David A. Norris, Brian L. Kotzin Journal of Investigative Dermatology

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Oligoclonal Expansion of Intraepidermal T Cells in Psoriasis Skin Lesions David A. Norris, Brian L. Kotzin Journal of Investigative Dermatology Volume 117, Issue 6, Pages 1546-1553 (December 2001) DOI: 10.1046/j.0022-202x.2001.01548.x Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Quantitative competitive PCR for T cell receptor chain. (A) Diagram of the construction of competitors used for quantitative competitive PCR analysis of TCRBV expression. Details of each TCRBV competitor construction are described in Materials and Methods. Arrows indicate the position of PCR primers used to amplify the competitor fragment. (B) Quantitative competitive PCR for TCRBC and TCRBV2 in the lesion 2 of psoriasis patient 3. A fixed amount of cDNA was added to compete with dilutions of the competitors. At least four competitor dilutions were used for each TCRBV or BC determination. PCR products were separated on a 2.5% agarose gel and the intensity of fluorescence quantified. (C) Logarithmic plots of quantitative competitive PCR titration and calculation of TCRBV percentage. The concentration of the target DNA in the sample was obtained when the ratio of competitor/target equals 1. Journal of Investigative Dermatology 2001 117, 1546-1553DOI: (10.1046/j.0022-202x.2001.01548.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 CDR3 length analysis of psoriasis patient 2 and a healthy control. The cDNA from CD8+ PBL of a healthy control (a) and CD8+ PBL of psoriasis patient 2 (b) as well as from the lesions of the patient (c) were amplified with 24 TCRBV specific primers paired with the fluorescein-labeled TCRBC primer. The amplified CDR3 variable regions were then separated by a 6% polyacrylamide sequencing gel and scanned with a FluorImager Two TCRBV were analyzed in each lane to conserve cDNA. The BV families were indicated above each set of samples with upper and lower number representing the spectra at top and bottom, respectively. Journal of Investigative Dermatology 2001 117, 1546-1553DOI: (10.1046/j.0022-202x.2001.01548.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Summary of CDR3 length analysis of psoriasis patients 2–5 and a healthy control. The CDR3 spectra were analyzed by ImageQuaNT and compared with the Gaussian distributions obtained from the CD4+ PBL of a healthy individual. Peaks that are 10% above their corresponding peaks in a normal Gaussian background were considered abnormal. The total percentage of the abnormal peaks in each BV family were grouped into <25%, 25–50%, and >50% (symbols for each group shown in figure). The white square corresponds to a normal Gaussian distribution. Typical histograms of each category are illustrated under the symbols. Journal of Investigative Dermatology 2001 117, 1546-1553DOI: (10.1046/j.0022-202x.2001.01548.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 CDR3 length analysis of psoriasis patient 6 (PP6). cDNA from CD4+ PBL, CD8+ PBL, and the epidermis of lesion center (L), lesion margin (L/N) and nonlesional skin (N) were amplified with TCRBV-BC primer pairs and separated by 6% sequencing gels. The gels were analyzed as described in the Materials and Methods. Journal of Investigative Dermatology 2001 117, 1546-1553DOI: (10.1046/j.0022-202x.2001.01548.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions