EpCAM Expressed by Murine Epidermal Langerhans Cells Modulates Immunization to an Epicutaneously Applied Protein Antigen  Takeshi Ouchi, Gaku Nakato, Mark C. Udey  Journal of Investigative Dermatology  Volume 136, Issue 8, Pages 1627-1635 (August 2016) DOI: 10.1016/j.jid.2016.04.005 Copyright © 2016 The Authors Terms and Conditions

Figure 1 EpCAM regulates claudin-1 expression in LCs. (a) Reduction of claudin-1 expression in EpCAM-deficient LCs in unperturbed epidermis as assessed by confocal microscopy. Bars = 20 μm (upper panels); bars = 5 μm (lower panels). (b) Quantification of claudin-1 content of EpCAM-deficient and control LCs from unperturbed epidermis and skin draining lymph nodes using flow cytometry. Mean fluorescence intensities (MFI) of claudin-1 in MHC class II+ (MHCII+) CD45+ LCs isolated from epidermis (left) or MHCIIhigh CD11c+ Langerin+ CD103− cells from skin draining lymph nodes (LN) (right) are depicted. Data shown is from n = 3 experiments. **P < 0.01 and ***P < 0.001 via Student’s t test as indicated. cKO, conditional knockout; EpCAM, epithelial cell adhesion molecule; LC, Langerhans cell; WT, wild type/control. Journal of Investigative Dermatology 2016 136, 1627-1635DOI: (10.1016/j.jid.2016.04.005) Copyright © 2016 The Authors Terms and Conditions

Figure 2 EpCAM-deficient LCs efficiently dock with epidermal TJ. (a) TJ docking points involving activated LCs and keratinocyte (KC) were identified as ZO-1high MHC class IIhigh (MHCIIhigh) Langerinhigh accumulations (arrowheads) visualized using confocal microscopy. Bars = 10 μm. (b) Quantification of LC TJ-docking efficiencies determined 16 hours after light tape stripping. Data presented are representative of those obtained with four mice (a) and aggregated from two independent experiments (b). BL, basal lamina; cKO, conditional knockout; EpCAM, epithelial cell adhesion molecule; LC, Langerhans cell; SG, stratum granulosum; TJ, tight junctions; WT, wild type/control; ZO-1, zonula occludens-1. Journal of Investigative Dermatology 2016 136, 1627-1635DOI: (10.1016/j.jid.2016.04.005) Copyright © 2016 The Authors Terms and Conditions

Figure 3 LC EpCAM is not required for uptake of external proteins by LCs. NHS-conjugated biotin was applied to the surface of tape-stripped skin containing control or EpCAM-deficient LCs and LC-associated biotin was localized 16 hours later using Alexa568-SA and confocal microscopy. (a) Biotin tracer was detected within dendrites that had penetrated tight junctions (TJ). Bars = 5 μm. (b) Internalization of biotin tracer demonstrated in displays of single dendrites in various orientations as streaks of fluorochrome (arrowheads) within dendrites below TJ. Bars = 1 μm. cKO, conditional knockout; EpCAM, epithelial cell adhesion molecule; LC, Langerhans cell; MHCII, MHC class II; NHS, N-hydroxysuccinimide; SG, stratum granulosum; WT, wild type/control. Journal of Investigative Dermatology 2016 136, 1627-1635DOI: (10.1016/j.jid.2016.04.005) Copyright © 2016 The Authors Terms and Conditions

Figure 4 Enhanced humoral and cellular immune responses to topically applied antigen in LC EpCAM cKO mice. (a) Serum Ova-specific IgG and IgE were quantified by ELISA 2 weeks after topical immunization with Ova. Aggregate data from two independent experiments with a total of n = 11 (wild type/control [WT] and LC EpCAM cKO) or n = 5 mice (phosphate buffered saline [PBS] patch WT). *P < 0.05 and **P < 0.01 using Student’s t test as indicated. (b, c) Antigen-specific T-cell proliferation was measured by injecting CFSE-labeled OT-II cells into mice 4 days after topical Ova immunization, recovering them 2 days later and quantifying the amount of CFSE in individual CD4+ B220− Vα2+ CD45.1+ T cells in skin draining lymph nodes (LN). Results shown are from one of two representative experiments involving n = 11 (WT and LC EpCAM cKO) and n = 2 mice/group (PBS patch WT). CFSE, carboxyfluorescein succinimidyl ester; cKO, conditional knockout; EpCAM, epithelial cell adhesion molecule; LC, Langerhans cell. Journal of Investigative Dermatology 2016 136, 1627-1635DOI: (10.1016/j.jid.2016.04.005) Copyright © 2016 The Authors Terms and Conditions

Figure 5 Exaggerated migratory activity of EpCAM-deficient LC during topical immunization. (a) Flow cytometric profiles of the skin-derived migratory dendritic cells (DCs) (MHC class IIhigh [MHCIIhigh] CD11cinter-to-high) present in skin draining lymph nodes (LN) from nontreated or Ova patch-immunized wild type/control (WT) and EpCAM cKO mice. MHCIIhigh CD11cinter-to-high DCs were analyzed for the expression of Langerin, CD11b, and CD103 using flow cytometry. Langerin+ CD103− DCs represent migratory LCs, whereas Langerin+ CD103+ DCs correspond to migratory dermal DCs. (b) Absolute numbers of lymph node DCs before, and after, topical immunization with Ova. Aggregate data from four independent experiments with a total of n = 8 mice/group (Ova-patched WT and LC EpCAM cKO) and n = 5 mice/group (nontreated WT and LC EpCAM cKO) are depicted. *P < 0.05 as determined by Student’s t test. cKO, conditional knockout; EpCAM, epithelial cell adhesion molecule; LC, Langerhans cell. Journal of Investigative Dermatology 2016 136, 1627-1635DOI: (10.1016/j.jid.2016.04.005) Copyright © 2016 The Authors Terms and Conditions