CD1d is Expressed on Dermal Dendritic Cells and Monocyte-Derived Dendritic Cells
2001; Elsevier BV; Volume: 117; Issue: 3 Linguagem: Inglês
10.1046/j.0022-202x.2001.01458.x
ISSN1523-1747
AutoresGianni Gerlini, Hans Peter Hefti, Martin Kleinhans, Brian J. Nickoloff, Günter Burg, Frank O. Nestlé,
Tópico(s)T-cell and B-cell Immunology
ResumoCD1 proteins are a family of cell surface molecules that present lipid antigens to T cells. We investigated skin dendritic cells and monocyte-derived dendritic cells for expression of CD1 molecules using a panel of 10 different monoclonal antibodies focusing on the recently described CD1d molecule. By immunohistochemical analysis, CD1d expression in normal human skin was restricted to dendritic appearing cells in the papillary dermis mainly located in a perivascular localization. Langerhans cells did not show detectable CD1d expression in situ. Epidermal/dermal cell suspensions analyzed by flow cytometry demonstrated distinct subpopulations of HLA-DR positive dermal dendritic cells expressing CD1a, CD1b, and CD1c. CD1d was expressed on HLA-DRbright dermal antigen-presenting cells in dermal suspensions (16% ± 3.6%), as well as on highly enriched dermal dendritic cells migrating out of skin explants (60.5% ± 8.0%). Migrated mature dermal dendritic cells coexpressed CD83 and CD1d. Western blot analysis on microdissected skin sections revealed the presence of a 50–55 kDa CD1d molecule in dermis, suggesting that CD1d is highly glycosylated in skin. Both immature and mature monocyte-derived dendritic cells cultured in autologous plasma expressed CD1d molecules. In contrast, culture in fetal bovine serum downregulated CD1d expression. In conclusion, antigen-presenting cells in skin express different sets of CD1 molecules including CD1d and might play a role in lipid antigen presentation in various skin diseases. Differential expression of CD1 molecules depending on culture conditions might have an impact on clinical applications of dendritic cells for immunotherapy. CD1 proteins are a family of cell surface molecules that present lipid antigens to T cells. We investigated skin dendritic cells and monocyte-derived dendritic cells for expression of CD1 molecules using a panel of 10 different monoclonal antibodies focusing on the recently described CD1d molecule. By immunohistochemical analysis, CD1d expression in normal human skin was restricted to dendritic appearing cells in the papillary dermis mainly located in a perivascular localization. Langerhans cells did not show detectable CD1d expression in situ. Epidermal/dermal cell suspensions analyzed by flow cytometry demonstrated distinct subpopulations of HLA-DR positive dermal dendritic cells expressing CD1a, CD1b, and CD1c. CD1d was expressed on HLA-DRbright dermal antigen-presenting cells in dermal suspensions (16% ± 3.6%), as well as on highly enriched dermal dendritic cells migrating out of skin explants (60.5% ± 8.0%). Migrated mature dermal dendritic cells coexpressed CD83 and CD1d. Western blot analysis on microdissected skin sections revealed the presence of a 50–55 kDa CD1d molecule in dermis, suggesting that CD1d is highly glycosylated in skin. Both immature and mature monocyte-derived dendritic cells cultured in autologous plasma expressed CD1d molecules. In contrast, culture in fetal bovine serum downregulated CD1d expression. In conclusion, antigen-presenting cells in skin express different sets of CD1 molecules including CD1d and might play a role in lipid antigen presentation in various skin diseases. Differential expression of CD1 molecules depending on culture conditions might have an impact on clinical applications of dendritic cells for immunotherapy. antigen-presenting cells autologous plasma dermal dendritic cells monocyte-derived dendritic cells Dendritic cells are professional antigen-presenting cells (APC) specialized for initiation of primary immune responses. Dendritic APC capture, process, and finally present protein antigens in the form of peptide major histocompatibility complexes (MHC) to effector T cells. For this purpose dendritic cells are well equipped with MHC, adhesion, and costimulatory molecules (Cella et al., 1997Cella M. Sallusto F. Lanzavecchia A. 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Isolation and characterization of a cDNA and gene coding for a fourth CD1 molecule.Proc Natl Acad Sci USA. 1989; 86: 252-256Crossref PubMed Scopus (103) Google Scholar;Yu and Milstein, 1989Yu C.Y. Milstein C. A physical map linking the five CD1 human thymocyte differentiation antigen genes.Embo J. 1989; 8: 3727-3732Crossref PubMed Scopus (58) Google Scholar). It has been demonstrated that the CD1d molecule enables cells to interact with natural killer T cells, a subset of lymphocytes able to produce high levels of interleukin-4 (IL-4) and interferon-γ (IFN-γ) upon stimulation (Exley et al., 1997Exley M. Garcia J. Balk S.P. Porcelli S. Requirements for CD1d recognition by human invariant Valpha24+ CD4-CD8- T cells.J Exp Med. 1997; 186: 109-120Crossref PubMed Scopus (478) Google Scholar;Kawano et al., 1997Kawano T. Cui J. Koezuka Y. et al.CD1d-restricted and TCR-mediated activation of valpha14 NKT cells by glycosylceramides.Science. 1997; 278: 1626-1629https://doi.org/10.1126/science.278.5343.1626Crossref PubMed Scopus (2157) Google Scholar;Brossay et al., 1998aBrossay L. Chioda M. Burdin N. Koezuka Y. Casorati G. Dellabona P. Kronenberg M. CD1d-mediated recognition of an alpha-galactosylceramide by natural killer T cells is highly conserved through mammalian evolution.J Exp Med. 1998; 188: 1521-1528Crossref PubMed Scopus (561) Google Scholar, Brossay et al., 1998bBrossay L. Naidenko O. Burdin N. Matsuda J. Sakai T. Kronenberg M. Structural requirements for galactosylceramide recognition by CD1-restricted NK T cells.J Immunol. 1998; 161: 5124-5128PubMed Google Scholar;Exley et al., 1998Exley M. Porcelli S. Furman M. Garcia J. Balk S. CD161 (NKR-P1A) costimulation of CD1d-dependent activation of human T cells expressing invariant V alpha 24 J alpha Q T cell receptor alpha chains.J Exp Med. 1998; 188: 867-876Crossref PubMed Scopus (168) Google Scholar). Although numerous studies have been carried out on the expression of group I CD1 molecules in skin (Furue et al., 1992Furue M. Nindl M. Kawabe K. Nakamura K. Ishibashi Y. Sagawa K. Epitope mapping of CD1a, CD1b, and CD1c antigens in human skin: differential localization on Langerhans cells, keratinocytes, and basement membrane zone.J Invest Dermatol. 1992; 99: 23S-26SCrossref PubMed Scopus (19) Google Scholar;Elder et al., 1993Elder J.T. Reynolds N.J. Cooper K.D. Griffiths C.E. Hardas B.D. Bleicher P.A. CD1 gene expression in human skin.J Dermatol Sci. 1993; 6: 206-213Abstract Full Text PDF PubMed Scopus (21) Google Scholar;Meunier et al., 1993Meunier L. Gonzalez Ramos A. Cooper K.D. Heterogeneous populations of class II MHC+ cells in human dermal cell suspensions. Identification of a small subset responsible for potent dermal antigen-presenting cell activity with features analogous to Langerhans cells.J Immunol. 1993; 151: 4067-4080PubMed Google Scholar,Meunier et al., 1996Meunier L. Vian L. Lagoueyte C. Lavabre B.T. Duperray C. Meynadier J. Cano J.P. Quantification of CD1a HLA-DR and HLA class I expression on viable human Langerhans cells and keratinocytes.Cytometry. 1996; 26: 260-264Crossref PubMed Scopus (16) Google Scholar;Nestle et al., 1993Nestle F.O. Zheng X.G. Thompson C.B. Turka L.A. Nickoloff B.J. Characterization of dermal dendritic cells obtained from normal human skin reveals phenotypic and functionally distinctive subsets.. 1993; 151 ([published erratum appears in J Immunol 152:376, 1994]): 6535-6545Google Scholar;Fivenson and Nickoloff, 1995Fivenson D.P. Nickoloff B.J. Distinctive dendritic cell subsets expressing factor XIIIa, CD1a, CD1b and CD1c in mycosis fungoides and psoriasis.J Cutan Pathol. 1995; 22: 223-228Crossref PubMed Scopus (34) Google Scholar;Larregina et al., 1997Larregina A.T. Morelli A.E. Kolkowski E. Sanjuan N. Barboza M.E. Fainboim L. Pattern of cytokine receptors expressed by human dendritic cells migrated from dermal explants.Immunology. 1997; 91: 303-313Crossref PubMed Scopus (22) Google Scholar) and monocyte-derived dendritic cells (mo-DC) (Kasinrerk et al., 1993Kasinrerk W. Baumruker T. Majdic O. Knapp W. Stockinger H. CD1 molecule expression on human monocytes induced by granulocyte-macrophage colony-stimulating factor.J Immunol. 1993; 150: 579-584PubMed Google Scholar), few data are available on the expression of CD1d by skin APC in humans (Bonish et al., 2000Bonish B. Jullien D. Dutronc Y. et al.Overexpresssion of CD1d by keratinocytes in psoriasis and CD1d-dependent IFN-gamma production by NK-T cells.J Immunol. 2000; 165: 4076-4085Crossref PubMed Scopus (188) Google Scholar). The aim of this study was therefore to investigate the expression of CD1 molecules on skin APC, focusing in particular on CD1d. We extended our investigation also to mo-DC, as these cells are classical in vitro models for immature and mature dendritic cells. Furthermore, mo-DC are increasingly used for immunotherapy of cancer such as melanoma (Nestle et al., 1998Nestle F.O. Alijagic S. Gilliet M. et al.Vaccination of melanoma patients with peptide- or tumor lysate pulsed dendritic cells.Nat Med. 1998; 4 (Comments in: Nat Med 4:269–270, 1998): 328-332Crossref PubMed Scopus (2686) Google Scholar). Here we show that in normal human skin dermal dendritic cells (DDC) both in vivo and in vitro express all members of the CD1 family, including CD1d. CD1d expression on mo-DC was influenced by the presence of culture factors such as autologous plasma (AP) or fetal bovine serum (FBS). In the presence of AP immature and mature mo-DC expressed CD1d, whereas downregulation of this molecule was induced by FBS. Thus mo-DC and DDC express surface molecules that facilitate the presentation of lipids and glycolipids and potential cross-talk with natural killer T cells. RPMI 1640 (Life Technologies, Eggenstain, Germany) and X VIVO 15 (BioWhittaker, Belgium) were supplemented with 1% penicillin/streptomycin (Gibco, Basel, Switzerland), 2% glutamin (Seromed, Berlin, Germany), and 10% heat-inactivated FBS. In some experiments FBS was replaced by 1% heat-inactivated (45 min at 56°C) AP. The following monoclonal (MoAb) or polyclonal antibodies were used: CD1a PE and CD1a (NA1/34, IgG2a) from Dako (Glostrup, Denmark); CD1b FITC (M-T101, IgG1) and CD86 FITC (2331 FUN-1, IgG1) from Pharmingen (San Diego, CA); CD1b (BCD1b3.1.6, IgG1) kindly provided by Dr. S.A. Porcelli; CD1c (L161, IgG1) and CD83 PE (HB15a, IgG2b) from Immunotech (Marseilles, France); CD1d (42.1, IgG1; D5, IgG2b; 51.1.3, IgG2b; 27.1, IgG1; rabbit anti-CD1d) kindly provided by Dr. S.P. Balk; CD1d (NOR3.2, IgG1) from Biosource International (Camarillo, CA); HLA-DR FITC, HLA-DR PE (L243, IgG2a), and CD80 PE (L307.4, IgG1) from Becton Dickinson (Mountain View, CA). Goat antimouse phycoerythrin (PE) and fluorescein isothiocyanate (FITC) from Dako were used as secondary antibodies for flow cytometry. Goat antimouse conjugated with Texas Red (Southern Biotechnology Associates, Birmingham, AL) was used for immunofluorescence staining. Peripheral blood mononuclear cells were obtained from buffy coats by Ficoll-Paque gradient (Amersham Pharmacia Biotech, Uppsala, Sweden) and frozen in 90% FBS and 10% dimethylsulfoxide until use. Dendritic cells were generated from peripheral blood mononuclear cells as previously described with minor modifications (Cella et al., 1997Cella M. Sallusto F. Lanzavecchia A. Origin maturation and antigen presenting function of dendritic cells.Curr Opin Immunol. 1997; 9: 10-16Crossref PubMed Scopus (1157) Google Scholar;Jonuleit et al., 1997Jonuleit H. Kuhn U. Muller G. et al.Pro-inflammatory cytokines and prostaglandins induce maturation of potent immunostimulatory dendritic cells under fetal calf serum-free conditions.Eur J Immunol. 1997; 27: 3135-3142Crossref PubMed Scopus (1010) Google Scholar;Nestle et al., 1998Nestle F.O. Alijagic S. Gilliet M. et al.Vaccination of melanoma patients with peptide- or tumor lysate pulsed dendritic cells.Nat Med. 1998; 4 (Comments in: Nat Med 4:269–270, 1998): 328-332Crossref PubMed Scopus (2686) Google Scholar). Briefly, 50 × 106 peripheral blood mononuclear cells were thawed and cultured in 10 cm diameter Petri dishes (Falcon-Becton Dickinson, Franklin Lakes, NJ) in complete RPMI with either 1% AP or 10% FBS for 30–45 min. After removing the floating cells and washing with phosphate-buffered saline (PBS) the adherent cells were cultured overnight in complete RPMI. The following day RPMI was removed and replaced with X VIVO 15 supplemented with 800 U per ml granulocyte macrophage colony stimulating factor (GM-CSF; Leucomax, Sandoz-Wander Pharma, Bern) and 1000 U per ml IL-4 (R&D System, Europe). At days 3 and 5 fresh medium supplemented with the cytokines was added. At day 6 the cells were stimulated with a maturation cytokine cocktail including tumor necrosis factor α (TNF-α) (10 ng per ml), IL-6 (1000 U per ml), and IL-1β (10 ng per ml) (R&D System, Europe). Cells were analyzed at different maturation days by flow cytometry. Skin dendritic cells were obtained from normal human skin tissue belonging to healthy patients undergoing breast reduction. The skin was incubated in Dispase type 2 (Boehringer-Mannheim, Mannheim, Germany) and RPMI 1:1. After 1 h at 37°C epidermis was separated from dermis, washed three times with PBS, and cut in small pieces (1–10 mm). A portion of the epidermal and dermal sheets was cultured in RPMI supplemented with 10% FBS for 2 d in order to allow the cells to migrate out as described previously (Nestle et al., 1993Nestle F.O. Zheng X.G. Thompson C.B. Turka L.A. Nickoloff B.J. Characterization of dermal dendritic cells obtained from normal human skin reveals phenotypic and functionally distinctive subsets.. 1993; 151 ([published erratum appears in J Immunol 152:376, 1994]): 6535-6545Google Scholar). The remaining epidermal sheets were incubated with trypsin (Biochrom, Berlin, Germany), whereas the dermal sheets were incubated with an enzyme cocktail consisting of collagenase, hyaluronidase (Sigma-Chemie, Switzerland), and DNAse (Boehringer-Mannheim, Mannheim, Germany) for 1 h at 37°C on a shaker in order to obtain a cell suspension. Fresh epidermal and dermal suspensions were either immediately analyzed by fluorescence-activated cell sorter (FACS) analysis or cultured for 2 d in RPMI supplemented with 10% FBS, 1000 U per ml IL-4, and 800 U per ml GM-CSF. After 2 d both migrated cells and cells in suspension were collected and used for FACS analysis. In addition cytospins (Shandon, Pittsburgh, PA) were also prepared using the migrated cells. Biopsy samples of normal skin (N = 10) were chosen from the cryopreserved tissue archived in the Department of Dermatology of Zurich. Representative 5–7 µm serial cryostat sections as well as migrated DDC spun down on cytospins were stained with the APAAP technique as described previously (Nestle et al., 1997Nestle F.O. Burg G. Fäh J. Wrone-Smith T. Nickoloff B.J. Human sunlight-induced basal-cell-carcinoma-associated dendritic cells are deficient in T cell co-stimulatory molecules and are impaired as antigen-presenting cells.Am J Pathol. 1997; 150: 641-651PubMed Google Scholar). Unconjugated primary polyclonal antibodies or MoAb, used at 10 µg per ml (except MoAb 27.1 supernatant at 1:1, MoAb D5 ascitis at 1:250), are described in Reagents and antibodies. Migrated DDC spun down on cytospins were fixed in acetone, incubated 15 min with normal rabbit serum (Dako Diagnostic, Switzerland), and stained with anti-CD1d (42.1, IgG1) followed by goat antimouse conjugated with Texas Red (Southern Biotechnology Associates) for 1 h. After three washing steps in PBS the anti-HLA-DR FITC-conjugated MoAb was added for an additional hour. Afterwards they were washed again, covered with a cover slip, and analyzed by microscopy (Leica Mikroscopie, Wetzlar, Germany) using the TCS4D confocal laser scanning system (Leica Lasertechnik, Heidelberg, Germany). All MoAb dilutions and washing steps were done in PBS containing 2% FBS. 1 × 105 cells per reaction were stained with the conjugated or unconjugated MoAb described in Reagents and antibodies followed by a secondary goat antimouse PE or FITC MoAb at 10 µg per ml. In order to block the Fc mediated unspecific binding cells were incubated with FBS and PBS (1:1) for 30 min at 4°C. To control for nonspecific staining the appropriate isotype controls were used. For single staining cells were labeled with anti-CD1 MoAbs. To identify dendritic cells cells were double stained with HLA-DR and CD1 MoAb. All the incubations were done at 4°C for 30 min. The specificity of the anti-CD1 MoAb was checked using C1R mock and C1R CD1d transfectant (kindly provided by Dr. S Balk, Boston) and C1R CD1a, CD1b, CD1c transfectants (kindly provided by Dr. S. Porcelli). Dermis from microdissected skin sections was boiled in sodium dodecyl sulfate sample buffer. Equal amounts of proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The Western blot was developed using D5 as anti-CD1d antibody (gift from Dr. S. Balk, Boston). C1R mock and C1R CD1d transfectant cell lines (kindly provided by Dr. S. Balk, Boston) were used as negative and positive controls, respectively. Cryostat sections were stained with hematoxylin-eosin and dermis microdissection was performed under light microscope visualization (magnification 200×) using a 30 gauge needle. Immuno histochemistry was performed on human skin sections using a panel of MoAb directed toward different members of the CD1 family. CD1a expression was mostly confined to dendritic appearing cells in the epidermis corresponding to Langerhans cells Figure 1a. There were few cells expressing CD1a in dermis. CD1b and CD1c expression was demonstrated on dendritic cells in dermis, mostly in a perivascular localization Figure 1b, c. These cells correspond to DDC as published previously (Lenz et al., 1993Lenz A. Heine M. Schuler G. Romani N. Human and murine dermis contain dendritic cells. Isolation by means of a novel method and phenotypical and functional characterization.J Clin Invest. 1993; 92: 2587-2596Crossref PubMed Scopus (261) Google Scholar;Meunier et al., 1993Meunier L. Gonzalez Ramos A. Cooper K.D. Heterogeneous populations of class II MHC+ cells in human dermal cell suspensions. Identification of a small subset responsible for potent dermal antigen-presenting cell activity with features analogous to Langerhans cells.J Immunol. 1993; 151: 4067-4080PubMed Google Scholar;Nestle et al., 1993Nestle F.O. Zheng X.G. Thompson C.B. Turka L.A. Nickoloff B.J. Characterization of dermal dendritic cells obtained from normal human skin reveals phenotypic and functionally distinctive subsets.. 1993; 151 ([published erratum appears in J Immunol 152:376, 1994]): 6535-6545Google Scholar). Investigation of CD1d expression was performed with a panel of six antibodies. Consistent and equivalent immunohistochemical staining was obtained with antibodies D5, 51.1.3, and polyclonal rabbit anti-CD1d, which revealed positive staining on large cells with dendritic processes in the dermis Figure 1d. Staining with the MoAb NOR 3.2 revealed strong staining of keratinocytes as well as a few dendritic cells in dermis as published previously (Bonish et al., 2000Bonish B. Jullien D. Dutronc Y. et al.Overexpresssion of CD1d by keratinocytes in psoriasis and CD1d-dependent IFN-gamma production by NK-T cells.J Immunol. 2000; 165: 4076-4085Crossref PubMed Scopus (188) Google Scholar). Most of these cells were located in the upper dermis in the papillary area and in a perivascular localization. We did not observe any CD1d expression on epithelial Langerhans cells. Cytospin preparations from migrated DDC and Langerhans cells revealed expression of CD1d in various intensities on DDC Figure 1e, f but not Langerhans cells (data not shown). There were DDC with strong expression (black arrow), intermediate expression (blue arrow), and no expression (green arrow) of CD1d. To further investigate the expression of CD1d on epidermal cells and DDC, cell suspensions were prepared from normal skin using a cocktail of enzymes as described above and analyzed by flow cytometry. Double staining with HLA-DR was performed to identify HLA-DR positive APC. In epidermal cell suspensions no expression of CD1b or CD1d was observed on HLA-DR positive cells (data not shown). CD1a and CD1c expression on HLA-DRbright cells was observed as previously described (Meunier et al., 1993Meunier L. Gonzalez Ramos A. Cooper K.D. Heterogeneous populations of class II MHC+ cells in human dermal cell suspensions. Identification of a small subset responsible for potent dermal antigen-presenting cell activity with features analogous to Langerhans cells.J Immunol. 1993; 151: 4067-4080PubMed Google Scholar; data not shown). In fresh dermal cell suspensions HLA-DR positive cells were found to express CD1a, CD1b, CD1c, and CD1d Figure 2a. These cells exhibited high forward and side scatter properties. Three different HLA-DR+ subsets based on their fluorescence intensity (bright, medium, dim) were identified Figure 2a. Expression of CD1d was observed in the HLA-DRbright population. Preferential expression of CD1d on the HLA-DRbright population indicates that CD1d expression in human dermis is mainly confined to activated dendritic cells. Within HLA-DR positive cells, the mean percentage of CD1 expression was calculated from three independent experiments: CD1a, 25% ± 3.6%; CD1b, 16.3% ± 4.0%; CD1c, 39.3% ± 3.0%; and CD1d, 16% ± 3.6%. A 2 d culture of fresh cell suspensions in complete medium with GM-CSF and IL-4 to induce maturation of dendritic cells did not change the percentages of CD1 positive cells (data not shown). To investigate CD1d expression on highly enriched dermal APC, cells migrating out of dermal fragments after 2 d culture were analyzed. DDC represented 70%-80% of the total population after metrizamide enrichment. These cells were highly HLA-DR positive and expressed CD83 Figure 2c, a marker of mature dendritic cells, as well as high levels of CD80 and CD86 (Nestle et al., 1993Nestle F.O. Zheng X.G. Thompson C.B. Turka L.A. Nickoloff B.J. Characterization of dermal dendritic cells obtained from normal human skin reveals phenotypic and functionally distinctive subsets.. 1993; 151 ([published erratum appears in J Immunol 152:376, 1994]): 6535-6545Google Scholar). HLA-DRbright cells coexpressed CD1b and CD1c. CD1d expression was found on 60.5% ± 8.0% of HLA-DRbright cells Figure 2b. Therefore, mature DDC express both classical MHC and nonclassical CD1 antigen-presenting molecules. To get an insight into the cellular distribution of CD1d molecules two-color immunofluoresence was performed on highly enriched DDC, obtained after migration out of dermal fragments. Cells were double stained with MoAb against HLA-DR and CD1d (42.1 MoAb) and were analyzed by confocal laser scanning microscopy. Strong expression of HLA-DR was observed on DDC, with HLA-DR containing compartments mostly localized close to or on the cell surface Figure 3a. Staining with anti-CD1d MoAb revealed a subset of cells that were CD1d positive Figure 3b. Colocalization of CD1d and HLA-DR (orange/yellow spots) was observed in compartments close to as well as on the cell surface Figure 3c, d. To further investigate the biochemical nature of CD1d expressed on dermal APC, Western blotting was performed on microdissected normal human dermis. CD1d protein was detected with antibody D5. CD1d in dermis was detected at a molecular weight of 50–55 kDa. This might be indicative of a highly glycosylated variant present in human dermis in the absence of a low or unglycosylated form as found in intestinal epithelium (Somnay Wadgaonkar et al., 1999Somnay Wadgaonkar K. Nusrat A. Kim H.S. Canchis W.P. Balk S.P. Colgan S.P. Blumberg R.S. Immunolocalization of CD1d in human intestinal epithelial cells and identification of a beta2-microglobulin-associated form.Int Immunol. 1999; 11: 383-392Crossref PubMed Scopus (68) Google Scholar). The corresponding band of the CD1d positive control cell line C1R CD1d was detected at 37 kDa, and appeared unglycosylated Figure 4. As a next step we investigated the expression of CD1 molecules on mo-DC. These cells correspond to DDC in vivo (Grassi et al., 1998Grassi F. Dezutter-Dambuyant C. McIlroy D. et al.Monocyte-derived dendritic cells have a phenotype comparable to that of dermal dendritic cells and display ultrastructural granules distinct from Birbeck granules.J Leukoc Biol. 1998; 64: 484-493PubMed Google Scholar) and are currently the favored in vitro study system that allows a discrimination between immature and mature dendritic cells (Banchereau et al., 2000Banchereau J. Briere F. Caux C. et al.Immunobiology of dendritic cells.Annu Rev Immunol. 2000; 18: 767-811Crossref PubMed Scopus (5616) Google Scholar). Furthermore they are increasingly used for immunotherapy of cancer. Therefore, CD1 expression in these cells might be of potential significance for basic research as well as clinical applications. Cells were cultured under FBS-free conditions using 1% AP as used in current clinical trials of dendritic cell vaccination. Under these conditions CD1a expression on immature and mature dendritic cells was weak to absent Figure 5. CD1b and CD1c expression was upregulated during in vitro differentiation from monocytes Figure 5. There was stable and equivalent expression of these molecules on immature (D6) and mature dendritic cells (D7 after addition of the maturation cocktail). Only few data are available on CD1d expression in human APC. Expression of CD1d was observed on monocytes as well as on mo-DC Figure 5. The mean fluorescence of mo-DC reached the highest level around day 4. Immature as well as mature dendritic cells expressed CD1d molecules indicating that the expression was not maturation dependent. Earlier studies on mo-DC including dendritic cell vaccination for patient treatment were performed in the presence of 10% FBS (Cella et al., 1997Cella M. Sallusto F. Lanzavecchia A. Origin maturation and antigen presenting function of dendritic cells.Curr Opin Immunol. 1997; 9: 10-16Crossref PubMed Scopus (1157) Google Scholar;Nestle et al., 1998Nestle F.O. Alijagic S. Gilliet M. et al.Vaccination of melanoma patients with peptide- or tumor lysate pulsed dendritic cells.Nat Med. 1998; 4 (Comments in: Nat Med 4:269–270, 1998): 328-332Crossref PubMed Scopus (2686) Google Scholar). Recently, FBS-free culture conditions using 1% AP were introduced (Thurner et al., 1999Thurner B. Roder C. Dieckmann D. et al.Generation of large numbers of fully mature and stable dendritic cells from leukapheresis products for clinical application.J Immunol Meth. 1999; 223 ([published erratum appears in J Immunol Meth April 224:211, 1999]): 1-15Crossref PubMed Scopus (447) Google Scholar). Mo-DC coming from the same patient were generated using AP or FBS as addition to the culture medium. CD1b and CD1c were expressed with the same pattern in both AP- and FBS-treated mo-DC (data not shown). In certain donors expression of CD1a was observed already in the starting population (day 0 monocytes). In contrast to mo-DC generated in AP, FBS-derived mo-DC expressed high levels of CD1a. The reverse was true for CD1d expression. Whereas mo-DC cultured in AP expressed high levels of CD1d, there was an important downregulation of CD1d during culture in FBS and in some cases it was completely absent on mature CD83-positive dendritic cells Figure 6. In conclusion there is a strong dependence of CD1 expression on dendritic cell culture conditions. The CD1 family of antigen-presenting molecules plays an important role in lipid antigen presentation as well as immune response to mycobacteria and tumors (Porcelli et al., 1998Porcelli S.A. Segelke B.W. Sugita M. Wilson I.A. Brenner M.B. The CD1 family of lipid antigen-presenting molecules.Immunol Today. 1998; 19: 362-368Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). In this study we focused on CD1d, a recently described member of this family, and show that CD1d expression in skin is largely restricted to dendritic appearing APC in dermis (DDC) apart from expression on keratinocytes in epidermis (Bonish et al., 2000Bonish B. Jullien D. Dutronc Y. et al.Overexpresssion of CD1d by keratinocytes in psoriasis and CD1d-dependent IFN-gamma production by NK-T cells.J Immunol. 