Mutational analysis of amino acid residues involved in IgE-binding to the Malassezia sympodialis allergen Mala s 11

The yeast Malassezia sympodialis, which is an integral part of the normal cutaneous flora, has been shown to elicit specific IgE- and T-cell reactivity in atopic eczema (AE) patients. The M. sympodialis allergen Mala s 11 has a high degree of amino acid sequence homology to manganese superoxide dismutase (MnSOD) from Homo sapiens (50%) and Aspergillus fumigatus (56%). Humoral and cell-mediated cross-reactivity between MnSOD from H. sapiens and A. fumigatus has been demonstrated. Taken together with the recent finding that human MnSOD (hMnSOD) can act as an autoallergen in AE patients sensitised to M. sympodialis, we hypothesized that cross-reactivity could also occur between hMnSOD and Mala s 11, endogenous hMnSOD thus being capable of stimulating an immune response through molecular mimicry. Herein we demonstrate that recombinant Mala s 11 (rMala s 11) is able to inhibit IgE-binding to recombinant hMnSOD and vice versa, indicating that these two homologues share common IgE-binding epitopes and providing an explanation at a molecular level for the autoreactivity to hMnSOD observed in AE patients sensitised to Mala s 11. Using molecular modelling and mapping of identical amino acids exposed on the surface of both Mala s 11 and hMnSOD we identified four regions each composed of 4-5 residues which are potentially involved in IgE-mediated cross-reactivity. Mutated rMala s 11 molecules were produced in which these residues were altered. Native-like folding was verified by enzymatic activity tests and circular dichroism. The rMala s 11 mutants displayed lower IgE-binding in comparison to wild-type rMala s 11 using plasma from AE patients. In particular, mutation of the residues E29, P30, E122 and K125 lowered the IgE-binding to Mala s 11. The results of this study provide new insights in the molecular basis underlying the cross-reactivity between Mala s 11 and hMnSOD.     

The allergenic yeast Malassezia furfur induces maturation of human dendritic cells

BACKGROUND: The yeast Malassezia furfur (M. furfur), present in the normal microflora of human skin, can act as an allergen that incites specific IgE reactivity and T cell proliferation in atopic dermatitis (AD) patients. The role of antigen presenting dendritic cells (DCs) in the onset and maintenance of AD is not well established. OBJECTIVE: The objective of the present study was to assess whether the interaction of M. furfur with human DCs will result in DC maturation, cytokine production and lymphocyte proliferation. METHODS: Monocyte-derived dendritic cells (MDDCs) were generated from human peripheral blood. Immature MDDCs were cultured with or without M. furfur or plastic beads, and with or without CD40L stimulation. Interaction of yeast cells by MDDCs was studied by time-lapse photography and cytokines were detected in culture supernatants with ELISA. The ability of MDDCs pre-incubated with M. furfur to induce proliferation in autologous lymphocytes was measured by [(3)H]-thymidine incorporation. RESULTS: Time-lapse photography showed that the majority of immature MDDCs internalized whole M. furfur yeast cells within 1 h. The presence of M. furfur induced maturation (CD83 expression) of MDDCs, and up-regulation of the costimulatory molecules CD80 and CD86. Production of TNF-alpha, IL-1 beta and IL-18 by MDDCs increased significantly (P < 0.05 for TNF-alpha and IL-1 beta, and P < 0.01 for IL-18) after the addition of M. furfur, while IL-10 and IL-12p70 levels remained unaltered. The CD40L-stimulated IL12p70 production by MDDCs was decreased in the presence of M. furfur (P < 0.05). Finally, immature MDDCs pre-incubated with M. furfur induced a proliferative response in autologous CD14-depleted peripheral blood mononuclear cells, in a dose-dependent manner. CONCLUSION: The data indicate that immature MDDCs can internalize the opportunistic yeast M. furfur. This process was associated with MDDC maturation, production of pro-inflammatory and immunoregulatory cytokines, which might favour induction of a Th2-type immune response, and a capacity to stimulate lymphocyte proliferation. This chain of events most likely contributes to the inflammatory reaction in AD.     

The Malassezia sympodialis Allergen Mala s 11 with Sequence Similarity to Manganese Superoxide Dismutase Induces Maturation and Production of Inflammatory Cytokines in Human Dendritic Cells

The chronic inflammatory skin disease atopic eczema (AE) affects almost 15% of the population in many countries today. The pathogenesis of AE is not fully understood. A combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. The yeast Malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific IgE and T-cell reactivity in patients with AE. Recently, we identified a novel major M. sympodialis allergen, designated Mala s 11 (22.4 kDa), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (MnSOD). Interestingly, Mala s 11 has a high degree of homology to human MnSOD. The aim of this study was to examine the effects of recombinant Mala s 11 on antigen-presenting dendritic cells. Monocyte-derived dendritic cells (MDDCs) from healthy blood donors were cultured with or without Mala s 11 for different time periods. It was found that the maturation marker CD83 and the costimulatory molecules CD80 and CD86 were upregulated on the MDDCs exposed to Mala s 11 for 24 h, as demonstrated by flow cytometry. Furthermore, coculture of MDDCs with Mala s 11 for 9 h induced an increased production of the inflammatory cytokines IL-6 (200-fold), TNF-α (100-fold) and IL-8 (sixfold), as detected by the cytometric bead array (CBA) analysis. Our results suggest that Mala s 11 affects the immune response through DC maturation and production of inflammatory cytokines. The potential cross-reactivity with human MnSOD needs to be explored and the exact role of Mala s 11 in the pathogenesis of AE assessed in clinical studies involving skin prick and atopy patch tests.     

Mitochondrial signal lacking manganese superoxide dismutase failed to prevent cell death by reoxygenation following hypoxia in a human pancreatic cancer cell line, KP4

One of the major characteristics of tumor is the presence of a hypoxic cell population, which is caused by abnormal distribution of blood vessels. Manganese superoxide dismutase (MnSOD) is a nuclear-encoded mitochondrial enzyme, which scavenges superoxide generated from the electron-transport chain in mitochondria. We examined whether MnSOD protects against hypoxia/reoxygenation (H/R)-induced oxidative stress using a human pancreas carcinoma-originated cell line, KP4. We also examined whether MnSOD is necessarily present in mitochondria to have a function. Normal human MnSOD and MnSOD without a mitochondrial targeting signal were transfected to KP4 cells, and reactive oxygen species, nitric oxide, lipid peroxidation, and apoptosis were examined as a function of time in air following 1 day of hypoxia as a H/R model. Our results showed H/R caused no increase in nitric oxide, but resulted in increases in reactive oxygen species, 4-hydroxy-2-nonenal protein adducts, and apoptosis. Authentic MnSOD protected against these processes and cell death, but MnSOD lacking a mitochondrial targeting signal could not. These results suggest that only when MnSOD is located in mitochondria is it efficient in protecting against cellular injuries by H/R, and they also indicate that mitochondria are primary sites of H/R-induced cellular oxidative injuries.     

Alteration of cellular phenotype and responses to oxidative stress by manganese superoxide dismutase and a superoxide dismutase mimic in RWPE-2 human prostate adenocarcinoma cells

To study biologic effects of increased manganese superoxide dismutase (MnSOD) on cell behavior, we overexpressed MnSOD in a human prostate cancer cell line RWPE-2 by cDNA transfection. Stable transfectants of MnSOD showed a two- to threefold increase in MnSOD protein and enzymatic activity and a decrease in growth rate with prolonged cell population doubling times. Western blot analysis showed a 1.5- to twofold increase in the cyclin-dependent kinase inhibitor p21(Waf1) in MnSOD transfectants. Overexpression of MnSOD resulted in a seven- to eightfold increase in reduced glutathione (GSH), 18- to 26-fold increase in oxidized glutathione (GSSG), and a two- to threefold decrease in the ratio of GSH to GSSG. MnSOD-overexpressing cells showed an increase in sensitivity to the cytotoxicity of buthionine sulfoximine, a glutathione-depleting agent, and vitamin C, but a decrease in sensitivity to sodium selenite. Treatment with a superoxide dismutase (SOD) mimic MnTMPyP resulted in similar effects of MnSOD overexpression on cell responses to vitamin C and selenium. These data demonstrate that overexpression of MnSOD or treatment with SOD mimics can result in antioxidant or prooxidant effects in cells, depending on the presence of other antioxidants and prooxidants. MnSOD also has redox regulatory effects on cell growth and gene expression. These findings suggest that MnSOD and SOD mimics have the potential for cancer prevention or treatment.     

Pimecrolimus does not affect the differentiation,maturation and function of human monocyte-derived dendritic cells,in contrast to corticosteroids

Clinically, corticosteroids (CS) are among the first line drugs in the therapy of autoimmune and allergic diseases and potently inhibit the activation of immune cells. However, due to their pleiotropic mode of action, the prolonged use of CS is generally associated with a range of undesirable side‐effects. In this study, we compared the activity of pimecrolimus, a novel immunomodulatory drug for the treatment of inflammatory skin disorders, and the CS dexamethasone (Dex) and beta‐methasone‐valerate (β‐MSV) in different in vitro assays addressing the cytokine‐induced differentiation and maturation of monocyte‐derived dendritic cells (M‐DC), the susceptibility of M‐DC to drug‐induced apoptosis and the potency of differentiated M‐DC to induce primary T cell activation. In contrast to pimecrolimus, Dex and β‐MSV strongly induced apoptosis of M‐DC precursors if added at the start of the DC differentiation culture. Flow cytometric analysis of surviving cells on day 6 of culture showed that the expression of several DC‐specific antigens such as CD1a, CD40 and CD80 was inhibited by 50% to 80% at concentrations between 1 nm and 10 nm of either Dex or β‐MSV. Furthermore, the presence of CS during the final maturation of M‐DC inhibited the synthesis of IL‐12p70, the expression of critical DC costimulatory molecules, such as CD83 and CD86 and impaired their ability to activate primary CD4⁺ T cell proliferation. In contrast, pimecrolimus did not inhibit the LPS‐induced secretion of IL‐12, surface expression of costimulatory molecules or the maturation of M‐DC into potent stimulators of T cells. Taken together, these data indicate that pimecrolimus does not interfere with the differentiation and viability of dendritic cells and their precursors or with the function of mature M‐DC to prime naïve T lymphocytes, and thus may have a lower potential than CS to interfere with DC‐mediated immunosurveillance.     

In vitro-generated viral double-stranded RNA in contrast to polyinosinic:polycytidylic acid induces interferon-alpha in human plasmacytoid dendritic cells

Double-stranded RNA (dsRNA) arises in the cytoplasm during viral replication and was shown to participate in the interferon (IFN)-alpha induction process. Besides the intracellular recognition, released dsRNA from dying, infected cells can function as a pathogen-associated molecular pattern (PAMP) for the innate immune system. In the present study, in vitro-generated dsRNA fragments of genomic sequences of Newcastle disease virus were used to induce IFN-alpha release in human peripheral blood mononuclear cells (PBMC), in immature myeloid dendritic cells (mDC) and in immature plasmacytoid DC (pDC). The extracellular administration of dsRNA fragments but not the application of the corresponding single-stranded RNA (ssRNA) strands led to an IFN-alpha production in PBMC. The synthetic dsRNA analogue polyinosinic acid : polycytidylic acid [Poly(I : C)] could only stimulate IFN-alpha production in enriched mDC but not in pDC. In contrast, dsRNA fragments induced IFN-alpha only in pDC. Complexation of dsRNA fragments with transfection reagents increased the efficiency of IFN-alpha induction and commuted ssRNA molecules into IFN-alpha inducers. However, stimulation of in vitro-generated murine Toll-like receptor 7 (TLR7) knockout DC and human TLR-transfected HEK293 cells with dsRNA fragments gave no evidences for the involvement of pDC-specific TLR7 or TLR9 in the observed IFN-alpha induction.     

Effect of antibodies to red cell superoxide dismutase on its activity in the blood of patients with rheumatoid arthritis

A relationship is revealed between the level of antibodies to superoxide dismutase and the activity, course, and form of a disease. The production of antibodies to red cell superoxide dismutase increases as the pathological process progresses. By reducing the resistance of erythrocyte membranes to reactive oxygen species, antibodies to red cell superoxide dismutase promote the development of anemia in patients with rheumatoid arthritis. The results indicate that inhibition of the active center of this enzyme by specific immunoglobulins is one of the causes of reduced activity of red cell superoxide dismutase or the lack of an appreciable increase in its activity during hyperproduction of reactive oxygen species. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 120, N ^o 10, pp. 391–393, October, 1995 Presented by A. B. Zborovskii, Member of the Russian Academy of Medical Sciences     

The role of heat shock protein (hsp70) in dendritic cell maturation: hsp70 induces the maturation of immature dendritic cells but reduces DC differentiation from monocyte precursors

Members of the heat shock protein (hsp70) family are either constitutively expressed (hsc70) or can be induced by hyperthermic stress (hsp70). Recombinant hsp70 (rhsp70) stimulates cytokine production from monocytes and enhances NK cell proliferation and cytotoxicity. Here we demonstrate that rhsp70 binds to immature dendritic cells (DC) derived from monocyte precursors and induces their maturation as evidenced by an increase in CD40, CD86 and CD83 expression. Immature DC stimulated to mature with rhsp70 show an enhanced ability to present tyrosinase peptide to specific CTL. Mature DC did not bind rhsp70, suggesting a down-regulation in the expression of its receptor. When rhsp70 was added to monocyte precursors at the same time as GM-CSF and IL-4 it reduced the differentiation of monocytes into DC as shown by a decrease in the level of CD40, CD83, CD86 and HLA-DR expression and an increase in CD14 expression. The constitutively expressed hsc70 had neither a stimulatory effect on the maturation of immature DC nor did it reduce the differentiation of monocytes into DC. These findings demonstrate the specific ability of rhsp70 to induce the maturation of immature DC. Therefore rhsp70 may be useful for its adjuvant like properties in DC based immunotherapy of certain tumors.     

Molecular and biochemical characterization of a unique mutation in CCS, the human copper chaperone to superoxide dismutase

Copper (Cu) is a trace metal that readily gains and donates electrons, a property that renders it desirable as an enzyme cofactor but dangerous as a source of free radicals. To regulate cellular Cu metabolism, an elaborate system of chaperones and transporters has evolved, although no human Cu chaperone mutations have been described to date. We describe a child from a consanguineous family who inherited homozygous mutations in the SLC33A1, encoding an acetyl CoA transporter, and in CCS, encoding the Cu chaperone for superoxide dismutase. The CCS mutation, p.Arg163Trp, predicts substitution of a highly conserved arginine residue at position 163, with tryptophan in domain II of CCS, which interacts directly with superoxide dismutase 1 (SOD1). Biochemical analyses of the patient's fibroblasts, mammalian cell transfections, immunoprecipitation assays, and Lys7Δ (CCS homolog) yeast complementation support the pathogenicity of the mutation. Expression of CCS was reduced and binding of CCS to SOD1 impaired. As a result, this mutation causes reduced SOD1 activity and may impair other mechanisms important for normal Cu homeostasis. CCS-Arg163Trp represents the primary example of a human mutation in a gene coding for a Cu chaperone.     

Endotoxin contamination in commercially available pokeweed mitogen contributes to the activation of murine macrophages and human dendritic cell maturation

Commercially available pokeweed mitogen (PWM) has been reported to activate macrophages, leading to production of proinflammatory cytokines and nitric oxide (NO). However, we found that polymyxin B (PMB), a specific inhibitor of endotoxin activity, inhibited the PWM-induced expression of proinflammatory cytokines and NO and the activation of Toll-like receptor 4 (TLR4). A kinetic-turbidimetric Limulus amebocyte lysate assay demonstrated that commercial PWM contained substantial endotoxin, over 10(4) endotoxin units/mg of the PWM. A PWM repurified by PMB-coupled beads no longer induced the expression of proinflammatory cytokines, TLR4 activation, or dendritic cell maturation. However, the repurified PWM remained able to induce proliferation of human lymphocytes, which is a representative characteristic of PWM. These results suggest that commercial PWM might be contaminated with a large amount of endotoxin, resulting in the attribution of misleading immunological properties to PWM.     

The interaction of human dendritic cells with yeast and germ-tube forms of Candida albicans leads to efficient fungal processing, dendritic cell maturation, and acquisition of a Th1 response-promoting function

T helper cell type 1 (Th1) cell-mediated immunity plays a critical role in protection against the opportunistic pathogen Candida albicans. Virulence of the fungus is closely associated with its ability to form germ-tubes (GT), the early phase of the dimorphic transition from the commensal yeast (Y) to the more invasive hyphal (H) form. In this study, we examined the functional outcome of the interaction of Y or GT forms with human dendritic cells (DCs), professional antigen-presenting cells, which are pivotal for initiation and modulation of T cell responses. DCs phagocytosed and killed Y and GT cells with a comparable efficiency, becoming able to trigger strong proliferative responses by Candida-specific, autologous T cell clones. Both fungal forms induced DC maturation, as indicated by up-regulation of CD83, CD80, CD86, CD40, and major histocompatibility complex classes I and II surface antigens. Chemokine receptors were also modulated in Candida-DCs, which showed increased CCR7/CXCR4 and decreased CCR5 expression. Y- and GT-activated DCs differed in the pattern of cytokine expression. In particular, GT cells, in common with fully differentiated H cells, induced significantly more elevated levels of interleukin (IL)-10 than Y cells. Nevertheless, Y-, GT-, or H-pulsed DCs secreted comparable amounts of IL-12p70. In addition, irrespective of the fungal form triggering DC activation, Candida-DCs acquired the ability to prime naive T lymphocytes with a defined Th1 phenotype. Overall, our findings highlight the induction of substantially similar functional patterns in human DCs encountering the different forms of growth of C. albicans, both seemingly activating the Th1-type immunity which is characteristic of the healthy human subjects, naturally immunized and protected against the fungus.     

In situ reduction of oxidative damage, increased cell turnover, and delay of mitochondrial injury by overexpression of manganese superoxide dismutase in a multistage skin carcinogenesis model

To study early subcellular pathologic changes of tumorigenesis in mouse skin and possible modulation by overexpression of the mitochondrial antioxidant enzyme manganese superoxide dismutase (MnSOD), skin keratinocytes from nontransgenic (Ntg) and transgenic (TgH) mice overexpressing MnSOD topically treated with one dose of 7,12-dimethylbenz(a)anthracene (DMBA) and a subsequent dose of 12-O-tetradecanoylphorbol 13-acetate (TPA) were analyzed in situ for levels of MnSOD and the oxidative damage product 4-hydroxy-2-nonenal (4HNE)-modified proteins using specific antibodies and immunogold electron microscopy. At all selected time points analyzed after TPA treatment, there was more MnSOD immunoreactive protein in mitochondria of keratinocytes of TgH mice than Ntg mice. Compared with untreated groups, there was a large increase in 4HNE-modified proteins at 6-24 h after TPA treatment, and this increase was larger in Ntg than TgH mice. Indices of mitosis and apoptosis of keratinocytes were greater in DMBA/TPA-treated TgH than Ntg mouse skin. Mitochondrial injury detected by transmission electron microscopy was delayed in keratinocytes of TgH compared with Ntg mice. The present study demonstrated that overexpression of MnSOD not only protected cells from oxidative damage, but also affected cell turnover kinetics. Thus, previously identified reduction in papilloma formation observed in TgH mice is correlated with mitochondrial events.     

Prothymosin alpha immunoactive carboxyl-terminal peptide TKKQKTDEDD stimulates lymphocyte reactions, induces dendritic cell maturation and adopts a beta-sheet conformation in a sequence-specific manner

Prothymosin alpha (ProTalpha) is a small acidic polypeptide with important immunostimulatory properties, which we have previously shown to be exerted by its carboxyl (C)-terminus. It exerts immunoenhancing effects through stimulation of monocytes via toll-like receptor (TLR) triggering. Here, we assayed the activity of synthetic peptides homologous to ProTalpha's C-terminus to stimulate lymphocyte functions, in particular natural killer cell cytotoxicity of peripheral blood mononuclear cells isolated from healthy donors. A synthetic decapeptide TKKQKTDEDD was identified as the most potent lymphocyte stimulator. The activity of this peptide was sequence-specific and comparable to that of the intact molecule, suggesting that ProTalpha's immunoactive segment encompasses the nuclear localization signal sequence of the polypeptide. Because ProTalpha stimulates immune responses in a monocyte-dependent manner, we further investigated whether the entire molecule and its peptide TKKQKTDEDD specifically act on monocytes and show that both can promote maturation of monocyte-derived dendritic cells (DC). Finally, knowing that, under specific conditions, ProTalpha forms amyloid fibrils, we studied the amyloidogenic properties of its C-terminal peptide segments, utilizing ATR FT-IR spectroscopy and transmission electron microscopy (negative staining). Although the peptide TKKQKTDEDD adopts an antiparallel beta-sheet conformation under various conditions, it does not form amyloid fibrils; rather it aggregates in globular particles. These data, in conjunction with reports showing that the peptide TKKQKTDEDD is generated in vivo upon caspase-cleavage of ProTalpha during apoptosis, strengthen our hypothesis that immune response stimulation by ProTalpha is in principle exerted via its bioactive C-terminal decapaptide, which can acquire a sequence-specific beta-sheet conformation and induce DC maturation.     

The effect of human placenta cytotrophoblast cells on the maturation and T cell stimulating ability of dendritic cells in vitro

The success of pregnancy depends upon regulatory mechanisms that allow the fetus to survive and develop to term in the uterus, despite maternal immune cells' awareness of paternal alloantigens. At least some of these specific mechanisms are mediated by the effect of fetal trophoblast cells. In the present study we examine the effect of human placental cytotrophoblast cells (CTCs) on the maturation of dendritic cells (DCs) in vitro. For that purpose, CTCs were isolated from samples of placentae at 5–11 weeks of gestation and co‐cultured with peripheral blood monocytes under conditions inducing DC maturation. CTC were shown to alter the morphology, phenotype and functional properties of DCs. As a result, a significant portion of cells acquire fibroblast‐like morphology and some of the cells retain the expression of CD14. DCs matured in the presence of CTCs do not differ from usual DCs in terms of CD80, CD83 and CD86 expression, as well as the ability to induce allogenic lymphocytes proliferation. However, CTCs reduce significantly the ability of DCs to stimulate interferon‐γ production and the loss of CD62L by T cells. The results obtained indicate that DCs may be involved in pregnancy‐associated changes of cytokine production and T cell migration.     

Altered expression and endocytic function of CD205 in human dendritic cells, and detection of a CD205-DCL-1 fusion protein upon dendritic cell maturation

CD205 (DEC-205) is a member of the macrophage mannose receptor family of C-type lectins. These molecules are known to mediate a wide variety of biological functions including the capture and internalization of ligands for subsequent processing and presentation by dendritic cells. Although its ligands await identification, the endocytic properties of CD205 make it an ideal target for those wishing to design vaccines and targeted immunotherapies. We present a detailed analysis of CD205 expression, distribution and endocytosis in human monocyte-derived dendritic cells undergoing lipopolysaccharide-induced maturation. Unlike other members of the macrophage mannose receptor family, CD205 was up-regulated upon dendritic cell maturation. This increase was a result of de novo synthesis as well as a redistribution of molecules from endocytic compartments to the cell surface. Furthermore, the endocytic capacity of CD205 was abrogated and small amounts of the recently identified CD205-DCL-1 fusion protein were detected in mature DC. Our results suggest that CD205 has two distinct functions -- one as an endocytic receptor on immature dendritic cells and a second as a non-endocytic molecule on mature dendritic cells -- and further highlight its potential as an immuno-modulatory target for vaccine and immunotherapy development.     

Melanoma cell lysate induces CCR7 expression and in vivo migration to draining lymph nodes of therapeutic human dendritic cells

We have previously reported a novel method for the production of tumour‐antigen‐presenting cells (referred to as TAPCells) that are currently being used in cancer therapy, using an allogeneic melanoma‐derived cell lysate (referred to as TRIMEL) as an antigen provider and activation factor. It was recently demonstrated that TAPCell‐based immunotherapy induces T‐cell‐mediated immune responses resulting in improved long‐term survival of stage IV melanoma patients. Clinically, dendritic cell (DC) migration from injected sites to lymph nodes is an important requirement for an effective anti‐tumour immunization. This mobilization of DCs is mainly driven by the C‐C chemokine receptor type 7 (CCR7), which is up‐regulated on mature DCs. Using flow cytometry and immunohistochemistry, we investigated if TRIMEL was capable of inducing the expression of the CCR7 on TAPCells and enhancing their migration in vitro, as well as their in vivo relocation to lymph nodes in an ectopic xenograft animal model. Our results confirmed that TRIMEL induces a phenotypic maturation and increases the expression of surface CCR7 on melanoma patient‐derived DCs, and also on the monocytic/macrophage cell line THP‐1. Moreover, in vitro assays showed that TRIMEL‐stimulated DCs and THP‐1 cells were capable of migrating specifically in the presence of the CCR7 ligand CCL19. Finally, we demonstrated that TAPCells could migrate in vivo from the injection site into the draining lymph nodes. This work contributes to an increased understanding of the biology of DCs produced ex vivo allowing the design of new strategies for effective DC‐based vaccines for treating aggressive melanomas.