Suction blistering the lesional skin of vitiligo patients reveals useful biomarkers of disease activity James P. Strassner, BS, Mehdi Rashighi, MD, Maggi.

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Suction blistering the lesional skin of vitiligo patients reveals useful biomarkers of disease activity James P. Strassner, BS, Mehdi Rashighi, MD, Maggi Ahmed Refat, MBBCh, MSc, Jillian M. Richmond, PhD, John E. Harris, MD, PhD Journal of the American Academy of Dermatology Volume 76, Issue 5, Pages 847-855.e5 (May 2017) DOI: 10.1016/j.jaad.2016.12.021 Copyright © 2016 American Academy of Dermatology, Inc. Terms and Conditions

Fig 1 Modified suction-blister technique. A, Graphic demonstrating T cells clustering near the epidermis, where the chemokine concentration is highest (top panel). After suction blistering, the epidermis forms the roof (blister), which becomes separated from the dermis (middle panel). T cells and chemokine at the site are present in the blister fluid and can be aspirated by fine needle aspiration (bottom panel). B, Representative photos from a patient with active vitiligo before blistering (left panel), the same site after blistering (middle panel), and of a nonlesional site (right panel). C, A study subject waits while the suction blisters form. Journal of the American Academy of Dermatology 2017 76, 847-855.e5DOI: (10.1016/j.jaad.2016.12.021) Copyright © 2016 American Academy of Dermatology, Inc. Terms and Conditions

Fig 2 CD8+ T-cell phenotyping and quantification. A, Representative flow plots of the T-cell infiltrate isolated from lesional, nonlesional, and healthy control skin. B, C-X-C motif chemokine receptor (CXCR) 3 expression on CD8+ T cells in lesional skin. C, Quantification of the CD8+ T-cell infiltrate of each sample normalized to 10,000 CD45+ cells for the defining group (left panel) and paired analysis of vitiligo subject samples (lesional vs nonlesional, right panel). E, Quantification of the CD8+ T-cell infiltrate of each sample normalized to 10,000 CD45+ cells for the validation group (left panel) and paired analysis of vitiligo subject samples (lesional vs nonlesional, right panel). D and F, Receiver operating characteristic curve for the defining group (D) and the validation group (F). Red dots mark threshold values described in the text. Journal of the American Academy of Dermatology 2017 76, 847-855.e5DOI: (10.1016/j.jaad.2016.12.021) Copyright © 2016 American Academy of Dermatology, Inc. Terms and Conditions

Fig 3 Chemokine protein levels in blister fluid. Quantification of C-X-C motif chemokine ligand (CXCL) 9 (A and C) or CXCL10 (B and D) in the blister fluid of the defining group (top panels) and validation group (bottom panels). Each panel is organized in the following manner: quantification of individual samples grouped by active lesional, active nonlesional, and healthy control (left), paired analysis of lesional and nonlesional blisters from vitiligo subjects (middle panel), and receiver operating characteristic curve (right). Red dots mark threshold values described in the text. Journal of the American Academy of Dermatology 2017 76, 847-855.e5DOI: (10.1016/j.jaad.2016.12.021) Copyright © 2016 American Academy of Dermatology, Inc. Terms and Conditions

Fig 4 Serum chemokine levels in patients with vitiligo and healthy controls. Serum levels of C-X-C motif chemokine ligand (CXCL) 9 and CXCL10 are reported for the defining group (A) and the validation group (B). C, Serum levels of CXCL9 (left) and CXCL10 (right) are compiled from both groups. D, Serum levels of CXCL11 (left) and IFN-γ (right). Journal of the American Academy of Dermatology 2017 76, 847-855.e5DOI: (10.1016/j.jaad.2016.12.021) Copyright © 2016 American Academy of Dermatology, Inc. Terms and Conditions

Fig 5 Concentrations of biomarkers CD8, CXCL9, and CXCL10 in a patient following conventional treatment. Subject began treatment at month 0, inconsistently followed treatment up to 4 months, and then consistently followed treatment from month 4 to month 8. A, T cells and chemokine protein reported at each visit (left panels). Representative slopes comparing lesional infiltrate to nonlesional infiltrate (right panels). B and C, CXCL9 concentration measured in the blister fluid (B) and serum (C) at each visit. D and E, CXCL10 concentration in the blister fluid (D) and serum (E) at each visit. Journal of the American Academy of Dermatology 2017 76, 847-855.e5DOI: (10.1016/j.jaad.2016.12.021) Copyright © 2016 American Academy of Dermatology, Inc. Terms and Conditions

Supplemental Fig 1 Gating strategy and flow controls. A, CD8+ and CD4+ cells were gated in the following manner: singlets, cell gate, live cells by dead stain exclusion, all CD45+ cells, and all CD3+ T cells. B, Stained whole blood was used to make the fluorescence minus one (FMO) controls to draw the C-X-C motif chemokine receptor (CXCR) 3 gate. Journal of the American Academy of Dermatology 2017 76, 847-855.e5DOI: (10.1016/j.jaad.2016.12.021) Copyright © 2016 American Academy of Dermatology, Inc. Terms and Conditions

Supplemental Fig 2 CD3 and CD4 quantification. A-C, Quantification of CD3+ and CD4+ cells normalized to CD45+ cells in the skin of vitiligo subjects and healthy controls. Results are shown separately for the defining group (A), validation group (B), and both data sets combined (C). D, Receiver operating characteristic analysis of compiled CD3 counts from both groups. Red dot signifies the normalized CD3 T-cell threshold value 6927, which achieves a sensitivity of 81.4% and specificity of 75.5%. Journal of the American Academy of Dermatology 2017 76, 847-855.e5DOI: (10.1016/j.jaad.2016.12.021) Copyright © 2016 American Academy of Dermatology, Inc. Terms and Conditions

Supplemental Fig 3 Skin C-X-C motif chemokine ligand (CXCL) 11 and interferon-γ (IFN-γ) protein concentration. Quantification of CXCL11 (A) and IFN-γ (B) protein concentration by enzyme-linked immunosorbent assay of blister fluid in vitiligo subjects and healthy controls. Journal of the American Academy of Dermatology 2017 76, 847-855.e5DOI: (10.1016/j.jaad.2016.12.021) Copyright © 2016 American Academy of Dermatology, Inc. Terms and Conditions