2000; 165: 4076-4085Crossref PubMed Scopus (188) Google Scholar). For comparison, we confirmed previous studies using an expanded panel of antibodies, demonstrating CD1a expression mostly on epidermal Langerhans cells, whereas CD1c was found on DDC Figure 1, 2 (Lenz et al., 1993Lenz A. Heine M. Schuler G. Romani N. Human and murine dermis contain dendritic cells. Isolation by means of a novel method and phenotypical and functional characterization.J Clin Invest. 1993; 92: 2587-2596Crossref PubMed Scopus (261) Google Scholar;Meunier et al., 1993Meunier L. Gonzalez Ramos A. Cooper K.D. Heterogeneous populations of class II MHC+ cells in human dermal cell suspensions. Identification of a small subset responsible for potent dermal antigen-presenting cell activity with features analogous to Langerhans cells.J Immunol. 1993; 151: 4067-4080PubMed Google Scholar;Nestle et al., 1993Nestle F.O. Zheng X.G. Thompson C.B. Turka L.A. Nickoloff B.J. Characterization of dermal dendritic cells obtained from normal human skin reveals phenotypic and functionally distinctive subsets.. 1993; 151 ([published erratum appears in J Immunol 152:376, 1994]): 6535-6545Google Scholar;Larregina et al., 1997Larregina A.T. Morelli A.E. Kolkowski E. Sanjuan N. Barboza M.E. Fainboim L. Pattern of cytokine receptors expressed by human dendritic cells migrated from dermal explants.Immunology. 1997; 91: 303-313Crossref PubMed Scopus (22) Google Scholar). Expression of CD1b on DDC was detectable with two different antibodies. CD1d was found to be mainly expressed on 16% ± 3.6% of HLA-DRbright cells in dermal cell suspensions, but was clearly absent on HLA-DRbright cells in epidermal cell suspensions. 60.5% ± 8.0% of cells expressed CD1d in highly enriched DDC, whereas CD1d expression was absent on Langerhans cells Figure 2. The fact that a distinct subpopulation of DDC expresses CD1d is in strong support of the hypothesis that there are phenotypically distinct subsets of DDC with otherwise homogeneous microscopic appearance and expression of adhesion, costimulatory, and HLA-DR molecules (Meunier et al., 1993Meunier L. Gonzalez Ramos A. Cooper K.D. Heterogeneous populations of class II MHC+ cells in human dermal cell suspensions. Identification of a small subset responsible for potent dermal antigen-presenting cell activity with features analogous to Langerhans cells.J Immunol. 1993; 151: 4067-4080PubMed Google Scholar;Nestle et al., 1993Nestle F.O. Zheng X.G. Thompson C.B. Turka L.A. Nickoloff B.J. Characterization of dermal dendritic cells obtained from normal human skin reveals phenotypic and functionally distinctive subsets.. 1993; 151 ([published erratum appears in J Immunol 152:376, 1994]): 6535-6545Google Scholar;Nestle and Burg, 1999Nestle F.O. Burg G. Dendritic cells: role in skin diseases and therapeutic applications.Clin Exp Dermatol. 1999; 24: 204-207https://doi.org/10.1046/j.1365-2230.1999.00456.xCrossref PubMed Scopus (9) Google Scholar). Interestingly, expression of all known members of the CD1 family was observed on DDC. This may be an advantage in the generation of immune response against tumor antigens or infectious agents such as mycobacteria. Mycobacterial antigens and glycolipids thereof have been shown to access distinct CD1 molecules at different intracellular sites (Schaible et al., 2000Schaible U.E. Hagens K. Fischer K. Collins H.L. Kaufmann S.H.E. Intersection of group I CD1 molecules and mycobacteria in different intracellular compartments of dendritic cells.J Immunol. 2000; 164: 4843-4852Crossref PubMed Scopus (104) Google Scholar). DDC subsets, as defined by differential expression of CD1 molecules, might play a distinct role in skin immune response. To get an insight into the cellular and subcellular distribution on DDC, CD1d was analyzed by confocal laser scanning microscopy. CD1d mostly colocalized to compartments that contained HLA-DR molecules Figure 3. These findings might suggest a common pathway for classical MHC-related and nonclassical CD1-related antigen presentation. Microdissected dermis contained CD1d with a molecular weight of 50–55 kDa Figure 4, indicating the presence of a glycosylated form of CD1d. Glycosylation is often considered to stabilize proteins and might allow for sustained expression of CD1d and prolonged presentation of CD1d-related antigens in human skin. CD1d expression in skin is of potential interest for skin biology as it is involved in the presentation of glycolipids to natural killer T cells. It has been shown that α-galactosylceramide, a synthetic acylphytosphingolipid originally isolated from a marine sponge, is presented by CD1d to the clonotypic vα24/vβ11 T cell receptor of natural killer T cells. Such cells were shown to have potent antitumor activity (Kawano et al., 1999Kawano T. Nakayama T. Kamada N. et al.Antitumor cytotoxicity mediated by ligand-activated human V alpha24 NKT cells.Cancer Res. 1999; 59: 5102-5105PubMed Google Scholar) and also to induce strong TH-1 bias in autoimmune diseases such as diabetes mellitus (Wilson et al., 1998Wilson S.B. Kent S.C. Patton K.T. et al.Extreme Th1 bias of invariant Valpha24JalphaQ T cells in type 1 diabetes.. 1998; 391 ([published erratum appears in Nature 399:84, 1999]): 177-181https://doi.org/10.1038/20005Google Scholar). Self glycolipids might represent potential autoantigens (Shamshiev et al., 1999Shamshiev A. Donda A. Carena I. Mori L. De Kappos L. Libero G. Self glycolipids as T-cell autoantigens.Eur J Immunol. 1999; 29: 1667-1675Crossref PubMed Scopus (251) Google Scholar). Human epidermis is rich in lipids and glycolipids (Wertz, 1992Wertz P.W. Epidermal lipids.Semin Dermatol. 1992; 11: 106-113PubMed Google Scholar). A disturbance in lipid production might lead to aberrant presentation of lipids and glycolipids by CD1d molecules in inflammatory and neoplastic skin conditions. CD1d expression has been demonstrated on psoriatic KC using the anti-CD1d antibody NOR3.2 (Nickoloff et al., 1999Nickoloff B.J. Wrone Smith T. Bonish B. Porcelli S.A. Response of murine and normal human skin to injection of allogeneic blood-derived psoriatic immunocytes: detection of T cells expressing receptors typically present on natural killer cells, including CD94, CD158, and CD161.Arch Dermatol. 1999; 135: 546-552Crossref PubMed Scopus (122) Google Scholar;Bonish et al., 2000Bonish B. Jullien D. Dutronc Y. et al.Overexpresssion of CD1d by keratinocytes in psoriasis and CD1d-dependent IFN-gamma production by NK-T cells.J Immunol. 2000; 165: 4076-4085Crossref PubMed Scopus (188) Google Scholar). This antibody might not always meet stringent staining criteria, however (Yong-Jun Liu, Stanford University, personal communication), or might recognize different CD1d epitopes compared to other available anti-CD1d antibodies. Here we have shown with a panel of anti-CD1d antibodies unequivocal expression on DDC. Therefore, expression of CD1d by keratinocytes and professional APC in human dermis might have implications for autoimmune type chronic inflammatory skin diseases, such as psoriasis. Mo-DC are currently the preferred in vitro study system for dendritic cells. They resemble DDC in phenotype and function (Grassi et al., 1998Grassi F. Dezutter-Dambuyant C. McIlroy D. et al.Monocyte-derived dendritic cells have a phenotype comparable to that of dermal dendritic cells and display ultrastructural granules distinct from Birbeck granules.J Leukoc Biol. 1998; 64: 484-493PubMed Google Scholar). Mo-DC may be generated in large numbers from peripheral blood. They allow the investigation of the impact of maturation stimuli on dendritic cell biology. Furthermore, these cells are increasingly used for the immunotherapy of cancers such as melanoma (Nestle et al., 1999Nestle F.O. Burg G. Dummer R. New perspectives on immunobiology and immunotherapy of melanoma.Immunol Today. 1999; 20: 5-7Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). We detected CD1d expression on monocytes and both mature and immature mo-DC cultivated in the presence of AP Figure 5. In contrast, when AP was replaced by FBS a downregulation of CD1d during the differentiation and maturation process was observed, whereas CD1a was strongly expressed Figure 6. Differential expression of CD1 molecules on dendritic cell preparations used for immunotherapy of cancer might have implications for the repertoire of T cells expanded during the vaccination process. In conclusion we have shown that CD1d reactivity in human dermis is confined to dermal dendritic APC in vivo and in vitro. CD1d in dermis is highly glycosylated and colocalizes with HLA-DR containing cellular compartments. Expression of CD1d on mo-DC used for immunotherapy depends on culture conditions but not on maturation stage. Expression of CD1d on dermal APC might have implications for presentation of lipids and glycolipids to effector T cells with potential relevance for autoimmune and anticancer immune response in skin. We would like to thank Steven Balk and Steven Porcelli for providing CD1 antibodies and cell lines as well as helpful advice. We thank also Mathias Höchli and Thomas Bächi from the Central Laboratory for Electronmicroscopy for help with confocal microscopy. Supported by grants from the Zürich Cancer League, Swiss Cancer League, the Robert Wenner Prize, the European Union, and "Stiftung für wissenschaftliche Forschung der Universität Zürich" to FON as well as a grant from the Ente Cassa di Risparmio di Firenze (Nr 446/1999) to GG.
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