CD4+CD25+FOXP3+ malignant T cells in Sézary syndrome are not necessarily functional regulatory T cells David A. Wada, MD, Mark R. Pittelkow, MD, Nneka.

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CD4+CD25+FOXP3+ malignant T cells in Sézary syndrome are not necessarily functional regulatory T cells David A. Wada, MD, Mark R. Pittelkow, MD, Nneka I. Comfere, MD, Lawrence E. Gibson, MD, Stephen M. Ansell, MD, PhD, Ryan A. Wilcox, MD, PhD Journal of the American Academy of Dermatology Volume 69, Issue 3, Pages 485-489 (September 2013) DOI: 10.1016/j.jaad.2010.01.013 Copyright © 2010 American Academy of Dermatology, Inc. Terms and Conditions

Fig 1 FOXP3+ cells are present in skin biopsy samples of two patients with Sézary syndrome. A, Photomicrograph of a skin biopsy specimen from patient 1. Scale bar represents 125 μm. Left, Hematoxylin–eosin stain; original magnification: ×200; inset, the infiltrate contains abundant CD4+ cells. Right, FOXP3+ cells (original magnification: ×200); inset, cells do not express CD25. B, Peripheral blood flow cytometry showed that Vβ-restricted neoplastic T cells did not have increased expression of FOXP3. C, Photomicrograph of a skin biopsy specimen from patient 2. Scale bar represents 125 μm. Left, Hematoxylin–eosin stain; original magnification: ×200; inset, the infiltrate contains high numbers of CD4+ cells. Right, FOXP3+ cells (original magnification: ×200); inset, cells are positive for CD25 (original magnification: ×200). D, Peripheral blood flow cytometry showed Vβ-restricted neoplastic T cells highly expressed FOXP3. E, Peripheral blood flow cytometry from a healthy (control) donor shows the CD4+CD25high population expresses FOXP3 at levels similar to those of patient 2, whereas the CD4+CD25– population does not express FOXP3 (similar to patient 1). Journal of the American Academy of Dermatology 2013 69, 485-489DOI: (10.1016/j.jaad.2010.01.013) Copyright © 2010 American Academy of Dermatology, Inc. Terms and Conditions

Fig 1 FOXP3+ cells are present in skin biopsy samples of two patients with Sézary syndrome. A, Photomicrograph of a skin biopsy specimen from patient 1. Scale bar represents 125 μm. Left, Hematoxylin–eosin stain; original magnification: ×200; inset, the infiltrate contains abundant CD4+ cells. Right, FOXP3+ cells (original magnification: ×200); inset, cells do not express CD25. B, Peripheral blood flow cytometry showed that Vβ-restricted neoplastic T cells did not have increased expression of FOXP3. C, Photomicrograph of a skin biopsy specimen from patient 2. Scale bar represents 125 μm. Left, Hematoxylin–eosin stain; original magnification: ×200; inset, the infiltrate contains high numbers of CD4+ cells. Right, FOXP3+ cells (original magnification: ×200); inset, cells are positive for CD25 (original magnification: ×200). D, Peripheral blood flow cytometry showed Vβ-restricted neoplastic T cells highly expressed FOXP3. E, Peripheral blood flow cytometry from a healthy (control) donor shows the CD4+CD25high population expresses FOXP3 at levels similar to those of patient 2, whereas the CD4+CD25– population does not express FOXP3 (similar to patient 1). Journal of the American Academy of Dermatology 2013 69, 485-489DOI: (10.1016/j.jaad.2010.01.013) Copyright © 2010 American Academy of Dermatology, Inc. Terms and Conditions

Fig 1 FOXP3+ cells are present in skin biopsy samples of two patients with Sézary syndrome. A, Photomicrograph of a skin biopsy specimen from patient 1. Scale bar represents 125 μm. Left, Hematoxylin–eosin stain; original magnification: ×200; inset, the infiltrate contains abundant CD4+ cells. Right, FOXP3+ cells (original magnification: ×200); inset, cells do not express CD25. B, Peripheral blood flow cytometry showed that Vβ-restricted neoplastic T cells did not have increased expression of FOXP3. C, Photomicrograph of a skin biopsy specimen from patient 2. Scale bar represents 125 μm. Left, Hematoxylin–eosin stain; original magnification: ×200; inset, the infiltrate contains high numbers of CD4+ cells. Right, FOXP3+ cells (original magnification: ×200); inset, cells are positive for CD25 (original magnification: ×200). D, Peripheral blood flow cytometry showed Vβ-restricted neoplastic T cells highly expressed FOXP3. E, Peripheral blood flow cytometry from a healthy (control) donor shows the CD4+CD25high population expresses FOXP3 at levels similar to those of patient 2, whereas the CD4+CD25– population does not express FOXP3 (similar to patient 1). Journal of the American Academy of Dermatology 2013 69, 485-489DOI: (10.1016/j.jaad.2010.01.013) Copyright © 2010 American Academy of Dermatology, Inc. Terms and Conditions

Fig 2 FOXP3+ cells in Sézary syndrome do not have T-cell suppressor activity. Conventional CD4+ T cells (Tconv) isolated from a healthy (control) donor were cultured alone or cocultured in triplicate in anti-CD3–coated 96-well plates (2×105 cells/well) with purified regulatory T cells (Treg) or malignant T cells from patient 2 at the ratios shown. Cells were pulse-treated with 1 μCi/well 3H-deoxyribonucleotide for the final 12 hours of a 72-hour culture before determination of thymidine incorporation. Journal of the American Academy of Dermatology 2013 69, 485-489DOI: (10.1016/j.jaad.2010.01.013) Copyright © 2010 American Academy of Dermatology, Inc. Terms and Conditions