We could recently show that the TAM receptor Axl is specifically induced during LC differentiation from human umbilical cord blood CD34+ hematopoietic progenitor/stem cells (15). Axl mRNA was directly induced by TGF-β1 in LC progenitor cells (15). In a first step we verified that Axl mRNA levels remain elevated after the 7d differentiation process ( Figure 1A ). Secondly, we investigated the expression of the TAM receptor ligands Gas6 and Protein S. Both ligands were expressed in CD34⁺ cells and in cells cultivated 7 days with LC promoting cytokines (GM-CSF, Flt3L, SCF, TNFα) ± TGF-β1 under serum free conditions ( Figure 1B ). Gas6 was highly expressed in CD34⁺ cells with a marked down-modulation during the differentiation process ( Figure 1B ). However, detectable levels of Gas6 and Protein S were present in TGF-β1 supplemented cultures until the culture-endpoint ( Figure 1B ). Notably, the human KC cell line HaCaT expressed high amounts of Gas6, as also observed in human skin (15). We previously established that Axl expression precedes the classical human LC markers CD1a and CD207 (Langerin), thus pointing to a functional involvement of this receptor tyrosine kinase during the LC differentiation process (15). Using retroviral over-expression vectors harboring Axl and IRES-EGFP, we could further enhance Axl surface expression during LC differentiation ( Figure 1C ). We observed a significant enhancement of CD207⁺ cells from the GFP positive Axl expressing LC fraction as compared to the empty control vector ( Figure 1D , right bar diagram). GFP negative fractions remained unaltered and served as an internal control ( Figure 1D , left bar diagram). No CD207 expression was observed in cultures infected with Axl or empty vector control without TGF-β1 addition ( Figure 1E ). Thus, Axl supports TGF-β1 dependent LC differentiation in vitro.

Taken this positive effect of Axl over-expression on the LC differentiation, we speculated that vitamin K could influence the activity of the endogenous ligands Gas6 and Protein S, which are constitutively expressed in our LC culture system ( Figure 1B ). The functionality of Gas6 and Protein S is regulated by post-translational vitamin K-dependent carboxylation (16). Indeed, addition of vitamin K to human CD34⁺ cell based TGF-β1 dependent in vitro LC differentiation cultures resulted in enhanced generation of large LC clusters, an indication for a positive effect on LC generation ( Figure 2A ). Accordingly, FACS analyses revealed increased percentages of CD1a, CD324 (Ecadherin) and CD207 positive ( Figures 2B, C ). This positive effect on LC generation was strictly TGF-β1 dependent, as omitting TGF-β1 in vitamin K cultures did not induce LCs ( Figure 2C right graphs). The changes of LC specific surface markers (i.e. CD207 and CD1a) are also reflected on the mRNA level as measured by real time RT-PCR ( Figure 2D ). Therefore, vitamin K promotes LC generation in vitro.

Warfarin antagonizes the recycling of endogenous vitamin K in the cell, thus inactivating vitamin K-dependent proteins (e.g. blood coagulation factors) (17). Expectedly, the addition of warfarin to LC cultures resulted in the opposite effect of vitamin K supplementation: LC numbers (CD207⁺ cells) were diminished; additionally the surface expression of CD1a was reduced ( Figure 2E ). Thus, vitamin K-dependent processes favor the development of human LCs in vitro.

Given the positive effect of vitamin K on LC generation and the here identified expression of the vitamin K-dependent receptor-ligand system Axl-Gas6-Protein S during LC differentiation ( Figures 1 , 2 ), we next investigated its direct involvement. We used a blocking antibody to silence Axl during human in vitro LC differentiation (15, 18). Antibodies against Axl, administered during the in vitro differentiation, efficiently blocked the positive effect of vitamin K on LC generation ( Figures 3A, B ). While warfarin reduced CD1a surface expression, vitamin K enhanced the levels of CD1a on LCs in an Axl dependent manner (compare Figures 2E , 3B , lower graph). Thus, TGF-β1 promotes in vitro LC differentiation through the early up-regulation of Axl, which is activated by its endogenous vitamin K-dependent ligands in these cultures.

Both Axl and its ligands (Gas6 and Protein-S) are implicated to have anti-inflammatory effects (19). We therefore investigated the impact of vitamin K on co-stimulatory molecule expression by in vitro generated LCs. Whereas vitamin K enhanced the expression of CD207 and CD1a ( Figure 2 ), the surface levels of the costimulatory molecule CD86, which is necessary for T-cell activation and survival, were significantly diminished ( Figure 4A ). Interestingly, this effect was Axl independent, suggesting a broader anti-inflammatory role of vitamin K in LCs ( Figure 4A ). Furthermore, the addition of vitamin K to LCs prior to their stimulation with TLR-ligands (e.g. LPS) or cytokines (e.g. TNFα) led to a blunted up-regulation of CD86 ( Figure 4B ).

In summary, these data indicate that vitamin K enhances LC differentiation, through Axl activation downstream of TGF-β1 signaling, while it simultaneously counteracts LC activation ( Figure 5 ).

Cord blood samples from healthy donors were collected during healthy full-term deliveries. CD34⁺ cells were isolated as described (26). CD14⁺ monocytes were isolated from peripheral blood of healthy donors as described (26). All procedures were carried out in accordance to the guidelines from the Medical University of Vienna Institutional Review Board for these studies. Informed consent was provided in accordance with the Declaration of Helsinki Principles.

Human stem-cell factor (SCF), thrombopoietin (TPO), tumor necrosis factor alpha (TNFα), granulocyte-macrophage colony-stimulating factor (GM-CSF), and fms-related tyrosine kinase 3 ligand (FLT3L) were obtained from PeproTech; transforming growth factor beta 1 (TGF-β1), interferon-alpha (IFN-α), mouse GM-CSF from Akron Biotech. Ultrapure LPS from Escherichia coli and Pam₃CSK₄ was purchased from InvivoGen.

CD34⁺ cord blood cells were cultured serum-free for 2–3 days under progenitor expansion conditions (Flt3L, SCF and TPO, each at 50 ng/ml) before sub-culturing with lineage-specific cytokines. LC cultures were described before (27). Briefly, CD34⁺ cells (5 × 10⁴ to 1 × 10⁵/ml per well) were cultured in 24-well tissue culture plates in serum-free CellGro DC medium (CellGenix) supplemented with GM-CSF (100 ng/ml), SCF (20 ng/ml), Flt3 (50 ng/ml), TNF-α (2.5 ng/ml) and TGF-β1 (1 ng/ml) for 7 days. Cultures were supplemented with GlutaMAX (2.5 mM; Gibco/Invitrogen) and penicillin/streptomycin (125 U/mL each). Vitamin K (Konakion, Roche) was added at 1μg/ml as indicated. Warfarin (Sigma) was added at 50 μg/ml when indicated. Axl blocking antibody from R&D systems (AF154) was used at a concentration of 5 μg/ml when indicated.

The AXL-IRES-GFP retroviral vector was a kind gift from Axel Ullrich (Max Planck Institute of Biochemistry, Martinsried, Germany). Transfection of packaging cell line phoenix-GP as well as infection of target cells was performed as previously described (28). In brief, to produce recombinant amphotropic retrovirus, vectors were transiently transfected into the packaging cell line Phoenix-GP (Gag-Pol) using a calcium-phosphate protocol (28). Phoenix-GP was cotransfected with an expression plasmid encoding gibbon ape leukemia virus (GALV) envelope (gift from D.B. Kohn, University of California, Los Angeles, Los Angeles, CA). Before gene transduction, fresh or thawed CD34⁺ cells were stimulated overnight in X-VIVO 15 medium supplemented with the cytokines SCF (50 ng/ml), FLT3L (50 ng/ml), and TPO (50 ng/ml). Afterwards, 1 ml of retroviral supernatant (harvested 36–48 h after transfection of packaging cells) was added to 4 × 10⁴ CD34⁺ HPCs in the presence of plate-bound RetroNectin (Takara Bio Inc.) using nontissue culture–treated 24-well plates (Cellstar; Greiner Bio-One GmbH) according to the instructions of the manufacturer. Infections were repeated two to three times at intervals of 12–24 h using fresh virus supernatants in the presence of cytokines SCF, FLT3L, and TPO. Within 60 h of the first transduction cycle, cells were harvested and re-cultured in LC lineage conditions.

RNA was isolated using RNeasy Mini Kit (Qiagen). 1 μg of RNA was reverse transcribed using Transcriptor First Strand cDNA Kit (Roche) and oligo(dT) primers. Quantitative PCR was run on Roche LightCycler (human samples) using the SYBR Green PCR Master Mix (Applied Biosystems) and indicated primers at 125 nM concentration. Results were analyzed using delta delta Ct method and presented as a fold of difference in mRNA level relative to HPRT (human samples) (29).

Flow cytometry staining and analysis were performed as described (28). Monoclonal antibodies (mAbs) of the following specificities were used: FITC-conjugated mAbs specific for CD1a, CD86 (BD Biosciences), Phycoerythrin (PE)-conjugated mAbs specific for CD207 (Immunotech), Allophycocyanin-conjugated Abs against CD1a (BD Biosciences) and CD324 (Biolegend); Pacific Blue conjugated mAbs against CD1a (Biolegend); biotinylated mAbs specific for CD86 (BD Biosciences). Second step reagent was streptavidin (SA)-PerCP (BD Biosciences). Axl was detected using mouse mAbs (R&D systems) followed with a PE conjugated anti mouse second step Ab (Dako). Flow cytometric analysis was performed using a LSRII instrument (BD Biosciences) and the FloJo software (Tree Star, Inc.).

Cells were lysed in lysis buffer containing 50 mM Tris-HCl pH 7.5, 1 mM EGTA, 1 mM EDTA, 1% (w/v) Triton X-100, 0.27 M sucrose, 0.1% 2-mercaptoethanol, protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Roche). Protein concentration was measured using Bradford reagent (Bio-Rad) and equal amount of protein (5 μg) in LDS sample buffer (Invitrogen) were subjected to electrophoresis on a polyacrylamide gel and transferred to PVDF membranes (Millipore). Membranes were blocked in TBS-T (50 mM Tris-HCl pH 7.5, 0.15M NaCl, and 0.25% (v/v) Tween-20) containing in 5% (w/v) BSA. The membranes were then immunoblotted overnight at 4°C with primary antibodies diluted 1000-fold in blocking buffer. The blots were washed six times with TBS-T and incubated for 1 h at room temperature with secondary HRP-conjugated antibodies diluted 5000-fold in 5% (w/v) skimmed milk in TBS-T. After repeating the washing steps, signal was detected with the enhanced chemiluminescence reagent and immunoblots were developed using an automatic film processor.

If not specified in figure legends, statistical analysis was performed using the paired or unpaired, 2-tailed Student t test; p-values of less than 0.05 were considered significant.