower numbers in spleen (2–3%) and lymph node (0.5–2%, depending on the location). The NKT cells found in lymph nodes display a less activated phenotype than those from spleen. Moreover, NKT cells from peripheral lymph nodes produce more IFN-γ and less IL-4 than NKT cells from spleen and mesenteric lymph nodes (3, 4). Studies to date have concentrated on the effects of i.v. injected synthetic glyoclipids, such as α-galactosylceramide (α-GalCer) on spleen and liver NKT cells (5, 6, 7). We were interested in determining whether skin DC can interact with NKT cells in peripheral lymph nodes.

Skin DC can be subdivided into three subsets by expression of markers and localization. They can be distinguished by their different expression of Langerin (8), CD103 (9), or epithelial cell adhesion molecule (Ep-CAM) (10). The epidermis harbors Langerhans cells (LC), which are Langerin+CD103Ep-CAM+. In the dermis we can discriminate dermal Langerin+ DC, which are Langerin+CD103+Ep-CAM and the dermal DC that are LangerinEpCam and mainly CD103 (11). Currently, it is not clear whether these subtypes of skin DC exert different functions in the skin immune system. LC and dermal DC have been shown to stimulate T cell responses; however, dermal DC are superior than LC in stimulating humoral responses (12).

Immunotherapy with DC against cancer has proven to be safe and effective, with some patients demonstrating partial to complete remissions (13, 14). Novel approaches aim at loading DC in situ with Ag by applying Ag in various formulations onto the skin (15, 16), thereby taking advantage of the ability of skin DC in inducing strong T cell responses. In our study, we investigated whether skin DC express CD1d molecules on their cell surface, and whether they can present glyolipid Ag to NKT cells. We were also interested in assessing the potency of the synthetic glycolipid α-GalCer as an adjuvant for immunization strategies through the skin against melanoma.

We used mice of inbred strains C57BL/6 (Charles River Laboratories), LangerinDTREGFP, and LangerinEGFP knockin mice expressing a diphtheria toxin receptor (DTR) and/or enhanced GFP (EGFP) under the Langerin promotor (17). All mice were bred at the animal facility of Innsbruck Medical University and used for experiments at 2–5 mo of age. All animal experiments were performed according to governmental guidelines.

Complete culture medium used was RPMI 1640 (PAA) supplemented with 10% heat-inactivated FBS (Lonza-BioWhittaker), 2 mM l-glutamine (Invitrogen Life Technologies), 50 μg/ml gentamicin (PAA), and 50 μM 2-ME (Sigma-Aldrich). The synthetic glycolipid α-GalCer was purchased from Alexis Biochemicals; OVA protein from Sigma-Aldrich; and peptide OVA257–264 (SIINFEKL) was provided by A. Eberhart (Biochemical Pharmacology, Innsbruck Medical University, Innsbruck, Austria). The B16.OVA cell line, generated by E. Lord and J. Frelinger (University of Rochester, Rochester, NY) (18), was provided by F. Ronchese (Malaghan Institute of Medical Research, Wellington, New Zealand).

Mouse ear skin was split into dorsal and ventral halves with forceps, and the dorsal halves (i.e., cartilage-free halves) were cultured for 3 days, as described earlier (19). Alternatively, epidermis and dermis were separated with the enzyme Dispase II (Roche Diagnostics), and the epidermal sheets were placed in culture for 3 days. Emigrated cells were harvested, and the remaining skin was used for immunofluorescence stainings. To prepare sheets for immunofluorescence from whole skin explants or fresh skin, ear halves were floated dermal side down on 0.5 M ammonium thiocyanate (Merck) for 20 min at 37°C. The epidermis was peeled off the dermis, and both parts were fixed in acetone for 20 min. Immunofluorescence stainings were performed with the following mAbs: anti-CD1d (clone 1B1; eBioscience), goat anti-rat-Alexa Fluor594 (Invitrogen-Molecular Probes), FITC-conjugated anti-Langerin (clone 929F3; Dendritics), and FITC-conjugated anti-MHC-class II (clone 2G9; BD Biosciences). The epidermal and dermal sheets were incubated with Abs for 1 h at 37°C. Migratory skin DC were stained for 30 min at 37°C after adhering them onto poly(l-lysine)-coated glass slides. Samples were viewed on a conventional Olympus epifluorescence microscope.

We routinely pooled the skin-draining lymph nodes (auricular, inguinal, and brachial) and ear skin from several mice per experiment to obtain sufficient cell numbers for FACS analysis. The following Abs were purchased from BD Biosciences: CD103 biotin (clone M290), CD40 PE (clone 23.2), CD19 FITC (clone ID3), CD69 biotin (clone H1.2F3), and CD3 PE Cy5 (clone 17A2). The following Abs were from eBioscience: CD11c PE Cy5 (clone N418), CD1d PE (clone 1B1), and MHC class II allophycocyanin (clone M5/114.15.2). Langerin FITC (clone 929F3; Dendritics) was used to identify LC after permeabilization with Cytofix/Cytoperm kit (BD Biosciences), according to manufacturer’s instruction. NKT cells were stained with α-GalCer-loaded CD1d tetramer (ProImmune) for 30 min on ice in the dark. After excluding CD19+ B cells, as recommended by the manufacturer’s protocol, NKT cells were identified as CD3+CD1d-tetramer+ cells. Dead cells were routinely excluded from analysis with 7-AAD staining (BD Biosciences). All Ab incubations were conducted on ice for 10 min. All samples were analyzed on a FACSCalibur using CellQuest software (BD Biosciences) and analyzed with FlowJo (Tree Star).

Migratory skin DC were obtained from whole and epidermal skin explant cultures. Lymph node DC were isolated from skin-draining lymph nodes that were digested with 0.5 mg/ml collagenase P (Roche Diagnostics) for 30 min at 37°C. The DC were enriched on a Nycodenz gradient and purified with CD11c microbeads (Miltenyi Biotec). For in vitro loading with α-GalCer, migratory skin DC and purified lymph node DC were loaded with 200 ng/ml α-GalCer overnight, and medium was supplemented with GM-CSF (200 U/ml) containing supernatant (transfected plasmacytoma cell line X38-Ag8; provided by A. Lanzavecchia, Bellinzona, Switzerland). For in situ loading with α-GalCer, mice were injected intradermally into both ears with PBS or 1 μg of α-GalCer in 50 μl vol per ear 4 h before onset of skin explant culture or 48 h before isolation of lymph node DC. Graded doses of DC in triplicates were cocultured with 5 × 104 NKT hybridoma cells (DN32.D3 (2), provided by V. Cerundolo, Oxford, U.K.) for 24 h, followed by measurement of IL-2 secretion (IL-2 ELISA; eBiosciences) as indicator for activation of NKT cells.

SIINFEKL/H-2Kb pentamer conjugated to PE was obtained from ProImmune. Groups of three mice were immunized either intradermally with PBS or 250 μg of OVA ± 1 μg of α-GalCer in 50 μl per ear (both ears injected) or i.v. with PBS or same amounts of OVA ± α-GalCer in 100 μl into the tail vein. A week after immunization, the auricular lymph nodes draining the immunization site and spleens were analyzed for the presence of SIINFEKL/H-2Kb pentamer+CD8+ T cells. The cells were stained with SIINFEKL/H-2Kb pentamer for 10 min at room temperature in the dark, followed by incubation with mAb against CD8α FITC (clone Ly-2) and CD19 allophycocyanin mAb (clone ID3; both BD Biosciences) for 10 min on ice. Unspecific binding of SIINFEKL/H-2Kb pentamer by dead cells and CD19+ B cells was excluded by gating on 7-AADCD19 cells before analysis, as recommended by manufacturer’s protocol.

Groups of three mice were immunized with PBS or 250 μg of OVA ± 1 μg of α-GalCer intradermally in 50 μl per ear (both ears injected). A week after immunization, mice were injected i.v. with a mixture of differentially CFSE (Invitrogen-Molecular Probes)-labeled spleen cells (20 or 200 nM) loaded with 10 or 100 nM OVA257–264 (OVA-peptide SIINFEKL), respectively, and unloaded spleen cells labeled with 10 μM chloromethyl-benzoyl-aminotetramethyl-rhodamine (CMTMR; Invitrogen-Molecular Probes). From each target cell population, we injected 3–4 × 106 cells, meaning a total of 9–12 × 106 target cells per mouse. We collected the auricular lymph nodes draining the immunization site and the blood 48 h after injection of target cells. Presence of viable injected target cells was determined by exclusion of 7-AAD (BD Biosciences)-positive dead cells. Percentage of killing was calculated using the formula, as described before (20): 100% − ((number of CFSE+ cells/number of CMTMR+ cells) × 100%). In some of the experiments, we depleted Langerin+ skin DC in LangerinDTREGFP mice by i.p. injection of 500 ng of diphtheria toxin (Sigma-Aldrich) in PBS 1 day before immunization. All LC and dermal Langerin+ DC in skin and lymph node undergo apoptosis within 24 h after toxin administration, and are therefore absent at the time of intradermal immunization (17). In another set of experiments, we cut off the immunization site 4 h after administration of OVA plus α-GalCer to prevent the migration of Ag-loaded skin DC to the draining lymph nodes, as described earlier (21).

Mice were injected s.c. into the flank with 105 B16.OVA tumor cells. Tumor challenge was 7 days after skin immunization (prophylactic setting), or 7 days before immunization (therapeutic setting). Five mice were used in each group. Tumor size was assessed three times per week by measuring the short and long tumor diameters using calipers. Measurements were stopped when mice reached approximately maximum tumor size (150 mm2) and had to be euthanized. Results are presented as percentage of survival of mice and are analyzed with Kaplan-Meier survival curves.

Results are presented as mean ± SD. Unpaired two-tailed Student’s t test was used to determine the statistical significance of differences between immunization with OVA ± α-GalCer. For the tumor experiments, we performed Kaplan-Meier analyses to determine percentage of survival. Possible outliers were detected by performing Grubbs test for significant outliers. Numbers of performed experiments are indicated in figure legends. Values of p were calculated with GraphPad Prism 5 (GraphPad). Probability values are expressed as the following: ∗∗∗, p < 0.001; ∗∗, p < 0.01; and ∗, p < 0.05.

Glycolipid Ags are presented on CD1d surface molecules by APCs to NKT cells. We were therefore interested in expression profiles of CD1d on the different skin DC subtypes. For this purpose, we stained epidermis and dermis before and after skin explant culture with a mAb against CD1d. LC expressed low levels of CD1d that were up-regulated during skin explant culture for 3 days. Emigrated skin DC from whole skin explants showed CD1d expression in a perinuclear localization because CD1d is internalized and recycles through endosomal/lysosomal compartments, as reported before (3). In the dermis, CD1d+ migratory skin DC that were either Langerin+ or Langerin were visible in lymphatic vessels (Fig. 1).

FIGURE 1.

Skin DC express CD1d in situ. Mouse ear skin was cultured for 3 days on medium. Epidermal and dermal sheets (before and after skin explant culture) as well as emigrated DC from whole skin explants were double labeled with anti-CD1d (red fluorescence) and anti-Langerin (green fluorescence). Staining with isotype control mAbs for CD1d and Langerin was negative. Results are representative for two experiments.

FIGURE 1.

Skin DC express CD1d in situ. Mouse ear skin was cultured for 3 days on medium. Epidermal and dermal sheets (before and after skin explant culture) as well as emigrated DC from whole skin explants were double labeled with anti-CD1d (red fluorescence) and anti-Langerin (green fluorescence). Staining with isotype control mAbs for CD1d and Langerin was negative. Results are representative for two experiments.

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The various subtypes of migratory skin DC can be differentiated as LC (Langerin+CD103), dermal Langerin+ DC (Langerin+CD103+), and dermal DC (Langerin partly CD103+). We examined CD1d expression on these different DC subtypes by flow cytometry after they had emigrated from whole skin explants. For these experiments, we used the LangerinEGFP mice in which all Langerin+ cells are EGFP+ (17). Two-thirds of the migratory skin DC are of epidermal origin, representing LC, and just a small proportion of Langerin+ cells (5–10%) was CD103+ dermal Langerin+ DC. The majority of the Langerin+ and Langerin DC emigrating from skin explants within 3 days expressed CD1d on their cell surface (Fig. 2,A). Interestingly, a higher proportion of LC migrating out of whole skin explants was positive for CD1d than the ones from epidermal explants, indicating that migration through the dermis further up-regulated CD1d on the surface of LC (Fig. 2,B). After in vitro culture of migratory skin DC for another 24 h with GM-CSF, even more skin DC expressed CD1d molecules; however, these results were not statistically significant (Fig. 2 C).

FIGURE 2.

Skin DC express CD1d upon migration and present the synthetic glycolipid α-GalCer to NKT cells. A, Ear skin from LangerinEGFP mice was cultured on medium for 3 days, and emigrated DC were examined for CD1d surface expression. Dot plot show cells gated on CD11c. Three CD11c+ skin DC subsets are shown, as follows: LangerinEGFP+CD103 LC, LangerinEGFP+CD103+ dermal DC, and LangerinEGFP dermal DC; gray line, isotype control; black line, CD1d staining. Data are representative of four experiments. B, The percentage of CD1d+ cells in the various migratory skin DC subsets derived from epidermal or whole skin explants is shown. Summary of four to five experiments; line indicates mean. C, Emigrated skin DC were analyzed immediately (d0) or cultured for an additional day in GM-CSF (d1). Percentages of CD1d+ DC are shown as a summary from three experiments, mean ± SD. D, Migratory LC from epidermal explants or migratory skin DC from whole skin explants (mix of LC and dermal DC) were loaded overnight with 200 ng/ml α-GalCer. After washing the DC, graded numbers of LC/DC in triplicates were cocultured with 5 × 104 NKT hybridoma cells (DN32.D3) for 24 h. IL-2 concentrations in the supernatant were measured with ELISA. Result is representative of two experiments; mean ± SD for triplicates is shown.

FIGURE 2.

Skin DC express CD1d upon migration and present the synthetic glycolipid α-GalCer to NKT cells. A, Ear skin from LangerinEGFP mice was cultured on medium for 3 days, and emigrated DC were examined for CD1d surface expression. Dot plot show cells gated on CD11c. Three CD11c+ skin DC subsets are shown, as follows: LangerinEGFP+CD103 LC, LangerinEGFP+CD103+ dermal DC, and LangerinEGFP dermal DC; gray line, isotype control; black line, CD1d staining. Data are representative of four experiments. B, The percentage of CD1d+ cells in the various migratory skin DC subsets derived from epidermal or whole skin explants is shown. Summary of four to five experiments; line indicates mean. C, Emigrated skin DC were analyzed immediately (d0) or cultured for an additional day in GM-CSF (d1). Percentages of CD1d+ DC are shown as a summary from three experiments, mean ± SD. D, Migratory LC from epidermal explants or migratory skin DC from whole skin explants (mix of LC and dermal DC) were loaded overnight with 200 ng/ml α-GalCer. After washing the DC, graded numbers of LC/DC in triplicates were cocultured with 5 × 104 NKT hybridoma cells (DN32.D3) for 24 h. IL-2 concentrations in the supernatant were measured with ELISA. Result is representative of two experiments; mean ± SD for triplicates is shown.

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The fact that migratory skin DC expressed CD1d upon migration suggested that skin DC can present glycolipids to NKT cells. Thus, we studied the ability of migratory skin DC to activate the NKT cell hybridoma DN32.D3 (2). We compared mixed skin DC populations from whole skin explants, consisting of LC, dermal Langerin+ DC, and dermal DC, with migratory LC derived from epidermal explants. Graded doses of DC were loaded with 200 ng/ml α-GalCer overnight, washed, and cocultured with NKT hybridoma cells for 24 h. The activation of the NKT cells was measured by the release of IL-2 into the culture medium. Similar levels of IL-2 were induced by the different skin DC populations in this assay, although LC derived from epidermal explants expressed less CD1d on their surface (Fig. 2 D). Migratory skin DC that were not loaded with the synthetic glycolipid α-GalCer did not stimulate detectable IL-2 secretion by NKT cells, suggesting that they were not able to present endogenous glycolipid Ags derived from the skin that are recognized by the NKT cell hybridoma (data not shown). When we titrated the amount of α-GalCer, all skin DC were still able to present as little as 50 ng/ml α-GalCer to NKT cells, although IL-2 production decreased proportionally with the amount of α-GalCer used (data not shown).

In the skin-draining lymph nodes, nearly all CD11c+ DC express CD1d. The migratory skin DC can be identified by their high expression of MHC class II. They can be further separated into Langerin dermal DC and Langerin+ DC of epidermal origin (CD103), or dermal origin (CD103+). We observed CD1d expression on all skin DC subtypes in the draining lymph nodes as well as on resident lymph node DC defined as MHC class IIlow cells. The levels of CD1d expression on the lymph node skin DC were comparable to migratory DC obtained from skin explants, with dermal DC showing the highest levels of CD1d (Fig. 3 A).

FIGURE 3.

Migratory skin DC and lymph node DC can present intradermally injected α-GalCer to NKTs in vitro. A, Skin-draining lymph nodes from LangerinEGFP mice were digested with collagenase. Dot plots show cells gated on CD11c. CD11c+MHC IIlow lymph node-resident DC and CD11c+MHC IIhigh migratory skin DC were analyzed for CD1d expression. Migratory skin DC were divided into the following three subsets: LangerinEGFP+CD103 LC, LangerinEGFP+ CD103+ dermal DC, and LangerinEGFP dermal DC; thin gray line, isotype control (applies for both histograms); CD1d staining on various DC subsets as indicated by arrows. Data are representative of two experiments. B, We injected PBS or 1 μg of α-GalCer intradermally into mouse ear skin. From some of the mice, whole skin explants were prepared 4 h later and cultured for 3 days. From the remaining mice, auricular lymph nodes draining the immunization site were digested 48 h after glycolipid application and CD11c+ DC were isolated with CD11c+ microbeads. Graded numbers of emigrated skin DC or purified CD11c+ lymph node DC in triplicates were cocultured with 5 × 104 NKT hybridoma cells (DN32.D3) for 24 h. IL-2 concentrations in the supernatant were measured with ELISA. Result is representative of two experiments; mean ± SD is shown for triplicates. C, Migratory skin DC from whole skin explants and lymph node DC isolated from skin-draining lymph nodes from untreated mice were loaded with 200 ng/ml α-GalCer overnight in vitro. After washing, the DC were cocultured with the NKT cell hybridoma cells for 24 h, and the secretion of IL-2 was measured by ELISA. Result is representative for three experiments. Mean ± SD is shown for triplicates.

FIGURE 3.

Migratory skin DC and lymph node DC can present intradermally injected α-GalCer to NKTs in vitro. A, Skin-draining lymph nodes from LangerinEGFP mice were digested with collagenase. Dot plots show cells gated on CD11c. CD11c+MHC IIlow lymph node-resident DC and CD11c+MHC IIhigh migratory skin DC were analyzed for CD1d expression. Migratory skin DC were divided into the following three subsets: LangerinEGFP+CD103 LC, LangerinEGFP+ CD103+ dermal DC, and LangerinEGFP dermal DC; thin gray line, isotype control (applies for both histograms); CD1d staining on various DC subsets as indicated by arrows. Data are representative of two experiments. B, We injected PBS or 1 μg of α-GalCer intradermally into mouse ear skin. From some of the mice, whole skin explants were prepared 4 h later and cultured for 3 days. From the remaining mice, auricular lymph nodes draining the immunization site were digested 48 h after glycolipid application and CD11c+ DC were isolated with CD11c+ microbeads. Graded numbers of emigrated skin DC or purified CD11c+ lymph node DC in triplicates were cocultured with 5 × 104 NKT hybridoma cells (DN32.D3) for 24 h. IL-2 concentrations in the supernatant were measured with ELISA. Result is representative of two experiments; mean ± SD is shown for triplicates. C, Migratory skin DC from whole skin explants and lymph node DC isolated from skin-draining lymph nodes from untreated mice were loaded with 200 ng/ml α-GalCer overnight in vitro. After washing, the DC were cocultured with the NKT cell hybridoma cells for 24 h, and the secretion of IL-2 was measured by ELISA. Result is representative for three experiments. Mean ± SD is shown for triplicates.

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Having determined that skin DC express CD1d and can present the synthetic glyoclipid Ag α-GalCer to NKT cells in vitro, we next investigated whether skin DC can be loaded in situ by intradermal injection of α-GalCer. For these experiments, we injected either PBS or 1 μg of α-GalCer intradermally into mouse ears and examined the presentation of glycolipids by migratory skin DC from whole skin explant cultures initiated 4 h after injection. Skin DC that emigrated from explants over the next 3 days were then cocultured with the NKT cell hybridoma DN32.D3 for 24 h. We also isolated CD11c+ DC from skin-draining lymph nodes 2 days after intradermal injection of α-GalCer, and assessed their stimulatory capacity in the same assay. DC isolated from lymph nodes presented α-GalCer less efficiently than migratory skin DC most probably caused by loss of α-GalCer through inefficient recirculation through the lymph nodes (Fig. 3,B). To underline this, we compared the stimulatory capacity of migratory skin DC derived from skin explant culture and DC isolated from lymph nodes from untreated mice after loading them in vitro with α-GalCer. The two DC populations stimulated similar levels of IL-2 production by NKT cells after in vitro loading (Fig. 3 C). These results indicate that glycolipids can be acquired in the skin and presented by migratory DC, and in the lymph node, by lymph node-resident DC.

We observed that lymph nodes draining the site of α-GalCer injection were enlarged in most of the experiments. Thus, we further investigated the effect of α-GalCer on the number and activation status of lymphocytes and DC in lymph nodes and spleen after application into the skin compared with α-GalCer injected into the blood. We injected either PBS or 1 μg of α-GalCer intradermally into mouse ears or i.v. into the tail veins of mice and analyzed the skin, skin-draining lymph nodes, and the spleen 24 and 48 h later. First, we examined the skin for activation of skin DC. When LC become activated in the epidermis, they express higher levels of MHC class II, which is localized at the cell surface. The number of LC also decreases, which can be visualized by immunofluorescence staining of epidermal sheets. After injection of α-GalCer into the dermis, we could not detect activation of LC in the epidermis or induction of emigration of LC (Fig. 4,A). Flow cytometry analysis of the skin-draining lymph nodes confirmed that there is no significant induction of migration of DC. We were unable to measure statistically significant changes in the percentage and number of lymph node-resident DC and migratory skin DC (Fig. 4,B and data not shown). In contrast, the percentages of NKT cells in lymph nodes and spleen were decreased 48 h after intradermal injection of α-GalCer in comparison with PBS. NKT cells present in skin-draining lymph nodes of naive mice express lower levels of the activation marker CD69 compared with NKT cells in the spleen. Upon intradermal or i.v. injection of α-GalCer, CD69 expression was up-regulated on NKT cells in lymph node and spleen, indicating that α-GalCer does indeed exert some systemic effect after injection into the skin (Fig. 5,A). The absolute numbers of NKT, T, and B cells in the skin-draining lymph nodes and spleen were not significantly changed when we summarized three different experiments, although some activation of T and B cells in the lymph nodes and spleen was observed after administration of α-GalCer by either route, as reflected by enhanced CD69 expression. Interestingly, DC in the skin-draining lymph nodes did not show up-regulation of CD40 expression, which is typically observed on spleen DC after i.v. injection of α-GalCer (Fig. 5 B).

FIGURE 4.

α-GalCer injected into the skin does not activate and induce migration of skin DC. Mice were injected with PBS or 1 μg of α-GalCer intradermally into ear skin, and epidermal sheets and lymph node cell suspensions were prepared 48 h later. A, Immunofluorescence stainings of MHC-class II+ LC in untreated or treated epidermis are shown in an overview (upper row) and higher magnification (lower row). Result is representative for two experiments. B, Numbers of various DC subsets draining the immunization site were determined by multiplying the percentage derived from FACS analysis with total cell numbers per lymph node. The mean ± SD of three experiments is shown.

FIGURE 4.

α-GalCer injected into the skin does not activate and induce migration of skin DC. Mice were injected with PBS or 1 μg of α-GalCer intradermally into ear skin, and epidermal sheets and lymph node cell suspensions were prepared 48 h later. A, Immunofluorescence stainings of MHC-class II+ LC in untreated or treated epidermis are shown in an overview (upper row) and higher magnification (lower row). Result is representative for two experiments. B, Numbers of various DC subsets draining the immunization site were determined by multiplying the percentage derived from FACS analysis with total cell numbers per lymph node. The mean ± SD of three experiments is shown.

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FIGURE 5.

Intradermally injected α-GalCer induces activation of lymphocytes in lymph nodes and spleen. Mice were injected with PBS or 1 μg of α-GalCer intradermally into ear skin or i.v. into tail veins, and 24 and 48 h later auricular lymph nodes and spleens were analyzed. A, NKT cells were identified as CD3+ and CD1d-tetramer+ cells after excluding CD19+ B cells. Percentages of NKT cells of CD19 cells in lymph nodes and spleen after intradermal injection of α-GalCer are presented as a summary of three different experiments; mean ± SD is shown. CD69 expression on NKT cells is shown for lymph node and spleen in the bottom panel. B, In lymph nodes and spleen, we determined the absolute numbers of CD1d-tetramer+CD3+ NKT cells, CD3+ T cells, and CD19+ B cells by multiplying the percentages derived from FACS analysis with total cell numbers per lymph node or spleen. The mean ± SD of three experiments is shown. The activation status of CD3+ T and CD19+ B cells by CD69 expression and of CD11c+ DC by CD40 expression was determined by FACS stainings. FACS plots are shown for the 24-h time point and are representative of three experiments (bottom panel).

FIGURE 5.

Intradermally injected α-GalCer induces activation of lymphocytes in lymph nodes and spleen. Mice were injected with PBS or 1 μg of α-GalCer intradermally into ear skin or i.v. into tail veins, and 24 and 48 h later auricular lymph nodes and spleens were analyzed. A, NKT cells were identified as CD3+ and CD1d-tetramer+ cells after excluding CD19+ B cells. Percentages of NKT cells of CD19 cells in lymph nodes and spleen after intradermal injection of α-GalCer are presented as a summary of three different experiments; mean ± SD is shown. CD69 expression on NKT cells is shown for lymph node and spleen in the bottom panel. B, In lymph nodes and spleen, we determined the absolute numbers of CD1d-tetramer+CD3+ NKT cells, CD3+ T cells, and CD19+ B cells by multiplying the percentages derived from FACS analysis with total cell numbers per lymph node or spleen. The mean ± SD of three experiments is shown. The activation status of CD3+ T and CD19+ B cells by CD69 expression and of CD11c+ DC by CD40 expression was determined by FACS stainings. FACS plots are shown for the 24-h time point and are representative of three experiments (bottom panel).

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Concomitant application of α-GalCer with protein Ag i.v. strongly increases cytotoxic CD8+ T cell responses (7). We wondered whether intradermal administration of α-GalCer together with the protein Ag OVA would equally improve T cell responses. We measured the numbers of endogenous CD8+ T cells with a SIINFEKL/H-2Kb pentamer in the lymph nodes draining the immunization site and in the spleen 1 wk after intradermal or i.v. injection of Ag. More pentamer+ CD8+ T cells were found in the lymph nodes, but not the spleens, of mice that were immunized with OVA plus α-GalCer intradermally in contrast to mice immunized with OVA alone. Only limited accumulation of CD8+ T cells was observed in lymph nodes when OVA plus α-GalCer was injected i.v., although accumulation was very clearly evident in the spleen, highlighting potential differences in locality of T cell proliferation dependent upon immunization route (Fig. 6,A). Next, we examined the effector function of CD8+ T cells with in vivo killing assays. One week after intradermal immunization, mice were injected with OVA peptide-pulsed target spleen cells to assess cytotoxic capacity of the induced response. Mice that were immunized with OVA alone showed no cytotoxic response. In contrast, mice that received α-GalCer together with OVA showed potent cytotoxic activity in the lymph nodes draining the immunization site (Fig. 6 B) and in the blood, although to a slightly lower extent (data not shown), indicating systemic T cell effector responses.

FIGURE 6.

α-GalCer induces proliferation and effector function in CD8+ T cells after intradermal application. A, Groups of three mice were immunized with PBS or 250 μg of OVA ± 1 μg of α-GalCer intradermally or i.v. One week later, lymph nodes draining the immunization site and spleens were analyzed for the percentage of Ag-specific CD8+ T cells as detected with SIINFEKL/H-2Kb pentamer. Each data point represents one mouse from a total of two to three experiments; mean is indicated by the line; id, intradermal. ∗∗, p < 0.01. B, Groups of three mice were immunized with PBS or 250 μg of OVA ± 1 μg of α-GalCer intradermally. One week later, peptide-loaded CFSE-labeled spleen cells and unloaded CMTMR-labeled spleen cells were injected i.v. into immunized mice as target cells. Percentage of specific in vivo killing was determined 48 h later in lymph nodes. Each data point represents one mouse from a total of three experiments; mean is indicated by the line. ∗∗∗, p < 0.001.

FIGURE 6.

α-GalCer induces proliferation and effector function in CD8+ T cells after intradermal application. A, Groups of three mice were immunized with PBS or 250 μg of OVA ± 1 μg of α-GalCer intradermally or i.v. One week later, lymph nodes draining the immunization site and spleens were analyzed for the percentage of Ag-specific CD8+ T cells as detected with SIINFEKL/H-2Kb pentamer. Each data point represents one mouse from a total of two to three experiments; mean is indicated by the line; id, intradermal. ∗∗, p < 0.01. B, Groups of three mice were immunized with PBS or 250 μg of OVA ± 1 μg of α-GalCer intradermally. One week later, peptide-loaded CFSE-labeled spleen cells and unloaded CMTMR-labeled spleen cells were injected i.v. into immunized mice as target cells. Percentage of specific in vivo killing was determined 48 h later in lymph nodes. Each data point represents one mouse from a total of three experiments; mean is indicated by the line. ∗∗∗, p < 0.001.

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To date, we observed that α-GalCer injected intradermally together with a protein Ag induces potent cytotoxic CD8+ T cell responses. We were interested to determine whether this killing activity can be targeted against tumors growing in the skin. In a prophylactic setting, mice were immunized with PBS or OVA ± α-GalCer intradermally 1 wk before we challenged the mice s.c. with a melanoma cell line that expresses OVA as a tumor model Ag (B16.OVA). The coapplication of α-GalCer completely prevented tumor growth as compared with immunization with OVA alone (Fig. 7,A). In a therapeutic setting, we first challenged mice with B16.OVA tumor cells, and then 1 wk later, when tumors are just palpable, we immunized mice intradermally. Addition of α-GalCer could inhibit tumor growth and significantly prolong survival of mice in this setting (Fig. 7 B).

FIGURE 7.

Coadministration of α-GalCer into the skin improves antitumor response. A, Prophylactic setting: groups of five mice were immunized with PBS or 250 μg of OVA ± 1 μg of α-GalCer intradermally. One week after immunization, mice were injected with 105 B16.OVA melanoma cells s.c. into the flank. Percentage of survival of tumor mice is shown from two experiments, total of 10 mice each group. B, Therapeutical setting: groups of five mice were injected with 105 B16.OVA melanoma cells s.c. into the flank. One week later, mice were immunized with PBS or 250 μg of OVA ± 1 μg of α-GalCer intradermally. Percentage of survival of tumor mice is shown from two experiments, total of 10 mice each group. ∗, p < 0.05; ∗∗, p < 0.01.

FIGURE 7.

Coadministration of α-GalCer into the skin improves antitumor response. A, Prophylactic setting: groups of five mice were immunized with PBS or 250 μg of OVA ± 1 μg of α-GalCer intradermally. One week after immunization, mice were injected with 105 B16.OVA melanoma cells s.c. into the flank. Percentage of survival of tumor mice is shown from two experiments, total of 10 mice each group. B, Therapeutical setting: groups of five mice were injected with 105 B16.OVA melanoma cells s.c. into the flank. One week later, mice were immunized with PBS or 250 μg of OVA ± 1 μg of α-GalCer intradermally. Percentage of survival of tumor mice is shown from two experiments, total of 10 mice each group. ∗, p < 0.05; ∗∗, p < 0.01.

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In the course of our study, we have observed that α-GalCer injected intradermally can be picked up and presented by skin DC emigrating from skin explants (Fig. 3). We investigated whether migratory skin DC were directly involved in the activation and induction of effector function in CD8+ T cells following intradermal immunization. For this purpose, we used mice in which Langerin+ DC (including LC and dermal Langerin+ DC) can be depleted with a single injection of diphtheria toxin. In LangerinDTREGFP mice, the diphtheria toxin receptor is expressed under the Langerin promoter and Langerin+ cells in the skin and lymph nodes undergo apoptosis within 24 h of administration of the toxin (17). We also examined responses to intradermal immunization in animals in which the skin immunization site was removed 4 h after immunization. Removal of the skin immunization site within 12 h has been shown to abrogate the migration of skin DC to the draining lymph nodes and inhibits T cell responses (21, 22). Within this short time, only Ag that diffuses through lymph and blood can be presented by lymph node DC; however, contribution by newly migrated skin DC can be ruled out. In diphtheria toxin-injected LangerinDTREGFP mice, the LC were still absent 1 wk after intradermal immunization with Ag (Fig. 8,A). In mice in which Langerin+ cells were depleted, the number of pentamer+CD8+ T cells induced by intradermal immunization with OVA plus α-GalCer was slightly decreased relative to controls. In contrast, mice in which the immunization site was completely removed 4 h after injection of OVA plus α-GalCer did not show statistically significant different proliferation of OVA-specific CD8+ T cells when compared with the control mice (Fig. 8,B). In vivo cytotoxicity assays demonstrated that in both skin DC depletion models the ability to kill Ag-loaded targets was unchanged, suggesting that migratory skin DC are not absolutely required for the induction of effector function in CD8+ T cells (Fig. 8 C). Thus, whereas we have shown that skin DC are capable of stimulating NKT cells, our evidence suggests that the adjuvant function of NKT cells on conventional T cell responses following intradermal application of glycolipids is exerted predominantly by DC already present in the draining lymph node.

FIGURE 8.

Migratory skin DC are not required for induction of cytotoxic response in CD8+ T cells. Groups of three LangerinDTREGFP mice were injected with PBS (control) or 500 ng of diphtheria toxin to deplete Langerin+ DC (depleted) 1 day before intradermal immunization with 250 μg of OVA plus 1 μg of α-GalCer. Groups of three C57BL/6 mice were immunized intradermally, and 4 h later we removed the immunization site (removal). A, One week after immunization, we stained epidermal sheets with a mAb against MHC class II to visualize LC in PBS or diphtheria toxin-injected mice. This result is representative for all experiments performed. B, At day 7, we investigated the lymph nodes draining the immunization site for the presence of Ag-specific CD8+ T cells with a SIINFEKL/H-2Kb pentamer. Each data point represents one mouse from a total of two experiments; mean is indicated by the line; ∗, p < 0.05. C, At day 7 after immunization, we injected peptide-loaded CFSE-labeled spleen cells and unloaded CMTMR-labeled spleen cells i.v. Percentage of specific in vivo killing was determined 48 h later in lymph nodes. Each data point represents one mouse from a total of three experiments. Mean is indicated by the line.

FIGURE 8.

Migratory skin DC are not required for induction of cytotoxic response in CD8+ T cells. Groups of three LangerinDTREGFP mice were injected with PBS (control) or 500 ng of diphtheria toxin to deplete Langerin+ DC (depleted) 1 day before intradermal immunization with 250 μg of OVA plus 1 μg of α-GalCer. Groups of three C57BL/6 mice were immunized intradermally, and 4 h later we removed the immunization site (removal). A, One week after immunization, we stained epidermal sheets with a mAb against MHC class II to visualize LC in PBS or diphtheria toxin-injected mice. This result is representative for all experiments performed. B, At day 7, we investigated the lymph nodes draining the immunization site for the presence of Ag-specific CD8+ T cells with a SIINFEKL/H-2Kb pentamer. Each data point represents one mouse from a total of two experiments; mean is indicated by the line; ∗, p < 0.05. C, At day 7 after immunization, we injected peptide-loaded CFSE-labeled spleen cells and unloaded CMTMR-labeled spleen cells i.v. Percentage of specific in vivo killing was determined 48 h later in lymph nodes. Each data point represents one mouse from a total of three experiments. Mean is indicated by the line.

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NKT cells are a unique population of T cells able to link adaptive and innate immunity. They recognize glycolipid Ags on CD1d presented by APCs, which leads to activation of DC and increased T cell responses. As a result, CD1d-binding glycolipids have gained a lot of interest recently and are currently being tested as adjuvants for immunotherapy to potentiate immune responses against tumors and pathogens. In most of the studies performed to date, the synthetic glycolipid α-GalCer was applied by the i.v. route and could improve immune responses against a protein Ag (6, 7). We were interested to determine whether α-GalCer can be used for immunization strategies through the skin and whether skin DC are involved in this process. We report in this study that skin DC express CD1d and present glycolipids on their surface to NKT cells in vitro. Intradermally applied α-GalCer induced activation of NKT, T, and B cells in the skin-draining lymph nodes. Accordingly, cytotoxic CD8+ T cell responses were enhanced that could be targeted against melanoma. For the induction of this immune response, migratory skin DC were not mandatory because in their absence NKT cells could still enhance cytotoxic T cell responses.

Although we have shown that migratory skin DC are capable of stimulating NKT cells in vitro, our evidence suggests that intradermally applied glycolipids are actually presented to NKT cells by lymph node DC. This finding was surprising in consideration of our observation that skin DC express CD1d and can present glycolipid Ags to NKT cells. We detected CD1d on the surface of all three skin DC subsets, with highest levels on dermal DC when they migrated out of skin explants or were isolated from skin-draining lymph nodes. Furthermore, CD1d is expressed on LC in the epidermis of mouse skin and up-regulated during skin explant culture, a model of skin inflammation. Skin DC were capable of activating NKT cell hybridoma cells very efficiently after loading with the glycolipid α-GalCer in vitro. From previous studies, it is known that CD1d molecules are mainly expressed on hemopoietic cells, for example, B and T cells, as well as DC (23, 24). In human skin, CD1d is mainly expressed on dermal DC, but can be up-regulated on keratinocytes in skin disorders such as psoriasis (25, 26). Our results are in contrast to the study by Gerlini et al. (25), in which CD1d expression was detected on dermal DC, but not on LC in human skin; however, this could be explained by a species-specific difference. Our findings support the hypothesis that skin DC possess the required molecules and are capable of presenting glycolipids to NKT cells.

We tested our hypothesis by injecting glycolipids intradermally and examining whether migratory skin DC can capture α-GalCer in situ and present it to NKT cells in vitro. Glycolipids injected into the skin were indeed presented very efficiently by migratory skin DC to NKT cells, and to a lesser extent by lymph node DC. This difference is most probably caused by the disappearance of α-GalCer from the lymph node through blood and lymph circulation in this experimental setting given that lymph node-resident DC express equal levels of CD1d and present α-GalCer to NKT cells as efficiently as skin DC when loaded in vitro. To dissect the contribution of migratory skin DC and lymph node DC in gylcolipid Ag presentation, we removed the immunization site 4 h after intradermal injection of Ag and α-GalCer. DC subsets migrate with different kinetics to the draining lymph nodes in case of inflammation, as, for example, induced by application of 1% FITC onto the skin. The dermal DC arrive within the first day, and their number peaks on day 2 after FITC application. In contrast, LC need longer for the way to the lymph node, and their numbers peak after 3–4 days (our unpublished observation) (17, 27, 28). The removal of the immunization site within 4 h prevents migration of all skin DC subsets, LC, and dermal DC, to the lymphatic tissue for presentation of Ag in draining lymph nodes (21, 22). In addition, we used mice in which Langerin+ DC can be depleted by injection of a diphtheria toxin (LangerinDTREGFP mice (17)), leading to ablation of LC and Langerin+ dermal DC in the skin and lymph node. The number of induced Ag-specific CD8+ T cells was slightly decreased after depletion of Langerin+ DC, but not affected by the removal of the immunization site. A possible explanation for this discrepancy could be that with the diphtheria toxin Langerin+ DC are depleted not just in the skin, but also in the lymph node. The resulting decrease in total DC number in the lymph node could be significant, because it has been reported that Langerin+ DC represent ∼40% of all CD11c+ DC in the lymph node (17). In contrast, the removal of the injection site does not affect the number of lymph node DC, but only prevents the influx of newly arriving skin DC into the lymph node. In the same experimental settings, we also measured cytotoxic CD8+ T cell responses that revealed unchanged killing activity in mice lacking newly migrated skin DC indicating that lymph node DC are sufficient to induce effector function in CD8+ T cells. The explanation for this is most likely that the intradermally applied Ag diffuses to the lymph node in the first few hours after immunization and is incorporated and presented by lymph node DC. We conclude that intradermally injected α-GalCer as a potent adjuvant induces cytotoxic CD8+ T cell responses in the draining lymph nodes independent of newly migrated skin DC; hence, lymph node DC seem to be sufficient for this process. It has been shown that protein Ag deposited into the skin diffuses into the draining lymph nodes within the first few hours and is presented by lymph node DC. However, skin DC migrating to the lymphatic tissue after Ag application can take part in the Ag presentation process and are necessary to sustain the T cell response (22). The application of α-GalCer as adjuvant seems to overcome this need for contribution of skin DC and can sustain the T cell response. The lack of Ag presentation by migratory skin DC in our in vivo setting is most probably due to the absence of inflammatory signals in the skin. This is underlined by the fact that the synthetic glycolipid Ag α-GalCer does not induce inflammation in the skin, or migration of skin DC. In the steady state, very few DC migrate from the skin to the draining lymph nodes; however, after induction of an inflammation, more skin DC appear in the lymphatic tissue (29). For induction of long-lasting T cell responses, the skin DC need to be activated, which can be achieved by tape stripping or application of imiquimod-containing Aldara cream (30, 31, 32). Thus, the few steady state migratory skin DC that might take part in the Ag presentation process after intradermal injection of OVA plus α-GalCer can be substituted by lymph node DC.

Several lines of evidence suggest that i.p. applied α-GalCer activates NKT cells in the liver and spleen, inducing up-regulation of CD69 and down-regulation of the TCR, making it difficult to detect NKT cells with glycolipid-loaded CD1d tetramers (33, 34, 35). We asked the question whether intradermally injected α-GalCer exerts its effect systemically or locally in the skin-draining lymph nodes. In our study, we observed that α-GalCer applied into the skin activates NKT cells in draining lymph nodes and the spleen, pointing out a systemic effect of α-GalCer. The activation marker CD69 was up-regulated on NKT cells; however, the percentage of NKT cells in the skin-draining lymph nodes and spleen was decreased. This is most probably an effect of the described down-regulation of TCRs (33, 34, 35). NKT cells rapidly release cytokines and chemokines that trans activate various cell types and change their numbers and activation (3, 36). We detected up-regulation of CD69 on T and B cells in lymph nodes and spleen after intradermal injection of α-GalCer, but their numbers did not significantly change. DC in the spleen expressed higher levels of CD40 after intradermal and i.v. application of α-GalCer. In contrast, we could not detect up-regulation of CD40 on the various subsets of DC in the skin-draining lymph nodes regardless of the administration route of α-GalCer. It has been reported that injection of α-GalCer into the blood induces a transient maturation of spleen DC as demonstrated by up-regulation of costimulatory molecules such as CD40, CD80, and CD86, and MHC class II molecules (6, 7, 37). However, the phenotype of lymph node DC was hardly changed when glycolipid Ag was injected into the blood, which is in agreement with our results (6). One explanation could be that DC in the skin-draining lymph nodes, especially migratory skin DC, already express very high levels of costimulatory molecules that, in contrast to spleen DC, could not be further up-regulated by NKT cells (29, 38), and this level of expression may already be sufficient for DC-NKT cell interaction.

Several lines of evidence suggest that i.v. injection of α-GalCer together with a protein Ag improves CD8+ T cell responses. Simultaneous application of the protein Ag OVA with α-GalCer induced proliferation of OVA-specific CD8+ T cells mediated by spleen DC and enhanced cytotoxic activity in vivo that could be targeted against tumors (6, 7, 37). In our study, immunization through the skin stimulated proliferation of OVA-specific CD8+ T cells in the draining lymph nodes, whereas i.v. immunization increased numbers of OVA-specific CD8+ T cells in the spleen. Thus, OVA-specific T cell proliferation was restricted to the lymphatic organ draining the immunization site, although intradermal α-GalCer demonstrated a systemic activation effect on T and B cells and DC. In regard to effector function, CD8+ T cells activated by intradermal immunization exerted killing systemically because we could measure cytotoxic responses against target cells in lymph nodes draining the immunization site and in the blood when α-GalCer was added as adjuvant for immunization. We conclude that intradermally injected α-GalCer diffuses through the blood into distant organs such as the spleen, where NKT, T, and B cells, as well as DC become activated. However, proliferation of OVA-specific CD8+ T cells occurs exclusively in skin-draining lymph nodes not in the spleen. Thereafter, the expanded pool of activated effector T cells recirculates through the blood to other tissues, where they can kill target cells. These results are underlined by findings demonstrating that antigenic peptides and α-GalCer have to be presented by the same DC to stimulate enhanced CD8+ T cell responses (39). The site of activation of T cells might be important for immunotherapy of tumors, because T cells are imprinted with a site-specific signature of homing receptors. By stimulating T cell in skin-draining lymph nodes, the T cells are likely to be programmed to home to the skin, which is desirable for treatment of skin cancers like melanoma (40).

NKT cells are being harnessed for immunotherapy against cancer (41) because CD1d-binding glycolipids are very potent adjuvants for induction of CD8+ T cell responses when applied together with Ag i.v., i.p., intranasally, or orally (6, 7, 37, 42). Other studies revealed that α-GalCer could be attractive for immunization strategies against infectious diseases. One study showed that the i.p. and intradermal application of α-GalCer together with irradiated sporozoites of Plasmodium yoelii activated CD8+ T cells that protected from subsequent malaria infections (43). Another report demonstrated that intradermal application of α-GalCer together with a DNA vaccine enhanced protection against Leishmania major infection (44). We demonstrate in this study that the α-GalCer can be used for intradermal immunization against melanoma with a similar adjuvant effect as application through the blood, peritoneum, oral cavity, or mucosa (6, 7, 37, 42). Effector CD8+ T cells were able to prevent tumor growth completely in a prophylactic setting and delay tumor growth in a therapeutic setting with B16-melanoma expressing the protein Ag OVA. This opens new ways for immunization strategies through the skin against melanoma. The idea of skin immunization is to load skin-resident DC with Ags in situ because they are known to be very potent APCs (15, 16). We have refined and developed a new immunization strategy, called epicutaneous immunization, in which we apply protein Ag in a cream onto barrier-disrupted skin. The resulting long-lasting cytotoxic T cell responses could be targeted against s.c. growing tumors (31). The knowledge of the ability of skin DC to present glycolipids to NKT cells encouraged us to test α-GalCer in our skin immunization strategy. Preliminary observations indicate that antitumor responses are indeed improved in this setting. We expect that CD1d-binding glycolipids as adjuvant will prove to be effective in skin immunization strategies and help to improve immmunotherapies against cancer. Furthermore, early clinical trials have already demonstrated that either α-GalCer alone or α-GalCer-loaded DC can be administered safely, with some evidence of immune activation. Combining this activity with tumor-specific vaccines may be a promising new way to treat cancer patients (45, 46, 47, 48).

We thank Nina Dickgreber from the Malaghan Institute of Medical Research, the staff of Department of Dermatology and Venerology of the Innsbruck Medical University, and Vincenzo Cerundolo and Jonathan Silk from the University of Oxford for useful suggestions and discussion. We appreciate the provision of the B16.OVA cell line by Drs. Edith Lord and John G. Frelinger, as well as the NKT cell hybridoma by Steve Porcelli and Albert Bendelac. Furthermore, we are grateful to Adrien Kissenpfennig and Bernard Malissen for making the LangerinDTREGFP and LangerinEGFP mice available for this study. We very much appreciate the help of Franz Koch and Rochus Sonnweber for animal husbandry and care. We are particularly indebted to P. Fritsch, Head of Department, for his continued support and encouragement.

The authors have no financial conflict of interest.

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 research grants from Innsbruck Medical University (MFI-9442 to P.S.; IFTZ-11 to F.S.) and the Austrian Science Fund (FWF-L120-B13 to C.H.T.). Temporary support for C.H.T. came from Center of Excellence in Medicine and IT/Kompetenzzentrum Medizin Tirol, project 3b. I.F.H. was supported by a New Zealand Health Research Council Sir Charles Hercus Fellowship.

3

Abbreviations used in this paper: DC, dendritic cell; α-GalCer, α-galactosylceramide; CMTMR, chloromethyl-benzoyl-aminotetramethyl-rhodamine; DTR, diphtheria toxin receptor; EGFP, enhanced GFP; LC, Langerhans cell; Ep-CAM, epithelial cell adhesion molecule.

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