Identification of a Novel CD160+CD4+ T-Lymphocyte Subset in the Skin: A Possible Role for CD160 in Skin Inflammation Sofia Abecassis, Jérôme Giustiniani,

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Identification of a Novel CD160+CD4+ T-Lymphocyte Subset in the Skin: A Possible Role for CD160 in Skin Inflammation Sofia Abecassis, Jérôme Giustiniani, Nicolas Meyer, Valérie Schiavon, Nicolas Ortonne, José A. Campillo, Martine Bagot, Armand Bensussan Journal of Investigative Dermatology Volume 127, Issue 5, Pages 1161-1166 (May 2007) DOI: 10.1038/sj.jid.5700680 Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 In situ phenotype of inflammatory and normal skin cellular infiltrates: detection of a CD160+ cell subset. Phenotype of cellular infiltrates of the skin established by immunohistochemistry (× 100) on AD skin (right column) and healthy skin (left column). In normal skin, there was a minor infiltrate of CD3+ (a), CD4+ (c) and CD8+ T cells (e). In AD skin, we found a marked dermal infiltrate made of CD3+ lymphocytes (b), mainly CD4+ T cells (d) accompanied by CD8+ cells (f). There were no NK cells, as CD56 stained only small nervous fibres of the dermis (g and h indicated by arrow heads). CD160+ cells were present both in the inflammatory infiltrate (j) and among resident cells of normal skin (i). Positively stained cells are indicated with arrows. Bar=0.25mm. Journal of Investigative Dermatology 2007 127, 1161-1166DOI: (10.1038/sj.jid.5700680) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Presence of CD160+ T-cells and CD160+CD4+ cells within the inflammatory skin infiltrates. Demonstrated by immunochemistry double staining (× 100). (a) CD160+ T cells infiltrating the dermis in AD. CD3 is stained in red and CD160 in brown. CD160+ T cells are indicated with arrows. (b) CD160+CD4+ cells in the papillary dermis of a PSO lesion are indicated with arrows. CD4 is stained in red and CD160 in brown. Bar=0.1mm. Journal of Investigative Dermatology 2007 127, 1161-1166DOI: (10.1038/sj.jid.5700680) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Detection of CD160 mRNA in healthy and inflammatory skin samples. (a) CD160 mRNA was identified by PCR analysis after mRNA extraction from frozen samples of healthy skin (HS1 and HS2) and AD lesions (AD1 and AD2). Positive and negative controls consisted respectively of PB-NK and CD4+ T-cells from a healthy subject. Detection of β-actin mRNA also served as a positive control for each experiment. (b) CD160 mRNA was identified by cDNA sequence analysis. Journal of Investigative Dermatology 2007 127, 1161-1166DOI: (10.1038/sj.jid.5700680) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Regulation of the CD160 expression by CD4+ T cells by IL2 and IL15 and not by IL-4. The expression of CD160 mRNA transcript in PB-CD4+ is induced by IL-2 and IL-15, but not IL-4. CD160 mRNA transcript was not detected in freshly isolated (1) PB-CD4+ nor after 6 days of culture in the presence of (2) IL-4, however it could be detected in PB-CD4+ cultured during 6 days in the presence of (3) IL-2, (4) IL-2 + IL-4, or (5) IL-15. Detection of β-actin mRNA served as positive control for each experiment. Journal of Investigative Dermatology 2007 127, 1161-1166DOI: (10.1038/sj.jid.5700680) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Expression and Function of CD160 on lymphocytes extracted from CD. (a) In vitro identification of the CD160+ CD4+ T-cell subset. Flow cytometry phenotype analysis of cells obtained from AD3 AD biopsy after cell expansion in vitro showed that CD160 was mostly expressed by CD4+ lymphocytes (b) CD160 acts as a co-receptor of CD3 induced proliferation of ex vivo cultured CD4+CD160+ T cells (AD3). Proliferation assay showing increased CD3-induced proliferation after CD160 stimulation as compared to controls (isotype-matched mAb IgM and goat anti-mouse antiboby (GAM)). BY55 is IgM anti-CD160 mAb. Journal of Investigative Dermatology 2007 127, 1161-1166DOI: (10.1038/sj.jid.5700680) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions