Epidermal TSLP expression and the presence of CD8⁺T cells indicate the occurrence of aGVHD at day 20 to 30 after HCT. (A) IF stainings of T cells (T cells: CD3⁺; CD4⁺ T cells: CD3⁺CD4⁺; CD8⁺ T cells: CD3⁺CD8⁺; and regulatory T cells: CD3⁺CD25⁺Foxp3⁺) in normal-appearing skin of HCT recipients at different time points prior to and after HCT. Patients who later developed aGVHD (n = 7) were compared with those who did not (no aGVHD, n = 7). Data are given either as absolute numbers of positive cells ± SD per mm (epidermis) or per mm² (dermis). (B) Epidermal IH taining of TSLP at different time points prior to or after HCT (prior conditioning, day −1 prior HCT, day +20-30 post-HCT) in a patient who later developed acute cutaneous GVHD compared with a patient who did not. The framed cells are shown in a larger magnification on the right-hand side of the figure. Slides were scanned using a TissueFAXS imaging system equipped with a Zeiss Axio Observer.Z1 microscope (Zeiss). Images were taken with a LD Plan-Neofluar objective (primary objective ×20/0.4, ocular objective ×10) using a PixeLINK PL-B623CF color digital camera (Zeiss), exported from the HistoQuest software (TissueGnostics GmbH) as tiff images, and processed in Adobe Photoshop CS5 (Adobe Systems). (C) The mean intensity of TSLP expression was evaluated using HistoQuest imaging analysis software (TissueGnostics GmBbH). All images were acquired and analyzed using identical hardware and software settings. *P < .05, ***P < .001.