Dermal Dendritic Cells Associated with T Lymphocytes in Normal Human Skin Display an Activated Phenotype Alexander D. McLellan, Axel Heiser, Rüdiger.

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Dermal Dendritic Cells Associated with T Lymphocytes in Normal Human Skin Display an Activated Phenotype Alexander D. McLellan, Axel Heiser, Rüdiger V. Sorg, David B. Fearnley, Derek N.J. Hart Journal of Investigative Dermatology Volume 111, Issue 5, Pages 841-849 (November 1998) DOI: 10.1046/j.1523-1747.1998.00375.x Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Flow cytometric analysis of activation and costimulatory antigens on fresh Langerhans cells, fresh dermal DC, and 2 d migratory dermal DC. Contour plots of (a) fresh Langerhans cells, (b) gradient enriched dermal DC following collagenase release (DDC), or (c) migratory dermal DC (mDDC) labeled with the indicated MoAb and FITC-SAM (horizontal axis) and HLA-DR-PE (vertical axis). The quadrant gates were set to contain >98% of negative control labeled cells. Ig con, representative negative control. The results are representative of at least three experiments performed. Journal of Investigative Dermatology 1998 111, 841-849DOI: (10.1046/j.1523-1747.1998.00375.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Forward and side scatter dot plots of dermal cells obtained either (a) directly following 1 h collagenase digestion or (b) following a single Lymphoprep density gradient cut. CD45+ leukocytes were backgated and are shown as black dots against total cells as gray dots. The typical positioning of myeloid and lymphoid gates used for size gating for flow cytometric analysis in this study are shown in (b). Journal of Investigative Dermatology 1998 111, 841-849DOI: (10.1046/j.1523-1747.1998.00375.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 MGG-stained cytospins of gradient-enriched collagenase released dermal mononuclear cell suspensions showing (a) cells displaying typical DC morphology, (b, c) vacuolated myeloid cells, probably macrophages, with multiple long cytoplasmic processes and often containing ingested melanin granules (c; arrow), (d) small dermal cells with typical lymphocyte morphology, and (e) fresh, activated dermal cells sorted for high density CMRF-44 expression. A melanophage present in the CMRF-44++ fraction (e) is arrowed. Scale bar: 10 μm. Journal of Investigative Dermatology 1998 111, 841-849DOI: (10.1046/j.1523-1747.1998.00375.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Allogeneic MLR stimulatory capacity of dermal and epidermal DC. (a–c) [3H]Thymidine uptake (y-axis; mean ± SD) by T lymphocytes stimulated for 5 d (□) or 7 d (•) by (a) collagenase-released gradient-enriched dermal cells (fDDC), (b) fresh Langerhans cells (fLC), or (c) 2 d migratory dermal DC (mDDC). (d) Allostimulatory activity of HLA-DQ++/CMRF-44++ (•) or HLA-DQ++/CMRF-44– dermal DC (□), sorted by flow cytometry, as determined in the 7 d MLR. For (a), (b), and (c), the y axis stimulator numbers were adjusted to show approximate numbers of HLA-DR+++ positive dermal DC or Langerhans cells per well, as determined from flow cytometric analyses. Representative of at least three experiments performed. Journal of Investigative Dermatology 1998 111, 841-849DOI: (10.1046/j.1523-1747.1998.00375.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Co-expression of CD1a with CMRF-44, CD83, or CD86 on HLA-DR+++ gated dermal DC. Collagenase-released gradient-enriched dermal DC populations were labeled with the MoAb indicated, followed by FITC-SAM (horizontal axis) and counterstained with CD1a-PE (vertical axis) and HLA-DR-QR (not shown). Contour plots were generated by gating myeloid sized, strongly HLA-DR positive cells. Representative of four experiments performed. neg con, negative control. Journal of Investigative Dermatology 1998 111, 841-849DOI: (10.1046/j.1523-1747.1998.00375.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Differentiation/activation antigens on dermal dendritic cells. Double immunohistochemical labeling of tissue macrophage (CD14, blue, first MoAb) with papillary dermal DC populations (arrowed, red, second MoAb) in en face sections of normal human skin labeled for (a) HLA-DR, (b) CMRF-44, (c) CD83, and (d) CD86. Scale bar: 10 μm. Data are from 3–8 different breast specimens. Journal of Investigative Dermatology 1998 111, 841-849DOI: (10.1046/j.1523-1747.1998.00375.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Leukocyte counts in the upper dermis. Density of CD14 positive macrophages, CD1a and HLA-DR positive DC, as well as activated CMRF-44, CD83, and CD86 positive DC as assessed by immunohistochemical analysis of the upper dermis of normal human breast skin. Total (▪) or CD14 negative (□) counts are plotted as means of 3–8 different donors expressed as leukocytes per mm2. Journal of Investigative Dermatology 1998 111, 841-849DOI: (10.1046/j.1523-1747.1998.00375.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Co-localization of activated DC with T lymphocytes in the upper dermis. Co-localization of CMRF-44+ (a, c, e) and CD83+ (b, d, f) dermal cells (red) with CD3+ T lymphocytes (blue) in the upper dermis (a, b), in periadnexal regions (c, d) and with CD25+ lymphocytes (blue) also in periadnexal regions (e, f). Co-localization of CMRF-44+ dermal DC (blue) with CD45RO+ lymphocytes (red) lining a sebaceous gland (g) and surrounding a hair follicle (h). The order of addition of the MoAb is given in the bottom left of each plate (first MoAb was revealed with fast blue, second with red AEC). Note that the first and second MoAb were reversed in (g) and (h) to avoid reaction of CD45RO with dermal DC. Scale bar: 10 μm. Data are from 3–7 different breast specimens. Journal of Investigative Dermatology 1998 111, 841-849DOI: (10.1046/j.1523-1747.1998.00375.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 9 Co-localization of activated DC with T lymphocytes in the upper dermis. The association of CD1a, CMRF-44, CD83 positive, and CD86 positive dermal cells with T lymphocyte populations identified by CD3 (▪) or CD8 (□) staining was qualified. The percentage of total dermal cells showing direct membrane abutment to T lymphocytes are plotted as mean values observed in 3–7 different breast skin specimens. Journal of Investigative Dermatology 1998 111, 841-849DOI: (10.1046/j.1523-1747.1998.00375.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions