CD24a Expression Levels Discriminate Langerhans Cells from Dermal Dendritic Cells in Murine Skin and Lymph Nodes Susanne Stutte, Bettina Jux, Charlotte.

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CD24a Expression Levels Discriminate Langerhans Cells from Dermal Dendritic Cells in Murine Skin and Lymph Nodes Susanne Stutte, Bettina Jux, Charlotte Esser, Irmgard Förster Journal of Investigative Dermatology Volume 128, Issue 6, Pages 1470-1475 (July 2008) DOI: 10.1038/sj.jid.5701228 Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 CD24a expression by DCs of the epidermis and dermis. Epidermis and dermis were separated by trypsin treatment and single-cell suspensions were analyzed by FACS. (a) Epidermal cells were stained for MHCII and CD207. Double positive cells were further analyzed for CD24a expression (filled graph). (b) CD24a expression in the dermis was analyzed in the population positive for MHCII and CD207 (filled graph), as well as in the MHCII+ CD207− fraction (black line). Gray line represents the isotype control. Data are representative of three independent experiments. Journal of Investigative Dermatology 2008 128, 1470-1475DOI: (10.1038/sj.jid.5701228) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 CD24a expression by sdDCs in LNs. Mice were shaved on the ventral skin and painted with FITC or solvent as control. A total of 24, 48, and 96hours after application, sdLNs and mesenteric LNs were harvested and cells were analyzed by FACS. (a) sdDCs can be identified as FITC-positive MHCIIhigh. Shown are the dot plots of cells isolated 24hours after application of FITC or solvent alone (control). (b) The FITC+ MHCIIhigh cells were analyzed for CD24a expression. The dot plot shows a representative CD24a staining pattern of MHCIIhigh cells 24hours after FITC application. The percentage of CD24high cells was calculated for all time points (n=3). (c) After staining of lymph node cells with Langerin and MHCII, the frequency of Langerin+ cells within the FITC+ MHCIIhigh fraction was calculated for all different time points after FITC application. (d) The dot plots are representative for the expression of CD24a and CD207 on FITC+ cells in sdLNs at the indicated time points. The frequencies shown in the figure are representative of three independent experiments. (e) The staining pattern of the whole sdLN cell suspension was analyzed at different time points. MHCII and CD24a expression is shown as well as the distribution of CD24ahigh and CD24alow/int cells within the MHCIIhigh population. One out of three independent experiments is depicted here. (f) Frequency of CD24low/int dDCs and CD24ahigh LCs within the MHCIIhigh cell population depending on the time after FITC application on the skin. (g) The mean fluorescence intensity of CD24a was compared between LCs in the epidermis and those in the sdLNs. *** indicates P<0.0001. Journal of Investigative Dermatology 2008 128, 1470-1475DOI: (10.1038/sj.jid.5701228) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Blood-derived CD8α+ DCs were analyzed for CD24a expression under steady-state conditions. sdLNs of untreated mice were harvested. DCs were enriched by density gradient centrifugation and stained for CD11c, CD8α, CD207, and CD24a. (a) Staining pattern for CD8α and CD11c after DC enrichment. The frequency of CD8α+CD11c+ DCs is indicated. (b) CD24a expression on gated CD11c+CD8α+CD207− cells (thin black line) and CD11c+CD8α+CD207+ cells (gray filled histogram) is compared with that of all CD11c+CD207+ cells (thick black line) considered as control. The dotted line represents the isotype control. Journal of Investigative Dermatology 2008 128, 1470-1475DOI: (10.1038/sj.jid.5701228) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Comparison of the CD40/CD11c expression pattern with CD207/CD24a staining. Ventral skin of C57BL/6 mice was painted with 200μl of 0.5% FITC to induce active migration of sdDCs to the sdLNs. After 48 and 96hours, sdLNs were harvested and DCs were enriched by density-gradient centrifugation, and stained for CD40, CD11c, CD24a, and CD207. (a) The representative 48-hour dot plot depicts the CD11c+CD40high (A) and CD11chighCD40high (B) populations. These cells were further analyzed for CD207 and CD24a expression at the indicated time points. (b) The percentages of CD24ahigh and CD207+ cells were calculated for population A (thick black line) and B (thin black line). Journal of Investigative Dermatology 2008 128, 1470-1475DOI: (10.1038/sj.jid.5701228) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions