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Published online before print February 20, 2007
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* INSERM U837 Université de Lille 2, IFR114, CHRU et Unité de Thérapie Cellulaire, Faculté de Médecine 1, Lille, Cedex, France;
Department of Medical Oncology, Erasme Hospital, Université Libre de Bruxelles, Brussels, Belgium; and
U774, IFR17, Institut Pasteur de Lille, Lille, France
3 Correspondence: INSERM U837, 1 Place Verdun, F-59045, Lille Cedex, France. E-mail: philippe.marchetti{at}lille.inserm.fr
ABSTRACT
Mild heat stress can modulate the activities of immune cells, including dendritic cells (DC) and theoretically, would constitute an innovative approach capable of enhancing the antitumor functions of DC. Therefore, we tested the effects of mild heat stress on the physiology and viability of human monocyte-derived DC, the major type of DC used in tumor immunotherapy trials. We first designed a heat-stress protocol consisting of repetitive, sublethal heat shocks throughout the generation of DC. Using this protocol, we observed that heat stress did not perturb the morphology and the phenotype of immature or mature DC or the capacities of immature DC to uptake antigens efficiently. It is noteworthy that in response to heat stress, mature DC produced higher levels of IL-12p70 and TNF-
, which are two cytokines involved in the stimulation of inflammatory reaction, whereas IL-10 production remained low. After heat-stress exposure, mature DC have the full ability to stimulate naive T cells with Th1 response polarization (high IFN-
and low IL-4 production) in an allogeneic MLR. It is interesting that heat stress enhanced the migratory capacities of DC in response to MIP-3ß/CCL19. Finally, heat stress partly protected DC from apoptosis induced by cytokine withdrawal. Overall, these findings validate the feasibility of improving immune response by heating human monocyte-derived DC and provide a strong rationale for using mild heat stress in combination with DC vaccination to increase antitumor response.
Key Words: heat shock • immunotherapy • temperature • hyperthermia • apoptosis • dendritic cells
INTRODUCTION
A growing body of evidence has demonstrated that dendritic cells (DC) are key actors in the diverse facets of immune regulation, including the induction of antitumor immune response [1 , 2 ]. Indeed, DC are the most potent APC. In an immature state, DC exhibit the capacities to capture and process tumor antigens with high efficiency. Subsequently, DC undergo maturation and migrate to lymphoid organs to stimulate T cells against specific antigens, e.g., tumor antigens, thus allowing the development of cell-mediated cytotoxicity toward tumor cells. During the last decade, the use of DC has been tested in clinical immunotherapy against cancer, and preliminary, positive results of vaccine therapy have already been reported. To perform cancer immunotherapy programs, high numbers of DC are required. For this purpose, methods have been developed to produce DC in vitro from precursor cells, including bone marrow, cord blood stem cells, and easily accessible blood monocytes. Monocyte-derived DC generated in vitro in the presence of IL-4 and GM-CSF are the major type of DC currently being used in tumor immunotherapy trials.
It has been shown that fever-like temperature changes might activate innate and also adaptive immunity [3 , 4 ]. Hyperthermia regulates L-selectin-dependent adhesion of lymphocytes to endothelium [5 ] and T cell proliferation and activation [6 ]. Moreover, mild heat stress can modify DC immune functions (for review, see ref. [7 ]). For instance, heat shock primes and matures mice DC in a heat shock protein (HSP)-independent manner [8 ] and enhances the migration of Langerhans cells from the epidermis to lymph nodes. The aim of this work was to characterize the consequences of heat-stress exposure on the phenotype, immune functions, and viability of human monocyte-derived DC, which play a pivotal role in immunotherapy against tumors, and enhancing their function through heat stress might be a key mechanism for the development of new cancer vaccines.
MATERIALS AND METHODS
Reagents
FITC-conjugated mAb used were anti-HLA-DR, anti-CD86, anti-CD14, and anti-CD1a (Becton Dickinson, Le Pont de Claix, France), as well as anti-HSP70 and anti-heat shock cognate protein 70 (HSC70; Tebu, Le Perray-en Yveline, France). PE-conjugated mAb included anti-CD80 and anti-IFN- (Becton Dickinson), in addition to anti-CD83 (Beckman Coulter, Villepinte, France). Isotype control mAb included FITC- and PE-labeled mouse IgG1 or IgG2a (Becton Dickinson). LPS from Escherichia coli Serotype 0111:B4 and propidium iodide (PI) were obtained from Sigma Chemical Co. (Saint Quentin Fallavier, France). ProSpec-Tany TechnoGene Ltd. (Le Perray en Yvelines, France) provided GM-CSF and IL-4, and R&D Systems (Abington, UK) provided MIP-3ß/CCL19. CFSE was obtained from Invitrogen (Cergy Pontoise, France), and the FITC-Annexin kit was from Becton Dickinson. Ionomycin and PMA were provided by Calbiochem (Fontenay sous bois, France). Finally, eBioscience (Montrouge, France) was the source of Brefeldin A.
Monocyte-derived DC generation
Blood monocytes from healthy donors were isolated from buffy coat, and the buffy coat product was diluted in RPMI 1640 (Life Technologies, Paisley, Scotland) and layered over a Ficoll gradient (Pharmacia, Upsala, Sweden). After centrifugation (400 g, 30 min), PBMC were harvested and washed. The PBMC were resuspended in 100 ml in 0.9 mg/ml NaCl. Monocytes were purified by elutriation using an Avanti J-20 XP elutriation centrifuge from Beckman Coulter. The elutriation process was performed at 2500 rpm rotor speed, and cells were loaded in the elutriation chamber at a counterflow rate of 11 ml/min. Then, the flow rate was increased to 20 ml/min to eliminate T cells. The monocyte-enriched fraction was harvested at a flow rate of 30 ml/min. To obtain a monocyte purity of >95%, elutriation was followed by an adhesion step: Monocytes (10–25x106) were cultured in a 75-cm2 flask for 2 h at 37°C in RPMI 10% FCS supplemented with antibiotics, nonessential amino acids, and 5 x 10–5 M 2-ME (complete RPMI). After intensive washes of the flask, monocytes were cultured in complete RPMI in the presence of GM-CSF (800 U/ml) and IL-4 (100 U/ml). On Day 3, GM-CSF and IL-4 were added and cultured further until Day 5. The DC were then harvested, washed, counted, and subjected to maturation in the presence of 1 µg/ml LPS for 18–24 h in six-well plates at 106 cells/ml. On Day 6, the cells were washed and cultured further for 4 h or 24 h until analysis.
Heat treatment
The DC heat-shock protocol consists of sequential, short heat shocks during the differentiation and maturation of DC (see Fig. 1C
). On Days 0 and 3, the culture flasks were dipped for 15 min in a fine-regulated water bath setup at 41°C. Then, GM-CSF and IL-4 were added to the cells. DC generated on Day 5 were heated at 41.5°C for 30 min and cultured in a six-well plate in the presence of LPS. After 8 h (Day 5.5), the cells were reheated at 41.5°C for 30 min. On Day 6, the cells were washed and heated at 41.5°C for 30 min and further cultured until analysis. The control plates contained nonheat-stressed cells.
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Cell surface immunophenotype
For two-color immunolabeling, the cells were washed twice in ice-cold PBS and incubated in 100 µl PBS containing appropriate fluorochrome-labeled mAb for 30 min on ice. The cells were washed twice with ice-cold PBS. At least 5000 cells were analyzed by flow cytometry (Beckman Coulter).
Evaluation of cytokine secretion
IL-12 (p70), TNF-, and IL-10 production by mature DC was measured in supernatants by ELISA (Diaclone, Besançon, France). IFN-, IL-4, and IL-10 production by T cells cocultured with DC was measured in supernatants by ELISA (Diaclone) after 5 days of coculture, followed by a 24-h stimulation with PMA (50 ng/ml) and ionomycin (2 µg/ml).
Cytokine intracellular staining
After 5 days of coculture of DC with allogeneic T cells, the cells were harvested, washed, and stimulated with PMA (50 ng/ml), ionomycin (2 µg/ml), and Brefeldin A (3 µg/ml) for 5 h. Then, the cells were fixed and permeabilized using the cytofix/cytoperm kit from Becton Dickinson according to the manufacturer’s recommendations. Finally, the cells were stained with appropriate fluorochrome-labeled mAb for 30 min on ice and washed. Five thousand events were analyzed by flow cytometry (Beckman Coulter).
Migration assay
Four hours after the last heat shock, DC were washed and resuspended in migration buffer HBSS, 1 mM CaCl2, 0.5 mM MgCl2, 1 mg/ml BSA (Sigma Chemical Co.), at a density of 2–3 x 106 cells/ml. Chemokine solution (600 µl) or buffer alone was added to individual wells of 24-well plates (Corning Life Science, Croissy, France) on ice. Immediately thereafter, Costar transwell devices with 5 µm pore size, polyvinylpyrrolidone-free polycarbonate membranes were inserted into the wells, and 100 µl of the cell suspension was layered on top of the membrane. Cells were allowed to attach to and transmigrate through the membrane for 2 h at 37°C. The fluid phase above the membrane was then removed, and transwell inserts were taken out of the wells. Nonmigrated cells were removed by scraping the filter, and the migrated, filter-bound cells were fixed with 40 mg/ml paraformaldehyde for 30 min at 4°C. The filters were mounted onto slides and embedded in a VectaShield medium containing DAPI (Vector Laboratories) for staining cells to enumerate the cells that had migrated through the filter. Migrated cells were counted on the bottom side of the filter in 10 randomly selected high power fields (hpf; original magnification, x630).
Allogenic T cell activation
Naive CD4+ T cells were purified using the CD4+ T cell isolation kit II from Miltenyi Biotec (Paris, France) and CD45RO beads (Miltenyi Biotech). Briefly, the PBMC were incubated with CD45RO microbeads and a cocktail of biotin-conjugated mAb against CD8, CD14, CD16, CD19, CD36, CD56, CD123, TCR-/
, and glycophorin A for 10 min at 4°C and then incubated with antibiotin microbeads for 15 min at 4°C. After washing the cells, the magnetically labeled, non-CD4+ T cells were depleted by retaining them on a MACS column in a magnetic field, and the CD4+ CD45RA+ naive T cells were recovered. After washing, the cells were frozen until use. Once thawed, CD4+ CD45RA+ naive T cells were labeled with CFSE. The cells were washed in RPMI without FCS and incubated with CFSE (2.5 µM) at 37°C for 10 min. Reaction was stopped by adding FCS. Then, the cells were washed extensively and resuspended at 106 cells/ml in a complete medium. In parallel, DC obtained 4 h after the end of the maturation were diluted at 100,000 cells/ml. A graded number of DC were dispensed in individual wells of 96-well, round-bottom plates and cocultured with 3 x 105 allogeneic, naive CD4+ T cells for 5 days. The cells were then harvested, and T cell proliferation was analyzed immediately by flow cytometry measuring the CFSE fluorescence intensity. In parallel, cells were restimulated with PMA (50 ng/ml) and ionomycin (2 µg/ml) for 24 h. Then, supernatants were collected to measure secreted cytokines. Cells were also restimulated with PMA (50 ng/ml), ionomycin (2 µg/ml), and Brefeldin A (3 µg/ml) for 5 h to analyze intracytoplasmic cytokine expression.
Induction and evaluation of apoptosis
DC were cultured and heated as described above. To induce apoptosis, DC were harvested on Day 5, washed extensively, and cultured further for 28 h in medium without GM-CSF and IL-4 (GM/IL-4). Apoptotic DC were detected using the FITC-Annexin kit (BD PharMingen, San Diego, CA, USA), according to the manufacturer’s instructions.
FITC-dextran assay
To evaluate the capacities for uptake of soluble antigens from the culture medium, DC were incubated with 1 mg/ml FITC-dextran (Sigma Chemical Co.) at 37°C for 1 h. As a negative control, cells were incubated under the same conditions at 4°C. After incubation, DC were washed twice in PBS and then analyzed by flow cytometry.
Statistical analysis
The results were analyzed using GraphPad Prism Version 3.00 (GraphPad Software, San Diego, CA, USA). For the analysis of cytokine production and migration capacities in nonheated and heated DC, a Wilcoxon-matched rank test for paired data was used. Values of P < 0.05 were considered statistically significant.
RESULTS
Effect of heat stress on the viability of DC
In a first series of experiments, we determined the conditions of heat exposure, which were just below a level that promoted DC death. Monocyte-derived DC, on Day 5 of culture, were exposed to temperatures of 40°C, 41°C, 41.5°C, 42°C, or 43°C for 15–90 min, followed by recovery at 37°C for 24 h. We observed that the impact of heat stress on the viability of DC was dependent on the temperature and duration of exposure: The DC did not lose viability upon exposure to 41.5°C for up to 30 min. However, exposure to 42°C or 43°C for 15 min was slightly lethal (Fig. 1A
), and beyond 60 min was highly toxic for DC. Extended incubation periods (24 h and 48 h), even at lower temperatures (i.e., 41°C and 41.5°C), decreased the viability of DC dramatically (data not shown).
As heat stress-induced HSP could play a crucial role in DC function, we also evaluated the impact of heat stress on HSP expression by immunofluorescence (Fig. 1B) . Short incubation periods of 15 min at 41°C allowed a higher expression of HSP27, -60, -70, and -90 on heated DC (data not shown). Nevertheless, to maintain a high level of HSP expression along the culture, repeated heat shocks were needed. Indeed, cells heated once on Day 0 or on Day 3 of the culture neither fully nor partially expressed HSP70 on Day 6, respectively. Conversely, cells heated once on Day 5 strongly maintained high levels of HSP70 (Fig. 1B) . No significant differences in the expression levels of HSC70, used as a control, were found after heat stress (Fig. 1B) . Based on these results, we designed a heat-stress protocol consisting of repetitive, sublethal heat shocks throughout ex vivo generation of DC (Fig. 1C) . This protocol was not toxic and allowed a high expression level of HSP70 on DC (data not shown). Thus, under the optimal conditions of heating defined above, we evaluated the effects of heat stress on the physiology of monocyte-derived DC.
Effect of heat stress on the morphology and phenotype of DC
Adherent monocytes cultured ex vivo with GM-CSF and IL-4 for 6 days differentiated from nonadherent cells, which displayed typical morphological features of DC, i.e., the presence of cytoplasmic extensions on the periphery and irregularly shaped nuclei (Fig. 2A
). Heat stress did not modify the morphological aspects of DC when compared with control DC (Fig. 2A)
. The expression of DC surface antigens was investigated by flow cytometry on immature (Fig. 2B)
and mature (Fig. 2C)
monocyte-derived DC, exposed or not exposed (control) to heat stress. More than 80% of nonadherent cells, generated ex vivo with GM-CSF and IL-4, exhibited CD1a, a specific marker of DC, were negative for the monocyte-macrophage marker CD14 and exhibited a phenotype of immature DC (low expression or absence of HLA-DR, CD80, CD86, and CD83). Heat stress did not modify the expression of cell-surface molecules of immature DC (Fig. 2B)
nor did it result in the maturation of DC or in the modification of the maturation-associated, phenotypical changes induced by LPS (Fig. 2C)
. Next, the endocytic capacities of immature, monocyte-derived DC were assessed by their ability to endocytose FITC-dextran. Immature, heated DC internalized a comparable amount of FITC-dextran particles [85%±10; 360±66 mean fluorescence intensity (MFI)] as control, immature DC (88%±10; 349±60 MFI; P=0.37). Overall, these results indicate that heat stress modifies neither the phenotype of DC nor the capacities of immature DC to uptake antigens efficiently.
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secretion, a proinflammatory cytokine; and IL-10, a cytokine that down-regulates DC functions. Immature DC did not produce these cytokines, and heat stress did not modulate the level of cytokines in the supernatant (not shown). Mature DC produced only low levels of IL-10, and differences between heated and unheated DC were undetectable (Fig. 3
). In contrast, mature DC produced a significant amount of IL- 12 p70 and TNF- (Fig. 3)
, and upon heat stress, the production of IL-12 p70 and TNF- increased significantly in the DC supernatants (Fig. 3)
. TNF- showed the most important increase, with an average of 2.5-fold increase in secretion from DC cultures after heat stress. Thus, it appears that heat stress significantly enhances the production of cytokines by mature monocyte-derived DC.
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production. Consistent with their ability to release a higher amount of IL-12 p70, a strong inducer of the Th1 differentiation [12
], heated DC significantly induced a higher amount of IFN- in comparison with control DC (Fig. 5C
, left panel). Moreover, the difference was clearly sustained through the restimulation of T cells with PMA/ionomycin (Fig. 5C
, right panel). It is worth noting that heated DC mainly increased the proportion of IFN--secreting CFSElow T cells, indicating that heated DC favor IFN- production by dividing T cells (Fig. 5D)
. In contrast, only a small amount of IL-4, a Th2 cytokine, was detected in the supernatant, and the level was not modified significantly by heat stress of DC (0.19±0.12 pg/ml for supernatant of control DC vs. 0.43±0.25 pg/ml for supernatant of heated DC; P=0.25). Taken together, although it induces the same levels of costimulatory molecule expression, heat stress enhances the capacities of LPS-pulsed DC to activate T cells and to promote a Th1-dominated response.
Heat stress reduced DC apoptosis
As it has been described that repetitive exposure to heat shock protects cells from apoptosis [13
], we tested the effect of heat stress on the apoptosis of DC induced by GM-CSF and IL-4 deprivation (Fig. 6
). GM-CSF/IL-4 withdrawal induced a significant increase of early apoptosis (Annexin V+ PI–). The number of necrotic cells (AnnexinV+ PI+) was only increased slightly. Heat stress had no toxic effect on DC and reduced the number of early apoptotic cells significantly in response to GM-CSF/IL-4 withdrawal (Fig. 6)
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The specific aim of the present study was to investigate whether the immunological function of human monocyte-derived DC generated ex vivo is potentiated by heat stress. We have established a heat-shock protocol consisting of successive, sublethal heat shocks throughout monocyte-derived DC culture. Our data indicate for the first time that human monocyte-derived DC are sensitive to heat stress and that the application of heat might constitute an alternative strategy to initiating a stronger immune response following DC vaccination for cancer therapy.
DC are APC, which play a critical role in stimulating or in silencing immune responses. Thus, it is tempting to control the direction of the immune response by manipulating DC. The differentiation and function of DC are regulated largely by exogenous factors such as UV- or -irradiation, microbial molecules, and heat shock [14
]. -Irradiation can result in DC maturation, but -irradiated DC reduced IL-12 production and are less effective in priming naive T cells [15
]. UV radiation delays migration of Langerhans cells into the draining lymph nodes [16
]. Mildly elevated temperatures associated with inflammation or fever have been linked to enhanced immunological functions and might constitute promising means to enhance the potency of DC-based, antitumor vaccines (for review, see ref. [7
]). Indeed, we have demonstrated that heat stress can significantly improve the immune response of human mature monocyte-derived DC by acting on several DC functions.
First, we observed that heat stress modulates cytokine secretion by monocyte-derived DC. Applied during DC maturation, heat favored TNF- production, a proinflammatory cytokine with antitumor effect, although the level of IL-10 remained low. It is well known that IL-10-producing DC are considered to be tolerogeneic DC involved in the establishment of tolerance favoring the growth of the tumor [17
]. The secretion of these two cytokines suggests that heated DC may favor an antitumor response. In parallel, mature DC secrete cytokines that contribute to the polarization of Th cells toward Th1 or Th2, depending on the type of the secreted cytokines. It is interesting that we observed a higher production of IL-12 p70 in heated monocyte-derived DC than in control cells. These data confirm those obtained previously with murine DC [18
]. As it is known that IL-12 favors differentiation of T cells into Th1 cells [19
], we further explored the effects of heat exposure on the Th1-polarizing capacities of monocyte-derived DC. We demonstrated that when exposed to heat stress, mature DC activated naive T cells with a high capacity to induce T cell proliferation and a Th1 response (high level of IFN-; trace of IL-4). Thus, heat stress exposure favors the Th1-polarizing capacities of human monocyte-derived DC and TNF--secreting DC, both important effects for the development of immunity against tumor cells in animal [20
] and human studies [21
].
In mice, the same effect has been described, i.e., a higher capacity of heated bone marrow-expanded DC (BM-DC), to induce a MLR [8 , 18 , 22 ] in a HSP90-dependent mechanism [22 ]. In addition, HSP60 was described to induce the maturation of murine BM-DC in vitro and to elicit a Th1-promoting phenotype [23 ]. Our heating protocol strongly induced the expression of HSP90 and HSP60 in cytoplasm (data not shown), suggesting the same role of these molecules in human DC as in mouse DC. However, these immunological effects could be related to the overexpression of HSP70 on heated DC. It has been reported recently that in humans, unlike in mice, HSP70 colocalized with MHC Class I molecules, suggesting a role for HSP70 in Class 1 antigen presentation [24 ]. In addition, another recent study shows that the increase in IL-12 production could be induced by HSP70, which binds and activates CD40 [25 ].
Second, in our study, mature DC exposed to heat stress displayed a higher capacity to migrate in response to CCL19/MIP-3ß in comparison with nonheated cells. CCL19/MIP-3ß is a chemokine playing a pivotal role in the migration of DC into the lymph nodes [9 ]. These results are consistent with the observation that in mice, fever-range, whole-body hyperthermia enhances the migration of Langerhans cells from the epidermis to the lymph node [26 ]. These cells also display a higher capacity to stimulate T cell proliferation in a MLR [27 ].
Despite these immunostimulating effects, it must be noted that our heat-shock protocol, consisting of several instances of successive, mild heat stress, did not allow human monocyte-derived DC to mature significantly (Fig. 2) . In contrast, longer exposure to heat leads to the maturation of mouse DC with a substantial increase in MHC I and II and costimulatory molecules such as CD80, CD86, and CD40 [8 , 22 ], which was not noted in humans (data not shown; refs. [24 , 25 ]). It is interesting that we did not observe any effect on immature DC in terms of phenotype (Fig. 2) , antigen-uptake capacities, or migration against CCL3 and CCL5 (data not shown). This suggests that our heating procedure does not affect precursors and their capacities to differentiate into DC in the presence of IL-4 and GM-CSF but rather differentiated DC.
Recently, others [24
] have described that heat shock induces an increase in the expression of the TLR-4 on monocyte-derived DC. TLR-4 recognizes microorganism-associated molecular patterns such as LPS. TLR-4 is required for LPS signaling in DC [28
]. In our study, DC underwent maturation with LPS stimulation. We can hypothesize that DC exposed to heat stress might express more TLR-4 on their surface, which might induce a higher response to LPS (i.e., increase in IL-12 and TNF- secretion, T cell activation, and capacities to migrate in response to CCL19).
As the longevity of DC may also play a role in regulating the strength of immune responses, we studied the effect of heat stress on apoptosis. Growth factor deprivation induced the aberrant exposure of phosphatidylserine residues on the outer plasma membrane leaflet of DC, a typical hallmark of apoptosis. We demonstrated that heat stress mediated significant protection, albeit transient (<24 h), against DC apoptosis (not shown). Recently, it has been shown that IL-12 and TNF- protect DC against apoptosis through an increase in antiapoptotic factors of the Bcl-2 family [29
30
31
32
]. However, we were unable to confirm this effect in our model, as no changes in Bcl-2 or Bclxl expression in heated DC were observed (data not shown).
For over the last decade, numerous preclinical and clinical trials (Phases I and II) have demonstrated that the injection of human monocyte-derived DC, pulsed with the relevant antigen, can induce a specific immune response in cancer patients and occasionally leads to the regression of metastatic tumors. However, the immune reaction is often transient with limited efficacy [33 , 34 ]. A number of mechanisms have been suggested for the lack of a sufficient immune response to control cancer growth in vivo, leading to escape of cancer cells from immunosurveillance and thus, limiting the efficacy of DC-based vaccines. Innovative approaches capable of enhancing antitumor functions of DC are therefore needed to overcome the limitations of current DC-based immunotherapy for cancer. Recently, it has been suggested that mild heat stress has the potential to improve the immune function of DC (for review, see ref. [7 ]). Our work offers new, experimental evidence supporting this concept and thus provides the rationale to improve the efficacy of DC-based immunotherapy in humans. The advantages of this approach are obvious in terms of cost and safety, given that exogenous material is injected together with DC. However, despite the obvious impact of repetitive heat shock on monocyte-derived DC functions and viability, it is difficult to speculate how these results can be extrapolated to the in vivo context. Consequently, we assume that modifications of procedure are needed to further increase immune induction strongly enough to use DC effectively for cancer immunotherapy. To optimize our protocol further, it will be important to control the initial population of monocytes used to generate DC. Indeed, a heterogeneity in the monocyte population, purified directly from buffy coat in terms of the number of apoptotic cells [35 ], has been described recently. In this case, heat stress might offer a promising way to improve the efficacy of monocyte-derived DC and could be beneficial for immunotherapy trials in cancer patients.
ACKNOWLEDGEMENTS
This work was supported by grants from the Ligue Contre le Cancer (Comité du Nord; to P. M.), the Société Française de Dermatologie (to P. M.), and the Cancéropole Nord Ouest. A. S. H-C. and L. C. received fellowships from the Cancéropole Nord Ouest and the Fondation pour la Recherche Médicale, respectively. Drs. Carolyn Straehle and Catherine Duez are gratefully acknowledged for helpful comments.
FOOTNOTES
1 These authors contributed equally to this work. 
Received May 24, 2006; revised January 24, 2007; accepted January 24, 2007.
REFERENCES
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) was analyzed using transwell chambers. DC (3x105) were seeded in the upper chamber in triplicate, and the number of migrated DC was analyzed by counting in 10 randomly selected hpf on the bottom side of the filter after 2 h. Data are from eight separate donors; circles represent single individuals, and horizontal bars represent group mean values. *, P < 0.05, between the two groups.
