Human skin punch biopsies were obtained under informed consent from normal healthy

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3 SUPPLEMENTAL METHODS Acquisition of human skin specimens. Human skin punch biopsies were obtained under informed consent from normal healthy volunteers (n = 30) and psoriasis patients (n = 45) under a protocol approved by the Stanford University Institutional Review Board and undertaken at the Stanford University Dermatology Clinic. Persons on systemic immunosuppressive agents, including steroids, were excluded from the study. Psoriasis patients considered for the study had a Psoriasis Area Severity Index (PASI) between 8.5 and 26 and a typical lesion of at least 1 cm suitable for biopsy. No topical steroids were applied to the lesion for at least two weeks prior to biopsy. At the study visit, two adjacent 4-mm biopsies were taken from the selected lesion, and a similar set was taken from a non-lesional site. Normal volunteers had two adjacent biopsies collected. PASI scores were determined for each patient at the study visit, and a lesion score determined. All lesion scores were between 5 and 11. All biopsies were immediately frozen in liquid nitrogen and stored at -80 o C. T cell proliferation assay CD4 + CD45RO + T cells (5 x 10 4 per well) were stimulated with beads coated with anti- CD3, anti-cd28 and anti-cd2 (T Cell Activation/Expansion Kit, Miltenyi Biotec) in the presence of IL-2 (100 U/ml) alone or with increasing concentrations of human IL-23 for 72 h and pulsed with 3 [H]-thymidine (TRK-T20, Amersham) for the final 6 h before harvesting to glass filters. 3 [H]-thymidine incorporation was determined by liquidscintillation counting with a Beckman β-counter.

4 RNA isolation. For real-time PCR or microarray analysis, total RNA was isolated by either of two methods. For tissue RNA isolation, organs were homogenized into RNA STAT-60 (Tel- Test) using a polytron homogenizer, and total RNA was extracted according to the manufacturer's instructions. After isopropanol precipitation, total RNA was re-extracted with phenol:chloroform:isoamyl alcohol (25:24:1) (Sigma-Aldrich) using phase-lock light tubes (Eppendorf). For cellular samples, RNA was isolated using the RNeasy method, according to the manufacturer s protocol (Qiagen). Total RNA (5 µg) was subjected to treatment with DNase (Roche Molecular Biochemicals) according to manufacturer's instructions to eliminate possible genomic DNA contamination. Real-time quantitative PCR for gene expression. DNase-treated total RNA was reverse-transcribed using Superscript II (Invitrogen) according to manufacturer's instructions. Primers were designed using Primer Express (PE Biosystems), or obtained commercially from Applied Biosystems. Real-time quantitative PCR on 10 ng of cdna from each sample was performed using either of two methods. In the first method, two gene-specific unlabelled primers were utilized at 400 nm in a Applied Biosystems SYBR green real-time quantitative PCR assay utilizing an ABI 7000, 7300 or 7900 instrument. In the second method, two unlabelled primers at 900 nm each were used with 250 nm of FAM-labeled probe (Applied Biosystems) in a TAQMAN TM real-time quantitative PCR reaction on an ABI 7000, 7300 or 7700 sequence detection system. The absence of genomic DNA contamination was confirmed using primers that recognize genomic region of the CD4 promoter. Quantities of

5 transcripts encoding ubiquitin (UBB) were measured in a separate reaction and used to normalize the data by the - Ct method 1 The following primers were used (Forward/Reverse, 5-3 ) : IL17A: CAACCGATCCACCTCACCTT GGCACTTTGCCTCCCAGAT IL17F: TGCCAGGAGGTAGTATGAAGCTT ATGCAGCCCAAGTTCCTACACT IL22: GCAGGCTTGACAAGTCCAACT GCCTCCTTAGCCAGCATGAA IL26: TTTGAGGTGTGGGTTGCTGTTA TCAACAGCTTGGGACAATGTTC IFNG: CTTTAAAGATGACCAGAGCATCCA ATCTCGTTTCTTTTTGTTGCTATTGA IL1B: AGCACCTCTCAAGCAGAAAACAT TTGCATGGTGAAGTCAGTTATATCC CCL20: CTGGCTGCTTTGATGTCAGTG GCAGTCAAAGTTGCTTGCTGC RORC: TGAGAAGGACAGGGAGCCAA CCACAGATTTTGCAAGGGATCA IL17RA: GCGCCCAGACCAGAAGAG CCCTTTAAGGTTGCGTAGAGTGA IL17RC: GCCTCTCAAAGACGATGTGCTA GAGGCTTTGCTGGGTAGTGAA IL10RB: GCAAACAACCCATGACGAAA TGAAGACCGAGGCCATGAG IL20RA: GGCCCGCAAACGTTACAGTA GGTCGTCGATGGCTCTTCC IL22RA: S100A7: ACACGGTCTACAGCATCGAGTATAA CGGTGACCCTGGCATAGTAGAG GCATGATCGACATGTTTCACAAATACAC TGGTAGTCTGTGGCTATGTCTCCC Primers for S100A8, S100A9, DEFB4, DEFB103A and DEFB2 were obtained from ABI. 1 Fehniger, T. A. et al. Differential cytokine and chemokines gene expression by human NK cells following activation with IL-18 or IL-15 in combination with IL-12: implications for the innate immune response. J. Immunol 162, (1999)

6 Intracellular cytokine staining T cells were stimulated with PMA and ionomycin in the presence of Golgi-plug for 4 h and were permeablized using Cytofix/Cytoperm reagents (BD Biosciences) according to manufacturer s instructions. Cells were incubated with anti-ifn-γ (BD Pharmingen) and anti-il-17a (DNAX, clone #12B12) and were washed. Cells producing IFN-γ and/or IL-17A were analyzed by flow cytometry. IL-17A ELISA and IL-17F electrochemiluminescence The Lower Limit of Quantitation of the human IL-17A Elisa was 1 pg/ml and has no cross reactivity to IL-17F up to 50 ng/ml which was the highest concentration tested. The Lower Limit of Quantitation of the human IL-17F electrochemiluminescence assay was 3 pg/ml and has no cross reactivity to IL-17A up to 50 ng/ml which was the highest concentration tested.