Matthew J. Deeths, Bart T. Endrizzi, Michelle L. Irvin, Lynne P

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Phenotypic Analysis of T-Cells in Extensive Alopecia Areata Scalp Suggests Partial Tolerance Matthew J. Deeths, Bart T. Endrizzi, Michelle L. Irvin, Lynne P. Steiner, Marna E. Ericson, Maria K. Hordinsky Journal of Investigative Dermatology Volume 126, Issue 2, Pages 366-373 (February 2006) DOI: 10.1038/sj.jid.5700054 Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 An example of one scalp biopsy analysis using dual laser flow cytometry. (a) Scatter plot demonstrates the lymphocyte gate with lymphocytes identified based on forward- and side-light scatter characteristics. (b) A T-cell-specific CD-3 stain is used to further gate out contaminating cells and to define the lymphocyte population. (c) Double-gated and back-graphed scatter plot identifies a homogeneous population of T-cells that falls into the region identical to that seen with control lymphocytes, and (d) stains with a bimodal distribution using anti-CD-4 antibody. Journal of Investigative Dermatology 2006 126, 366-373DOI: (10.1038/sj.jid.5700054) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 T-cell lL-2 expression in EAA scalp. (a) Scatter plot showing CD-8 and lL-2 expression on T-cells. Histograms showing IL-2 expression by (b) CD-8-positive and (c) CD-8-negative T-cells. Journal of Investigative Dermatology 2006 126, 366-373DOI: (10.1038/sj.jid.5700054) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 IFN-γ expression measured in cells stained as inFigure 2. Journal of Investigative Dermatology 2006 126, 366-373DOI: (10.1038/sj.jid.5700054) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Summary of CD-3 and CD-4 staining of scalp T-cells in control and EAA patients. (a) The percentages of CD-3-positive T-cells in EAA scalp specimens are fewer than seen in control scalp specimens; however, (b) the T-cell subset distribution is the same in both the groups. Journal of Investigative Dermatology 2006 126, 366-373DOI: (10.1038/sj.jid.5700054) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 CD-69 expression in T-cells from EAA scalp suggests recent antigen exposure. (a) Histogram showing CD-69 staining (heavy line) and isotope control staining (light line) in CD-3-gated T-cells from EAA scalp. (b) CD-69 expression is similar for CD-4 and non-CD-4 cells. Journal of Investigative Dermatology 2006 126, 366-373DOI: (10.1038/sj.jid.5700054) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Cytokine expression phenotype of scalp T-cells from 12 EAA patients and six controls. The CD-8-negative helper T-cell production of IFN-γ was minimal in both control and EAA (P=0.54) scalp biopsy specimens. (a) The percentage of EAA CD-8 cells producing IFN-γ showed more variation than control subjects (P=0.04), but was decreased on average. The CD-8-negative T-cell production of IL-2 was higher in controls compared to EAA scalp biopsy specimens (P=0.003). (b) The CD-8-positive T-cell production of IL-2 was comparable between controls and EAA (P=0.07) scalp biopsy specimens. (c) The CD-8 (P=0.09) and non-CD-8 T-cell (P=0.11) production of tumor necrosis factor α (TNF-α) was higher in control scalp biopsy specimens compared to EAA scalp biopsy specimens, but these differences were not statistically significant. Journal of Investigative Dermatology 2006 126, 366-373DOI: (10.1038/sj.jid.5700054) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Immunohistochemical staining for IFN-γ with a peroxidase assay (brown) and hematoxylin nuclear staining (blue). (Upper panel) On days 3, 7, and 12 post-transvaginal inoculation of SIV virus, draining lymph nodes were harvested and fixed. These samples were then embedded in paraffin and 5–6μm sections were taken for immunohistochemical staining. Images (original magnification: × 4 and × 20) are presented for each time point. The sample on day 12 is a positive control for immune cell production of IFN-γ. (Lower panel) Four-millimeter punch biopsy specimens from the parietal scalp were fixed with 8% paraformaldehyde, paraffin-embedded, and stained for IFN-γ expression with the same methodology used for the positive control in the upper panel. Control scalp biopsy data are on the left; three sample pairs from EAA subjects are on the right. No significant IFN-γ expression was observed in either the control or subject biopsy specimens. Also, there was no propensity for IFN-γ expression localizing around hair follicles. All sections were visualized at original magnification: × 4 (bar=250μm) and original magnification: × 20 (bar=50m). Journal of Investigative Dermatology 2006 126, 366-373DOI: (10.1038/sj.jid.5700054) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions