Gianni Gerlini, Hans Peter Hefti, Martin Kleinhans, Brian J

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CD1d is Expressed on Dermal Dendritic Cells and Monocyte-Derived Dendritic Cells Gianni Gerlini, Hans Peter Hefti, Martin Kleinhans, Brian J. Nickoloff, Günter Burg, Frank O. Nestle Journal of Investigative Dermatology Volume 117, Issue 3, Pages 576-582 (September 2001) DOI: 10.1046/j.0022-202x.2001.01458.x Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Expression of CD1 molecules, including CD1d, on skin dendritic APC. Representative immunohistochemical staining of normal skin. (a) Epidermal dendritic cells and DDC (arrow) stained with anti-CD1a (NA1/34). (b) -(d) Dendritic dermal APC express CD1b (b, BCD1b3.1.6), CD1c (c, L161), and CD1d (d, polyclonal rabbit antibody). (e) -(f) A subset of cultivated DDC express CD1d. Immuno histochemical staining of migrated DDC using anti-CD1d antibody (e, 51.1.3). There is strong (f, black arrow), intermediate (blue arrow), and negative (green arrow) expression of CD1d. Note the clustering of small lymphocytes with CD1d positive DDC (black arrow). Scale bar: (a–e) 50 μm; (f) 10 μm. Journal of Investigative Dermatology 2001 117, 576-582DOI: (10.1046/j.0022-202x.2001.01458.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 CD1d expression on fresh and migrated DDC. (a) HLA-DRbright cells express CD1d in fresh dermal cell suspension. Two-color staining of dermal cell suspension analyzed by flow cytometry shows different HLA-DR+ cell subsets: CD1a (25% ± 3.6% of the total HLA-DR+ population), CD1b (16.3% ± 4.0%), CD1c (39.3% ± 3.0%), and CD1d (16% ± 3.6%). (b) Migrated DDC express CD1d. Two-color staining of migrated DDC analyzed by flow cytometry: 60.5% ± 8.0% of DDC were positive for CD1d. (c) Migrated mature CD83 positive DDC express CD1d. Journal of Investigative Dermatology 2001 117, 576-582DOI: (10.1046/j.0022-202x.2001.01458.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 HLA-DR and CD1d molecules are colocalized in the same cellular compartments. Immunofluorescence staining of DDC analyzed by confocal laser scanning microscopy: (a) HLA-DR-FITC; (b) CD1d-Texas Red (42.1); and (c, d) double staining. Arrows show double positive cells (c, yellow/orange). Note at higher magnification the colocalization of HLA-DR and CD1d represented by yellow/orange spots (d). Scale bars: 10 µm. Journal of Investigative Dermatology 2001 117, 576-582DOI: (10.1046/j.0022-202x.2001.01458.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 A high molecular variant of CD1d is present in human dermis. Western blot analysis of microdissected dermal sections using CD1d antibody (D5). C1R mock (–) and C1R CD1d transfectants (+) were used as negative and positive controls. A, B, C indicate different dermal samples. Note the presence of a CD1d molecule with apparent molecular weight 50–55 kDa. Journal of Investigative Dermatology 2001 117, 576-582DOI: (10.1046/j.0022-202x.2001.01458.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 CD1 expression on mo-DC generated in the presence of AP. Monocytes were cultured in the presence of IL-4 and GM-CSF for 6 d in 1% AP. At day 6 they were stimulated with a maturation cocktail containing IL-6, TNF-α, and IL-1β for 24 h. At days 0, 2, 4, 6, and 7 cells were analyzed for CD1 molecule expression. Values represent the mean fluores cence. Mo-DC generated in AP display a low level of CD1a expression, whereas CD1b and CD1c molecules are strongly upregulated during maturation. CD1d is constantly expressed during culture. Journal of Investigative Dermatology 2001 117, 576-582DOI: (10.1046/j.0022-202x.2001.01458.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 CD1d expression is downregulated in the presence of FBS. Effects of FBS versus AP on CD1a and CD1d expression were evaluated. Mo-DC cultured in FBS upregulate CD1a expression whereas CD1d expression is downregulated. Addition of AP instead of FBS to the culture medium leads to low or undetectable CD1a expression on mo-DC, whereas CD1d expression is detectable throughout the culture period. Journal of Investigative Dermatology 2001 117, 576-582DOI: (10.1046/j.0022-202x.2001.01458.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions