Nature Immunology: doi: /ni Supplementary Figure 1. Cytokine pattern in skin in response to urushiol.
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1 Supplementary Figure 1 Cytokine pattern in skin in response to urushiol. Wild-type (WT) and CD1a-tg mice (n = 3 per group) were sensitized and challenged with urushiol (uru) or vehicle (veh). Quantitative real-time PCR was performed to analyze cytokine gene expression in the ear tissues obtained on day 2 after challenge. Results are presented as fold increases over vehicle-treated ear tissues from wild-type mice. Results are representative of two independent experiments (mean ± s.e.m).
2 Supplementary Figure 2 CD1a suppresses contact hypersensitivity mediated by IFN- -producing cells. Wild-type (WT) and CD1a-tg mice (n = 3 per group) were sensitized with 0.5% DNFB by painting on the abdomen on day 0, and challenged with either 0.2% DNFB or vehicle (veh) on ears on day 5 after sensitization. (a) Ear swelling after challenge. (b) Frequencies of Gr-1 hi CD11b hi granulocytes (left panel), CD4 + or CD8 + T cells among live CD45 + TCR + cells (middle panel), and IL- 17A + or IFN- + cells among TCR + cells (right panel). (c) Absolute cell numbers of CD8 + or CD4 + T cells, IFN- - or IL-17A-producing cells in ears. * P < 0.05, ** P < 0.01, *** P < 0.001, using unpaired t-test. Results are representative of three independent experiments (mean ± s.e.m).
3 Supplementary Figure 3 V TCR subfamily profile in skin and thymus. (a-e) Ear cells or thymocytes were isolated from wild-type (WT) or CD1a-tg mice on day 2 after urushiol C15:2 or vehicle challenge and analyzed for 15 different V TCR subfamilies using flow cytometry. Relative contribution of each V + cell subset among CD45 + CD3 + CD4 + T cells is presented in bar graphs. (a) V TCR repertoire in urushiol-treated skin. (b) Frequencies of IL-17A + cells among V 2 + or V 4 + CD4 + T cells in ear. (c) Frequencies of V 2 + or V 4 + cells in ears from CD1a-tg versus CD1a-tg mice injected with anti-cd1a antibody. (d) V TCR repertoire in vehicle-treated skin. (e) V TCR repertoire in thymus. * P < 0.05, ** P < 0.01; NS, not significant, using unpaired t-test. Results are representative of two to three independent experiments (mean ± s.e.m, a,c,d).
4 Supplementary Figure 4 Electron-density shots of urushiol in the antigen-binding cleft of CD1a. (a) Fo-Fc electron density map (yellow orange) of urushiol contoured at 2.2 level. (b) 2Fo-Fc electron density map (blue) of urushiol contoured at 0.8 level.
5 Supplementary Table 1. Data collection and refinement statistics
6 Supplementary Table 2. CD1a contacts with Urushiol
7 Supplementary Table 3. Flow cytometry antibody list Name Clone Dilution Vendor anti-cd3ε 145-2C11 1:200 BioLegend anti-cd8α :400 BioLegend anti-cd4 GK1.5 1:400 BioLegend anti-cd :200 BioLegend anti-ifn-γ XMG1.2 1:200 BioLegend anti-cd1a HI149 1:200 BioLegend anti-il-17f 9D3.1C8 1:200 BioLegend anti-il-22 Poly5164 1:200 BioLegend anti-γδtcr GL3 1:200 BioLegend anti-cd11b M170 1:200 BioLegend anti-gr-1 RB6-8C5 1:200 BioLegend anti-f4/80 BM8 1:200 BioLegend anti-cd11c N418 1:200 BioLegend anti-il-17a ebio17b7 1:200 ebioscience anti-cd207 ebiol31 1:200 ebioscience anti-cd103 2E7 1:200 ebioscience anti-rorγt B2D 1:200 ebioscience anti-mhc class II M5/ :200 ebioscience Anti-CD16/CD32 2.4G2 1µg/ml Bio-XCell
8 Supplementary Table 4. Primers for qpcr Mouse Actb, forward Actb, reverse Il1b, forward Il1b, reverse Il22, forward Il22, reverse Tnf, forward Tnf, reverse Il23, forward Il23, reverse Il10, forward Il10, reverse Il17a, forward Il17a, reverse 5 -TCCAGCCTTCCTTCTTGGGTATGGA-3 5 -CAGGCAGGCAGTATCACTCA-3 5 -CAGGCAGGCAGTATCACTCA-3 5 -AGGTGCTCATGTCCTCATCC-3 5 -CCGAGGAGTCAGTGCTAAGG-3 5 -GTAGGGCTGGAACCTGTCTG-3 5 -CCGATGGGTTGTACCTTGTC-3 5 -AGATAGCAAATCGGCTGACG-3 5 -CCAGCGGGACATATGAATCT-3 5 -TGTGGGTCACAACCATCTTC-3 5 -CCAAGCCTTATCGGAAATGA-3 5 -TCTCACCCAGGGAATTCAAA-3 5 -AGCTGGACCACCACATGAAT-3 5 -AGCATCTTCTCGACCCTGAA-3 Human IL22, forward IL22, reverse IL17A, forward IL17A, reverse IFN-γ, forward IFN-γ, reverse 5 -GCTTGACAAGTCCAACTTCCA-3 5 -GCTCACTCATACTGACTCCGT-3 5 -TCCCACGAAATCCAGGATGC-3 5 -GGATGTTCAGGTTGACCATCAC-3 5 -TCGGTAACTGACTTGAATGTCCA-3 5 -TCCTTTTTCGCTTCCCTGTTTT-3
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