Gene Expression Time Course in the Human Skin during Elicitation of Allergic Contact Dermatitis  Malene B. Pedersen, Lone Skov, Torkil Menné, Jeanne D. Johansen, Jørgen Olsen  Journal of Investigative Dermatology  Volume 127, Issue 11, Pages 2585-2595 (November 2007) DOI: 10.1038/sj.jid.5700902 Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 CA score plot. Plot of CA scores for the first and second CA axes. The projections of samples from control subjects, early (0 and 7hours) patient samples and late (48 and 96hours) patient samples are indicated by circles. A single late patient sample (P7_48.CEL) is projected together with the early patient group. The genes corresponding to the probe sets that define the positive and negative directions of the CA axis were analyzed for overrepresentation of terms for biological processes. Overrepresented Go IDs and terms (compared to the list of genes defining the other end of the same axis) are written in continuation of the axes. Journal of Investigative Dermatology 2007 127, 2585-2595DOI: (10.1038/sj.jid.5700902) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Hierarchical clustering of genes involved in the elicitation of ACD. Hierarchical clustering of the 74 probe sets that were significantly differentially expressed in at least one time point from the patient dataset, as determined by ANOVA. In the cluster diagram, each row represents a probe set and each column represents a biopsy. The number written immediately after the letter (P for patients) refers to the person and the number after the underscore refers to the time point. For example, P7_7 is data from patient no. 7 from the 7-hour time point. The colours indicate the experession level: red highest, green lowest. Journal of Investigative Dermatology 2007 127, 2585-2595DOI: (10.1038/sj.jid.5700902) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Expression of CCL19 and CCR7 in nickel-exposed skin from allergic subjects. Punch biopsies of 4mm were taken from a nickel-allergic subject at the time points 0, 7, 48, and 96hours following the exposure of skin to nickel during a patch test. The biopsies were taken from the skin beneath the patch test. The biopsies from the time points (a) 0hour, (b) 7hours, and (c and d) 96hours after nickel exposure were analyzed with a CCL19 antibody. (c) At the 96-hour time-point staining deep dermal structures are visible. (d) At a higher magnification the staining appears cloudy around cells with an irregular morphology. (e) The expression of CCR7 was analyzed in a biopsy from a control subject and (f) from an allergic subject 96hours after nickel exposure. The staining was found in cells scattered in and beneath (e) the epidermis and in the dermis. In some areas the CCR7-positive cells were arranged in clusters in (f) the deep dermis. Bar=250μm. Journal of Investigative Dermatology 2007 127, 2585-2595DOI: (10.1038/sj.jid.5700902) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 CCL19 mRNA is expressed in the deep dermis beneath the area of nickel exposure during elicitation of ACD. A 4mm punch biopsy from the 48-hour time point of the time-series experiment shown in Figure 3 was sectioned in parallel to the epidermal surface. The sections were pooled in fractions and RNA extracted. Every tenth section was retained for haematoxylin/eosin staining. The micrograph (bar=100μm) on the left is a section from the 96-hour time point. The micrographs (bar=100μm) on the right are haematoxylin/eosin-stained sections of the 48-hour biopsy (Figure 3 legend) corresponding to section 10 (700μm from the surface), section 20 (140μm from the surface), and section 40 (280μm from the surface). Sections 1–9 were pooled as fraction 1 (epidermis fraction), sections 11–20 were pooled as fraction 2 (upper dermis fraction), and sections 31–100 were pooled as fraction 3 (lower dermis fraction). RNA was extracted and converted into cDNA. Copy numbers of the indicated transcripts were measured in the fractions by quantitative real-time PCR. The transcript for glyceraldehyde 3-phosphate dehydrogenase was used for normalization. The CCL19 and CCR7 transcripts could not be detected in the fractions 1 and 2 (indicated by asterisks), but were detectable in fraction 3 corresponding to the lower dermis fraction. Journal of Investigative Dermatology 2007 127, 2585-2595DOI: (10.1038/sj.jid.5700902) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions