Targeted delivery of a vaccine protein to Langerhans cells in the human skin via the C-type lectin receptor Langerin.

Human skin is a preferred vaccination site as it harbors multiple dendritic cell (DC) subsets, which display distinct C-type lectin receptors (CLR) that recognize pathogens. Antigens can be delivered to CLR by antibodies or ligands to boost antigen-specific immune responses. This concept has been established in mouse models but detailed insights into the functional consequences of antigen delivery to human skin DC in situ are sparse. In this study, we cloned and produced an anti-human Langerin antibody conjugated to the EBV nuclear antigen 1 (EBNA1). We confirmed specific binding of anti-Langerin-EBNA1 to Langerhans cells (LC). This novel LC-based vaccine was then compared to an existing anti-DEC-205-EBNA1 fusion protein by loading LC in epidermal cell suspensions before coculturing them with autologous T cells. After restimulation with EBNA1-peptides, we detected elevated levels of IFN-γ- and TNF-α-positive CD4+ T cells with both vaccines. When we injected the fusion proteins intradermally into human skin explants, emigrated skin DC targeted via DEC-205-induced cytokine production by T cells, whereas the Langerin-based vaccine failed to do so. In summary, we demonstrate that antibody-targeting approaches via the skin are promising vaccination strategies, however, further optimizations of vaccines are required to induce potent immune responses.

Peptides from allergenic lipocalins bind to formyl peptide receptor 3 in human dendritic cells to mediate TH2 immunity.

BACKGROUND: How TH2-mediated allergic immune responses are induced is still under investigation. OBJECTIVE: In an in vitro system we compared the effect of lipocalin allergens and nonallergenic homologues on human monocyte-derived dendritic cells (DCs) to investigate how they polarize naive CD4+ TH cells. Microarray data gained with these DCs showed a significant difference in expression of formyl peptide receptors (FPRs). Activation of FPR3 in human monocyte-derived DCs leads to inhibition of IL-12 production. Low concentrations of IL-12 during T-cell priming biases immune responses toward TH2. We hypothesize that binding of allergenic lipocalins to FPR3 might be a mechanism for induction of allergic immune responses. METHODS: We examined whether lipocalins and FPR3 colocalize within the cells by using confocal microscopy. With calcium mobilization assays of FPR3-transfected HEK 293 cells, we measured FPR3 signaling in response to allergenic and nonallergenic lipocalins. Silencing of FPR3 in DCs and pretreatment with an antagonistic peptide were used to assess the function of FPR3 in TH2 induction. RESULTS: FPR3 and lipocalins colocalize in the same vesicles in DCs. Cathepsin S-digested allergenic lipocalins, but not digestion products of nonallergenic homologues, activate FPR3 signaling. FPR3 silencing in DCs or pretreatment with an antagonistic peptide restores IL-12 and induces IL-10 expression by DCs treated with lipocalin allergens, attenuating the TH2 bias and inducing IL-10 production in cocultured TH cells. CONCLUSION: We describe a novel molecular mechanism for induction of TH2-mediated allergic immune responses.

Allergenic Can f 1 and its human homologue Lcn-1 direct dendritic cells to induce divergent immune responses.

Why and when the immune system skews to Th2 mediated allergic immune responses is still poorly characterized. With two homologous lipocalins, the major respiratory dog allergen Can f 1 and the human endogenous, non-allergenic Lipocalin-1, we investigated their impact on human monocyte-derived dendritic cells (DC). The two lipocalins had differential effects on DC according to their allergenic potential. Compared to Lipocalin-1, Can f 1 persistently induced lower levels of the Th1 skewing maturation marker expression, tryptophan breakdown and interleukin (IL)-12 production in DC. As a consequence, T cells stimulated by DC treated with Can f 1 produced more of the Th2 signature cytokine IL-13 and lower levels of the Th1 signature cytokine interferon-γ than T cells stimulated by Lipocalin-1 treated DC. These data were partially verified by a second pair of homologous lipocalins, the cat allergen Fel d 4 and its putative human homologue major urinary protein. Our data indicate that the crosstalk of DC with lipocalins alone has the potential to direct the type of immune response to these particular antigens. A global gene expression analysis further supported these results and indicated significant differences in intracellular trafficking, sorting and antigen presentation pathways when comparing Can f 1 and Lipocalin-1 stimulated DC. With this study we contribute to a better understanding of the induction phase of a Th2 immune response.

In human monocyte derived dendritic cells SOCS1 interacting with CYTIP induces the degradation of CYTIP by the proteasome.

CYTIP (cytohesin interacting protein) is an intracellular molecule induced in dendritic cells during maturation. CYTIP modulates the binding intensity of the adhesion molecule LFA-1. If dendritic cells are silenced for CYTIP they keep longer contacts with T-cells resulting in a lower T cell stimulation. We identified Suppressor of cytokine signaling-1 (SOCS-1) as a binding partner for CYTIP in human monocyte derived dendritic cells. In Western blot analyses we found that CYTIP expression is down regulated at later time points, starting at about 72 hours after induction of maturation. To investigate a possible role for SOCS-1 in taking CYTIP to the degradation machinery of the cell we measured endogenous CYTIP protein levels in mature dendritic cells transfected with SOCS-1 encoding plasmid in quantitative Western blot analyses. We observed lower amounts of endogenous CYTIP in mature dendritic cells transfected with SOCS-1 encoding plasmid compared with untransfected dendritic cells. Experiments with the proteasome-inhibitor Bortezomib/Velcade® show stable CYTIP expression levels in dendritic cells. In addition, we show that CYTIP in dendritic cells matured for 48 hours is ubiquitinated and thus ready for degradation. We here describe a newly identified binding partner of CYTIP, SOCS-1, and confirm its function in regulating the degradation of CYTIP by the proteasome.

Cytip regulates dendritic-cell function in contact hypersensitivity.

Cytohesin-interacting protein (Cytip) is induced during dendritic cell (DC) maturation and in T cells upon activation. It has also been shown to be involved in the regulation of immune responses. Here, we evaluated the functional consequences of Cytip deficiency in DCs using Cytip knockout (KO) mice. No difference in DC subpopulations in the skin draining lymph nodes (LNs) was found between Cytip KO mice and their wild-type counterparts, excluding a role in DC development. To investigate the function of Cytip in DCs in vivo, we used 2,4,6-trinitrochlorobenzene (TNCB)-induced contact hypersensitivity (CHS) as a model system. In the sensitization as well as in the elicitation phase, DCs derived from Cytip KO mice induced an increased inflammatory reaction indicated by more pronounced ear swelling. Furthermore, IL-12 production was increased in Cytip KO bone marrow-derived DCs (BMDCs) after CpG stimulation. Additionally, Cytip-deficient DCs loaded with ovalbumin induced stronger proliferation of antigen-specific CD4(+) and CD8(+) T cells in vitro. Finally, migration of skin DCs was not altered after TNCB application due to Cytip deficiency. Taken together, these data suggest a suppressive function for Cytip in mouse DCs in limiting immune responses.

The adaptor protein Bam32 in human dendritic cells participates in the regulation of MHC class I-induced CD8+ T cell activation.

The B lymphocyte adaptor molecule of 32 kDa (Bam32) is strongly induced during the maturation of dendritic cells (DC). Most known functions of Bam32 are related to the signaling of the B cell receptor for Ag. Because DC do not express receptors specific for Ags, we aim at characterizing the role of Bam32 in human monocyte-derived DC in this study. Our results show that binding of allogeneic T cells to mature DC causes accumulation of Bam32 on the contact sites and that this translocation is mimicked by Ab-mediated engagement of MHC class I. Silencing of Bam32 in mature monocyte-derived DC results in an enhanced proliferation of CD8(+) T cells in an Ag-specific T cell proliferation assay. Further studies identify galectin-1 as an intracellular binding partner of Bam32. Regulating immune responses via regulatory T cell (Treg) modulation is one of the many immunological activities attributed to galectin-1. Therefore, we assayed mixed leukocyte reactions for Treg expansion and found fewer Treg in reactions stimulated with DC silenced for Bam32 compared to reactions stimulated with DC treated with a nontarget control. Based on our findings, we propose a role for Bam32 in the signaling of MHC class I molecules in professional Ag-presenting DC for the regulation of CD8(+) T cell activation. It is distinct from that of MHC class I recognized by CD8(+) T cells leading to target [corrected] cell death. Thus, our data pinpoint a novel level of T cell regulation that may be of biological relevance.

Cybr, CYTIP or CASP: an attempt to pinpoint a molecule's functions and names.

Over the last decade several groups, including ourself, have published a series of findings on a molecule expressed in leukocytes. The molecule was termed Cybr, CYTIP or CASP for its functions and PSCDBP for its binding properties. In this review we attempt to chronicle and combine the findings on the molecule to gain an overview of its features.

Sphingosine-1-phosphate receptor type-1 agonism impairs blood dendritic cell chemotaxis and skin dendritic cell migration to lymph nodes under inflammatory conditions.

SEW2871 is a potent sphingosine-1-phosphate receptor type-1 (S1P(1))-selective agonist that induces peripheral lymphopenia through sequestration of lymphocytes into secondary lymphoid organs, similar to the non-selective sphingosine-1-phosphate (S1P) receptor agonist FTY720. FTY720 has been reported to interfere with human dendritic cell (DC) effector functions and both FTY720 and SEW2871 have been shown to modulate murine DC trafficking in vivo. Little is known about the possible effects of SEW2871 on human and murine DC functions. Here, we demonstrate that in contrast to FTY720, SEW2871 does not induce down-regulation of S1P(1) in human DCs and thus does not exert a functional antagonism at S1P(1). Notably, the compound was found to impair chemotaxis of immature and mature human DCs in vitro, possibly by interfering with the activation of p44/p42 and p38 mitogen-activated protein kinase signaling pathways. Comparative FACS analyses show that SEW2871 mediates CD18 down-regulation on mature human DCs. The influence on DC migration could be confirmed with in vivo assays using BALB/c mice in which SEW2871 impairs the migration of CD11c+ DC and CD207+ Langerhans cells (LC) to the draining lymph nodes (LNs) under inflammatory conditions. These results suggest that the S1P-S1P(1) axis might not only control lymphocyte trafficking but also play a pivotal role in DC migration from the skin to LN.

Dendritic cells regulate T-cell deattachment through the integrin-interacting protein CYTIP.

When T cells are primed by dendritic cells (DCs) to initiate antigen-specific immune responses screening for matching antigen receptor-MHC/peptide pairs takes place in DC-T-cell conjugates. For an immune response DC-T-cell conjugates formed during priming events need to dissolve. Although detailed knowledge on molecules involved in the conjugate formation is available, dissolving of them has not been considered to be an active process. Here, we identify CYTIP (cytohesin-interacting protein) to mediate DC-T-cell deattachment. CYTIP, which is induced during maturation of DCs, shortly accumulates to the contact zones with T cells within the first hour of coculture. Specific silencing of CYTIP results in stronger adhesion of DCs to T cells and to fibronectin. When a need for deattachment is created in a T-cell priming assay by only partially loading DCs with antigen, CYTIP silencing causes reduced priming capacity. Thus, CYTIP allows DCs to actively control DC-T-cell interactions.

Langerhans cells are strongly reduced in the skin of transgenic mice overexpressing follistatin in the epidermis.

Activins are members of the transforming growth factor-beta (TGF-beta) family and are important for skin morphogenesis and wound healing. TGF-beta1 is necessary for the population of the epidermis with Langerhans cells (LC). However, a role for activin in LC biology is not known. To address this question, we analyzed skin from transgenic mice overexpressing the activin antagonist follistatin in the epidermis. Using immunofluorescence, we observed a striking decrease in the number of LC in the epidermis of transgenic mice in comparison to wild-type mice. Nevertheless, these LC expressed normal levels of major histocompatibility complex (MHC)-class II and Langerin/ CD207 in situ. In explant cultures of whole ear skin the number of dendritic cells (DC), which migrated into the culture medium, was reduced. This reduction was even more pronounced in cultures of epidermal sheets. Virtually all emigrated cutaneous DC displayed typical morphology with cytoplasmic "veils", showed translocation of MHC-class II to the surface membrane, and expressed the maturation marker 2A1. Thus, cutaneous DC from transgenic mice seemed to mature normally. These results demonstrate that overexpression of follistatin in the epidermis affects LC trafficking but not maturation and suggest a novel role of the follistatin-binding partner activin in LC biology.

Tetrahydro-4-aminobiopterin attenuates dendritic cell-induced T cell priming independently from inducible nitric oxide synthase.

Formation of NO by NO synthases (NOSs) strictly depends on tetrahydrobiopterin. Its structural analog, tetrahydro-4-aminobiopterin, is an inhibitor of all NOS isoenzymes, which prolongs allograft survival in acute murine cardiac rejection and prevents septic shock in the rat. In this study, we show that murine bone marrow-derived dendritic cells treated with tetrahydro-4-aminobiopterin had a reduced capacity to prime alloreactive murine T cells in oxidative mitogenesis. Checking for a possible influence on LPS-induced dendritic cell maturation, we found that tetrahydro-4-aminobiopterin down-regulated MHC class II expression and counteracted LPS-induced down-regulation of ICOS ligand, while expression of CD40, CD86, CD80, B7-H1, and B7-DC remained unchanged. Tetrahydro-4-aminobiopterin also reduced activation of CD4(+) T cells isolated from mice overexpressing an OVA-specific TCR by OVA-loaded murine bone marrow-derived dendritic cells, thus indicating that its effect on MHC class II expression is involved in attenuating T cell activation. In line with affecting dendritic cell function and T cell activation, tetrahydro-4-aminobiopterin impaired production of proinflammatory cytokines and the Th1 response. With regard to cell survival, tetrahydro-4-aminobiopterin induced efficient apoptosis of murine T cells but not of murine dendritic cells. Experiments with cells from inducible NOS (iNOS) knockout mice and with N(6)-(1-iminoethyl)-L-lysine, a specific inhibitor of iNOS, ruled out participation of iNOS in any of the observed effects. These findings characterize attenuation of T cell stimulatory capacity of murine bone marrow-derived dendritic cells as an immunosuppressive mechanism of tetrahydro-4-aminobiopterin that is not related to its iNOS-inhibiting properties.

The immunomodulator FTY720 interferes with effector functions of human monocyte-derived dendritic cells.

The potent immunomodulator FTY720 elicits immunosuppression via acting on sphingosine 1-phosphate receptors (S1PR), thereby leading to an entrapment of lymphocytes in the secondary lymphoid tissue. To elucidate the potential in vitro effects of this drug on human monocyte-derived DC, we used low nanomolar therapeutic concentrations of FTY720 and phosphorylated FTY720 (FTY720-P) and investigated their influence on DC surface marker expression, protein levels of S1PR and DC effector functions: antigen uptake, chemotaxis, cytokine production, allostimulatory and Th-priming capacity. We report that both FTY720 and FTY720-P reduce chemotaxis of immature and mature DC. Mature DC generated in the presence of FTY720 or FTY720-P showed an impaired immunostimmulatory capacity and reduced IL-12 but increased IL-10 production. T cells cultured in the presence of FTY720- or FTY720-P-treated DC showed an altered cytokine production profile indicating a shift from Th1 toward Th2 differentiation. In treated immature and mature DC, expression levels for two S1PR proteins, S1P1 and S1P4, were reduced. We conclude that in vitro treatment with FTY720 affects DC features that are essential for serving their role as antigen-presenting cells. This might represent a new aspect of the overall immunosuppressive action of FTY720 and makes DC potential targets of further sphingolipid-derived drugs.

In vitro treatment of dendritic cells with tacrolimus: impaired T-cell activation and IP-10 expression.

BACKGROUND: High doses (10(-6)-10(-8) M) of tacrolimus (FK506) were reported to induce a type-2 T-helper cell (Th2)-promoting function in developing dendritic cells (DC). We used a therapeutic dose (2.4 x 10(-9 )M) of tacrolimus to investigate its effect on human monocyte-derived DC. METHODS: Using untreated and treated immature and mature DC we compared T cell-activating capacity, surface marker expression, T cell and DC cytokine profile and transcription of genes coding for a panel of DC function-related molecules. RESULTS: Tacrolimus-treated mature DC had reduced T-cell stimulatory capacity. Although interleukin (IL)-12 production of DC was impaired, they did not promote Th2 development as T cells activated by tacrolimus-treated DC produced less interferon (IFN)-gamma, IL-4 and IL-10. The up-regulation of the T-cell activation marker CD69 and the production of IL-2 were impaired. In addition, tacrolimus-treated DC produced less IP-10 (CXCL10), which is known to be involved in allograft rejection. Other molecules related to DC function remained unchanged. CONCLUSIONS: Tacrolimus treatment reduces the ability of DC to stimulate T cells and the impaired production of DC-derived IP-10 (CXCL10) and IL-12 might play a role in the immunosuppressive action of tacrolimus.

Adenosine slows migration of dendritic cells but does not affect other aspects of dendritic cell maturation.

We report the induction and reduction of adenosine receptor A2a and A3 mRNAs, respectively, during maturation of human monocyte-derived dendritic cells. Adenosine, an immunomodulatory molecule, is unstable in vitro; therefore we tested a stable agonist, 5'-(N-ethylcarboxamido)-adenosine, to explore the effect of adenosine receptor activation on dendritic cell function. We clearly show that adenosine receptor engagement affects the migratory activity of dendritic cells in three distinct settings. In human skin explant culture experiments the emigration of epidermal and dermal dendritic cells was diminished by the addition of 5'-(N-ethylcarboxamido)-adenosine. In a murine contact hypersensitivity assay 5'-(N-ethylcarboxamido)-adenosine caused a reduction in the numbers of epidermal and dermal dendritic cells arriving in the draining lymph node. In a chemotaxis assay of human dendritic cells in response to macrophage inflammatory protein 3beta (MIP-3beta)/CCL19, adenosine caused a delay in transmigration. Expression of a number of molecules involved in dendritic cell migration (CCR5, MIP-3beta/CCL19, and MDR-1) was reduced. Importantly, all other features of dendritic cells tested--phenotype, antigen uptake, cytokine production, T cell activation, and the T cell subset induction--remained unchanged. Dendritic cells carry antigens from the periphery to secondary lymphoid organs, where initiation of immune responses occurs. Increased adenosine release may modulate immune responses by delaying the encounter of antigen-loaded dendritic cells with T cells.

Low tetrahydrobiopterin biosynthetic capacity of human monocytes is caused by exon skipping in 6-pyruvoyl tetrahydropterin synthase.

Biosynthesis of (6 R )-5,6,7,8-tetrahydro-L-biopterin (H(4)-biopterin), an essential cofactor for aromatic amino acid hydroxylases and NO synthases, is effectively induced by cytokines in most of the cell types. However, human monocytes/macrophages form only a little H(4)-biopterin, but release neopterin/7,8-dihydroneopterin instead. Whereas 6-pyruvoyl tetrahydropterin synthase (PTPS) activity, the second enzyme of H(4)-biopterin biosynthesis, is hardly detectable in these cells, PTPS mRNA levels were comparable with those of cell types containing intact PTPS activity. By screening a THP-1 cDNA library, we identified clones encoding the entire open reading frame (642 bp) as well as clones lacking the 23 bp exon 3, which results in a premature stop codon. Quantification of the two mRNA species in different cell types (blood-derived cells, fibroblasts and endothelial cells) and cell lines showed that the amount of exon-3-containing mRNA is correlated closely to PTPS activity. The ratio of exon-3-containing to exon-3-lacking PTPS mRNA is not affected by differential mRNA stability or nonsense-mediated mRNA decay. THP-1 cells transduced with wild-type PTPS cDNA produced H(4)-biopterin levels and expressed PTPS activities and protein amounts comparable with those of fibroblasts. We therefore conclude that exon 3 skipping in transcription rather than post-transcriptional mechanisms is a major cause of the low PTPS protein expression observed in human macrophages and related cell types.

Attenuation of cell adhesion in lymphocytes is regulated by CYTIP, a protein which mediates signal complex sequestration.

An important theme in molecular cell biology is the regulation of protein recruitment to the plasma membrane. Fundamental biological processes such as proliferation, differentiation or leukocyte functions are initiated and controlled through the reversible binding of signaling proteins to phosphorylated membrane components. This is mediated by specialized interaction modules, such as SH2 and PH domains. Cytohesin-1 is an intracellular guanine nucleotide exchange factor, which regulates leukocyte adhesion. The activity of cytohesin-1 is controlled by phospho inositide-dependent membrane recruitment. An interacting protein was identified, the expression of which is upregulated by cytokines in hematopoietic cells. This molecule, CYTIP, is also recruited to the cell cortex by integrin signaling via its PDZ domain. However, stimulation of Jurkat cells with phorbol ester results in re-localization of CYTIP to the cytoplasm, and membrane detachment of cytohesin-1 strictly requires co-expression of CYTIP. Consequently, stimulated adhesion of Jurkat cells to intracellular adhesion molecule-1 is repressed by CYTIP. These findings outline a novel mechanism of signal chain abrogation through sequestration of a limiting component by specific protein-protein interactions.

A novel role for IL-3: human monocytes cultured in the presence of IL-3 and IL-4 differentiate into dendritic cells that produce less IL-12 and shift Th cell responses toward a Th2 cytokine pattern.

Dendritic cells (DC) derived from plasmacytoid precursors depend on IL-3 for survival and proliferation in culture, and they induce preferentially Th2 responses. Monocytes express not only GM-CSF receptors, but also IL-3Rs. Therefore, we examined whether IL-3 had an effect on the functional plasticity of human monocyte-derived DC generated in a cell culture system that is widely used in immunotherapy. DC were generated with IL-3 (instead of GM-CSF) and IL-4. Yields, maturation, phenotype (surface markers and Toll-like receptors), morphology, and immunostimulatory capacity were similar. Only CD1a was differentially expressed, being absent on IL-3-treated DC. In response to CD40 ligation DC generated in the presence of IL-3 secreted significantly less IL-12 p70 and more IL-10 compared with DC grown with GM-CSF. Coculture of naive allogeneic CD4(+) T cells with DC generated in the presence of IL-3 induced T cells to produce significantly more IL-5 and IL-4 and less IFN-gamma compared with stimulation with DC generated with GM-CSF. These data extend the evidence that different cytokine environments during differentiation of monocyte-derived DC can modify their Th cell-inducing properties. A hitherto unrecognized effect of IL-3 on DC was defined, namely suppression of IL-12 secretion and a resulting shift from Th1 toward Th2.