Josette Péguet-Navarro

Gangliosides from Human Melanoma Tumors Impair Dendritic Cell Differentiation from Monocytes and Induce Their Apoptosis

Gangliosides are ubiquitous membrane-associated glycosphingolipids, which are involved in cell growth and differentiation. Most tumor cells synthesize and shed large amounts of gangliosides into their microenvironment, and many studies have unraveled their immunosuppressive properties. In the present study we analyzed the effects of GM3 and GD3 gangliosides, purified from human melanoma tumors, on the differentiation of monocyte-derived dendritic cells (MoDC). At concentrations close to those detected in the sera from melanoma patients, both gangliosides dose-dependently inhibit the phenotypic and functional differentiation of MoDC, as assessed by a strong down-regulation of CD1a, CD54, CD80, and CD40 Ags and impaired allostimulatory function on day 6 of culture. Furthermore, GM3 and GD3 gangliosides decreased the viable cell yield and induced significant DC apoptosis. Finally, addition of GD3 to differentiating DC impaired their subsequent maturation induced by CD154. The resulting DC produced low amounts of IL-12 and large amounts of IL-10, a cytokine pattern that might hamper an efficient antitumor immune response. In conclusion, the results demonstrate that gangliosides impair the phenotypic and functional differentiation of MoDC and induce their apoptosis, which may be an additional mechanism of human melanoma escape.

In vitro primary sensitization of hapten‐specific T cells by cultured human epidermal Langerhans cells—a screening predictive assay for contact sensitizers

Background The need to develop predictive tests which could identify potential allergens has been recognized for many years. There is as yet no accepted in vitro method for the assessment of contact sensitizers.

Human epidermal Langerhans cells express the mannose‐fucose binding receptor

Sugar receptors are being increasingly implicated in host‐pathogen interactions because of their specific recognition of carbohydrates of microorganisms. The aim of this study was to identify sugar receptors expressed on the surface of human epidermal Langerhans cells (LC). To this end, binding of a panel of fluorescent neoglycoproteins to human epidermal LC was analyzed by quantitative flow cytofluorometry after standardization with calibrated beads. We demonstrate that fresh human LC are the only cells isolated from healthy epidermis which express a membrane receptor specific for fucose‐bovine serum albumin (BSA) and mannose‐BSA. Quantitative analysis of mannose‐BSA or fucose‐BSA binding showed non‐linear Scatchard plots, denoting the presence of high and moderate affinity binding on the LC surface. The binding parameters of these two ligands were not significantly different. Mannan, the yeast mannose‐rich polysaccharide, fucose‐BSA, mannose‐BSA and free fucose are strong competitors of the three known ligands of the mannose receptor, i.  e. fucose‐BSA, mannose‐BSA and fluorescein isothiocyanate dextran. The amount of mannose‐BSA and fucose‐BSA bound to LC was 1.5‐fold higher at 37 °C than at 4 °C, suggesting an internalization process. Antibodies raised against the human macrophage mannose receptor strongly stained CD1a‐positive LC but not CD1a‐negative population. Taken together, our data demonstrate that fresh human LC are the only cells in the epidermis to express a fucose‐mannose receptor on their surface.

Binding and in vitro modulation of human epidermal Langerhans cell functions by substance P.

Substance P (SP) is distributed in both the central and peripheral nervous system. It has various effects on immunocompetent cells, such as macrophages and lymphocytes. The aim of our study was to search for the presence of SP receptors (SP-R) on human cutaneous Langerhans cells (LC), and to determine the effects of SP on LC immunological functions in a model of mixed epidermal cell-lymphocyte reaction (MELR). Radioligand binding studies showed that LC-enriched epidermal cell suspensions reversibly bound SP, and that the specific binding increased with the percentage of LC. Functional assays showed that SP had no effect when added at concentrations from 10–6M to 10–12M to the MELR. The addition of SP at concentrations of 10–4M and 10–5M was able to inhibit the allogeneic T-cell response (98.3 ± 1.8% and 92.8 ± 8.9% inhibition, respectively) without modifying the cell viability. This inhibition was through an effect of SP on both T-cell and LC function. We conclude that SP has receptors on LC and may inhibit antigen presentation.

