Tumor Necrosis Factor-α- and IL-4-Independent Development of Langerhans Cell-Like Dendritic Cells from M-CSF-Conditioned Precursors Jean-Baptiste Barbaroux,

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Tumor Necrosis Factor-α- and IL-4-Independent Development of Langerhans Cell-Like Dendritic Cells from M-CSF-Conditioned Precursors Jean-Baptiste Barbaroux, Wing-Hong Kwan, Jean-Pierre Allam, Natalija Novak, Thomas Bieber, Wolf H. Fridman, Richard Groves, Chris G. Mueller Journal of Investigative Dermatology Volume 126, Issue 1, Pages 114-120 (January 2006) DOI: 10.1038/sj.jid.5700023 Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Generation of DDL-DC from CD34+ hematopoietic progenitors. (a) Schematic overview of the culture conditions to obtain CD1a+, CD14+, double positive and negative cells at day 5 and a majority (72%) of CD14+ cells at day 11. (b) Phenotype analysis of CD14+-purified DDL-DC using the indicated antibodies. Expression of all markers was detected at the cell surface. Specific labeling is shown in black histograms and isotype controls in white histograms. Journal of Investigative Dermatology 2006 126, 114-120DOI: (10.1038/sj.jid.5700023) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 DDL-DC differentiate into CD1a+ LangerinHi cells. DDL-DC were cultured for 3 days in GM-CSF/TGFβ, alone or in the presence of IL-4 or TNFα. (a) Representative phenotype analysis of CD1a/Langerin expression. (b) The percentage of CD1a+ LangerinHi cells, electronically gated as shown in (a) and obtained in GM-CSF/TGFβ in the presence or absence of 1ng/ml IL-4 or 1ng/ml TNFα, is shown for each donor. Significance was evaluated using the t-tailed Student's test. (c) Kinetics of differentiation of DDL-DC into CD1a+ LangerinHi cells in GM-CSF/TGFβ in the presence or absence of IL-4. The percentage of CD1a+ LangerinHi cells was measured every 24hours for 3 days and expressed as the mean±SE of three experiments. Journal of Investigative Dermatology 2006 126, 114-120DOI: (10.1038/sj.jid.5700023) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 GM-CSF/TGFβ induces differentiation of CCL20-responsive CCR6+ cells. (a) CCR6 expression of DDL-DC before and after a 24-hour culture in GM-CSF/TGFβ in the presence or absence of IL-4. Specific labeling is shown in gray histograms and isotype controls in white histograms. (b) Measurement of calcium flow in response to CCL20 or CCL2 ligation expressed as nm of mobilized calcium per 106 cells. The arrow indicates the time of chemokine addition. The cells were as in (a). The experiment was performed twice using different donor CD34+ cells with similar results. (c) GM-CSF/TGFβ- or GM-CSF/TGFβ +IL-4-cultured DDL-DC were tested for chemotactic migration in response to increasing concentrations of CCL20. The number of migrated cells is expressed as the mean±SE of three independent experiments. Journal of Investigative Dermatology 2006 126, 114-120DOI: (10.1038/sj.jid.5700023) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 TNFα and IL-4 inhibit LC differentiation, but simultaneous addition of both cytokines diminishes their inhibitory effect. (a) DDL-DC cultured for 3 days in GM-CSF/TGFβ in the presence or absence of 0.5ng/ml TNFα or 1ng/ml IL-4, or in the presence of both TNFα and IL-4, were analyzed for CD1a/Langerin expression. The results of three experiments are shown. The CD1a+ LangerinHi population is gated. (b) Representation of the mean percentage±SE of the CD1a+ LangerinHi cells for the three experiments. The CD1a+ LangerinHi cells were defined as located within the gate of (a). The doses (ng/ml) of TNFα and IL-4 are indicated below the histograms. Significance was calculated using the t-tailed Student's test (*P<0.05). (c) Representation of the geometric mean±SE intensity index of fluorescence of Langerin expression in the upper right quadrant of (a). The doses of TNFα and IL-4 are indicated below the histograms. Significance was calculated using the t-tailed Student's test (*P<0.05). Journal of Investigative Dermatology 2006 126, 114-120DOI: (10.1038/sj.jid.5700023) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 GM-CSF/TGFβ-cultured cells mature in response to LPS/anti-CD40. (a) DDL-DC cultured for 3 days in GM-CSF/TGFβ in the presence or absence of TNFα or IL-4, or in the presence of both TNFα and IL-4, were visualized by light microscopy and analyzed for expression of cell-surface Langerin versus CCR6, CCR7, HLA-DR, CD86, CD83, and CD80. The FACS plots for CCR6, CCR7, and CD83 are representative of five experiments and, for CD86, CD80, and HLA-DR, are representative of two experiments. (b) The same cells, as shown in (a), were stimulated for 24hours with LPS/anti-CD40, visualized by light microscopy, and analyzed for cell-surface marker expression. For all flow cytometry analyses, expression levels were obtained after gating on total live cells, identified by forward and side scatter profiles. Journal of Investigative Dermatology 2006 126, 114-120DOI: (10.1038/sj.jid.5700023) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Transmission electron microscopy visualizes Birbeck granules. DDL-DC were cultured in GM-CSF/TGFβ for 6 days and processed for transmission electron microscopy. About 10/100 cells analyzed at a magnification of × 30,000 showed rod-shaped Birbeck granules (inset, lower left). Journal of Investigative Dermatology 2006 126, 114-120DOI: (10.1038/sj.jid.5700023) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions