Thymic stromal lymphopoietin converts human epidermal Langerhans cells into antigen- presenting cells that induce proallergic T cells Susanne Ebner, PhD,

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Thymic stromal lymphopoietin converts human epidermal Langerhans cells into antigen- presenting cells that induce proallergic T cells Susanne Ebner, PhD, Van Anh Nguyen, MD, Markus Forstner, Yui-Hsi Wang, PhD, Dolores Wolfram, MD, Yong-Jun Liu, MD, PhD, Nikolaus Romani, PhD Journal of Allergy and Clinical Immunology Volume 119, Issue 4, Pages 982-990 (April 2007) DOI: 10.1016/j.jaci.2007.01.003 Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Phenotype of LCs activated with TSLP. LCs were identified by their CD1a expression. Fluorescence of CD1a-gated cells is depicted in the histograms. Filled histograms represent staining of experimental antibodies; open histograms are isotype controls. A, Freshly isolated LCs were cultured with medium alone, with TSLP, and with GM-CSF for 48 hours. TSLP markedly increased the percentage of CD83-expressing LCs. One representative experiment of 3 complete experiments (ie, with all antibodies) is shown in the histograms. CD83 analyses were performed in 4 additional experiments. B, Maturation-associated molecules CD40, 86, 83, 205, and CCR7 were expressed at high levels already in the absence of TSLP. Only CD80 expression was further enhanced by TSLP. One representative experiment of 5 is shown. C, Migratory LCs are highly positive for HLA-DR and CD1a, and there is no differential expression of OX-40L. Journal of Allergy and Clinical Immunology 2007 119, 982-990DOI: (10.1016/j.jaci.2007.01.003) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 TSLP improves viability of cultured LCs and enhances migration of human skin emigrants. Highly enriched LCs were cultured side by side in the presence or absence of TSLP (A) or in the presence of TSLP or GM-CSF (B) for 2 days. Cell yields, that is, percent surviving LCs of LC plated at the onset of culture are indicated (n = 8; P = .0078 for control vs TSLP; P = .0078 for GM-CSF vs TSLP). C, Dispase-procured epidermal sheets were cultured side by side in the presence or in the absence of TSLP for 3 days. LCs that had migrated into the culture medium were counted (n = 7, P = .0156). D, Cultures were conducted as in C, but for a total of 4 days (n = 4, NS). Journal of Allergy and Clinical Immunology 2007 119, 982-990DOI: (10.1016/j.jaci.2007.01.003) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Naïve CD4 T-cell proliferation induced by TSLP-treated migratory LCs (A) and by freshly isolated LCs (B). Different numbers of LCs were cocultured with naïve allogeneic T cells. Freshly isolated LCs cultured in the presence of TSLP or GM-CSF stimulate T cells more efficiently (B). One representative experiment of 3 each is shown. Journal of Allergy and Clinical Immunology 2007 119, 982-990DOI: (10.1016/j.jaci.2007.01.003) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 CCL17/TARC production by TSLP-treated migratory LCs in response to CD40 ligation. Epidermal explants were cultured for 3 days in the absence or presence of TSLP and in the presence of a neutralizing anti-TSLP antibody. The resulting mature LCs were cocultured with CD40 ligand-expressing cells, and supernatants were assayed for CCL17. In response to CD40 ligation, TSLP-treated migratory LCs produced significantly more CCL17/TARC than LCs without TSLP treatment (n = 5, P < .05). CD40 ligation of LCs that had migrated in the absence of TSLP or in the presence of anti-TSLP antibody did not induce increased CCL17 secretion. Journal of Allergy and Clinical Immunology 2007 119, 982-990DOI: (10.1016/j.jaci.2007.01.003) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 TSLP-treated migratory LCs induce T cells to make less IFN-γ and IL-10 but more IL-4, 5, and 13. A, Naive T cells were cocultured with allogeneic mature LCs that had migrated from epidermal sheets in the absence (control) or presence of TSLP or a neutralizing anti-TSLP antibody (–TSLP+Ab) for 3 days. Differences between control- and TSLP-LC–stimulated T-cell cytokine levels were significant for IL-4, IL-13, IL-10, IFN-γ, and TNF-α (see text). The increase in IL-5 was not statistically significant. Individual donors are marked by identical symbols across stimulation conditions. B, Intracellular cytokine staining of T cells confirmed the cytokine secretion profiles measured by ELISA. Data are representative for 1 of 6 independent experiments. Journal of Allergy and Clinical Immunology 2007 119, 982-990DOI: (10.1016/j.jaci.2007.01.003) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions