Fig. 1.

Characterization of EpCAM-deficient LC in LC/EpCAM-cKO mice. (A) Demonstration in situ via immunofluorescence microscopy of EpCAM deletion in LC in LC/EpCAM-cKO mice. Epidermal sheets from LC/EpCAM-cKO (KO) and Cre-negative (WT) animals were stained with anti-EpCAM and anti-MHC Class II mAb and visualized using immunofluorescence microscopy (Scale bar: 50 μm.) Note residual EpCAM expression in hair follicles in WT and KO mice. (B) Demonstration via flow cytometry of EpCAM deletion in LC in LC/EpCAM-cKO mice. Flow cytometric analysis of CD45⁺ MHC Class II⁺ Langerin⁺ cells (LC) and CD45⁻ MHC Class II⁻ cells (KC) in epidermal cell suspensions from LC/EpCAM-cKO (KO) and WT animals. n = 3 mice per group in three experiments; thick line, KO; thin line, WT; gray line with shading, isotype control. EpCAM expression levels are depicted as geometric mean fluorescence intensities (MFI). (C) Increases in LC densities in LC/EpCAM-cKO mice. LC were enumerated by counting MHC Class II⁺ cells in epidermal sheets from KO and WT mice (five random fields per mouse; n = 5 mice per group in six experiments.) *P < 0.005. DETC (TCR γ/δ-positive cells) were quantified in epidermal sheets from KO and WT mice (five random fields per mouse, n = 3 mice per group in a single experiment). (D) T-cell–stimulatory activity of EpCAM-deficient LC. Epidermal cells from KO and WT mice were cocultured with MHC-mismatched (BALB/c) naive T cells (n = 3 mice per group in three experiments using triplicate measurements in each experiment). Numbers of LC per well were determined using flow cytometry to assess LC frequency in epidermal single-cell suspensions. T-cell proliferation was measured by quantifying cleavage of WST-1 using a spectrophotometric assay.