In vitro human T cell sensitization to haptens by monocyte-derived dendritic cells

We previously reported that in vitro primary sensitization of hapten-specific T cells by cultured human epidermal Langerhans cells (LC) provides an alternative approach to discriminate strong contact sensitizers from irritants (Krasteva et al., 1996; Moulon et al., 1993). However, this LC-based immunoassay was limited by the availability of human skin samples. In the present study, we used monocyte-derived dendritic cells (DC) to analyse the autologous proliferative T cell response to several allergens. Monocytes were purified from the peripheral blood of healthy donors and cultured for 6-8 days in the presence of GM/CSF and IL-4 and then for 2 days in the presence of GM/CSF and TNFalpha. The resulting cells exhibited the phenotype of mature DC, as assessed by the strong expression of HLA-DR, CD80, CD83 and CD86 antigens. We showed that trinitrophenyl (TNP)-treated mature DC induced a significant T cell proliferative response in all experiments, while fluorescein isothiocyanate (FITC) gave positive results in about half of them. The prohaptens eugenol and isoeugenol induced significant proliferation in one out of eight and in four out of 12 experiments, respectively. Interestingly, in 16 assays T cells never proliferated in the presence of sodium lauryl sulfate (SLS)-treated DC. Thus, this in vitro model allows discrimination between strong contact sensitizers and irritants. It might be very useful, therefore, for restriction of animal experimentation.

Lack of demonstrable effect of cyclosporin A on human epidermal Langerhans cell function.

SummaryThere is controversy about whether Cyclosporin A (CsA) affects antigen-presenting cell function. Within the skin, Langerhans cells (LC) are very potent antigenpresenting cells. We investigated the effect of CsA on alloantigen presentation by human LC using the in vitro mixed skin-cell lymphocyte reaction (MSLR). MSLR (6 day cultures) were performed in round-bottomed microplates and lymphocyte proliferation was assessed by 3H-thymidine incorporation during the final 18 h of culture. When CsA was added into the wells a dose-dependent inhibition of T-cell proliferation occurred. Similar results were obtained when crude or LC-enriched epidermal cells (EC) were incubated for 2 h in the presence of CsA and extensively washed. The inhibition caused by CsA treatment of EC was not overcome by the addition of indomethacin. However, when CsA-treated EC were added to a fresh MSLR, T-cell proliferation was impaired. Furthermore, supernatants from CsA-treated EC, that had been kept for 6 days in culture medium, were able to inhibit the T-cell proliferative assay. These supernatants were found to contain CsA by a radioimmunoassay. From these results, it is clear that inhibition of MSLR obtained after CsA pulsing of EC suspensions can be explained by a release of the drug into the supernatant and thus by a direct effect on T cells. These findings contrast with recent reports showing a direct effect of CsA on human LC function.

Effects of CD40 ligation on human keratinocyte accessory function.

Abstract CD40/CD40 ligand interactions are known to play a key role in the development of immune reactions, especially by enhancing the costimulatory function of professional antigen-presenting cells (APC). Little is known, however, about the role this receptor plays on occasional APC, i.e. cells that are induced to express MHC class II molecules following an inflammatory process. In this study, we used CD40 ligand-transfected cells to analyze the effect of CD40 ligation on the phenotype, as well as accessory function, of human keratinocytes. We found that CD40 ligation enhanced ICAM-1 expression and did not upregulate HLA-DR, CD80 or CD86 expression on IFN-γ-treated keratinocytes. CD40 triggering was not sufficient to generate primary allogeneic T-cell responses even in the presence of anti-CD28 monoclonal antibody (mAb). Moreover, CD40 ligation, in the presence or not of IFN-γ, did not alter the accessory function of keratinocytes in PHA- or superantigen-induced T-cell activation. The lack of effect on the T-cell response was confirmed in blocking experiments using anti-CD40 mAbs. Collectively, these results suggest that CD40-CD40 ligand interactions on nonprofessional APC may amplify the inflammatory reaction without providing a mitogenic signal to the T cells.

CIS-UROCANIC ACID FAILED TO AFFECT IN VITRO HUMAN LANGERHANS CELL ALLOSTIMULATORY FUNCTION

Abstract— ‐Urocanic acid (UCA) represents the major ultraviolet B (UVB, 290–320 nm)‐absorbing component of the skin. Trans‐UCA is naturally produced in the stratum corneum and converts to the cis isomer upon UVB irradiation. In this study, we examined the effect of purified cis‐UCA (about 99% of cis isomer) on the human Langerhans cell (LC) allostimulatory function by using the mixed epidermal cell‐lymphocyte reaction (MELR). We found that addition of increasing amounts (6.5–400 μg/mL) of purified cis‐UCA or (rara‐UCA did not modify the T‐cell response supported by enriched LC (eLC: 8–25% LC) as well as purified LC (pLC: 70–90% LC) suspensions. Because cis‐UCA had no effect on the allostimulatory function of untreated LC, we investigated whether this compound could modify T‐cell proliferation induced by UVB‐irradiated LC. The UVB exposure of eLC or pLC to 100 J/m2 significantly inhibited the capacity of both suspensions to mount a T‐cell response. However, addition of cis‐UCA did not potentiate this UVB‐induced immunosuppression. The eLC or pLC were then incubated with cis‐UCA for 18 h at 37°C and washed before adding to allogeneic T cells. The obtained proliferative response was similar to that induced by control LC incubated in medium alone, demonstrating that pretreatment with cis‐UCA did not alter human LC function. In conclusion, these results strongly suggest that cis‐UCA has no direct effect on human LC antigen‐presenting function.

In vitro determination of sunscreen immune protection factors.

Abstract The present study was aimed at determining immune protection factors (IPFs) for sunscreens. Human skin explants from donors of phototype II-III were treated, or not, with sunscreens with increasing sun protection factors (SPF 4, 8, 15 and 30), or their respective vehicles. Explants were submitted, or not, to increasing doses of UVB irradiation (312 nm). After an 18-h incubation at 37 °C, epidermal cells were recovered through trypsinization and tested in a mixed epidermal cell/T lymphocyte reaction. The UVB dose providing 50% immunosuppression (D50%) was determined graphically. We first demonstrated a large difference in the individual response to UVB, as assessed by the D50% in the absence of any topical treatment (mean 1615 ± 839 J/m 2 from 14 experiments with values ranging from 500 to 3200 J/m 2 ). For all the tested sunscreens, the D50% values were significantly higher than those obtained without sunscreens or with their respective vehicles ( P < 0.01), thus demonstrating their immunoprotective effect. IPFs were determined as the ratio of the D50% in the presence of sunscreen to that with vehicle alone. Although they displayed important individual variations, IPFs ranked according to the sunscreen SPFs.

Human in Vitro T Cell Sensitization Using Hapten-Modified Epidermal Langerhans Cells

Epidermal Langerhans cells (LC), a population of non-lymphoid dendritic cells which constitutively express MHC class II molecules, have been demonstrated to playa key role in the development of contact hypersensitivity reactions by picking up the haptens within epidermis and migrating to draining lymph nodes where antigen presentation to specific T cells occurs l. Recent studies reported that after 2-3 day in vitro incubation, LC undergo profound phenotypic changes and acquire an interdigitating cell appearance2,3. In the murine system, at least in some strains of mice, the incubated LC become substantially more potent accessory cells than fresh LC, while they are relatively inefficient in processing of protein antigens4. It has been thus suggested that cultured LC may represent the in vitro counterparts of antigen-bearing LC that have migrated to regional lymph nodes5. Although previous papers have reported in vitro sensitization of naive human T cells to haptens, these studies used peripheral blood cells as antigen presenting cells (APC)6,7. In the present study, we analysed the capacity of both freshly isolated and 2-day incubated human LC to elicit in vitro primary T cell sensitization to the hapten TNP.