The composition of the cellular infiltrate differs between the diverse types of cutaneous GVHD. (A-B) Quantitative in situ analysis of the cellular infiltrate within aGVHD, clGVHD, and csGVHD skin lesions and normal skin from HCs. Single and multicolor IF stainings were performed with the following markers: pan-leukocytes: CD45⁺; T cells: CD3⁺; CD4⁺ T cells: CD3⁺CD4⁺; CD8⁺ T cells: CD3⁺CD8⁺; regulatory T (Treg) cells: CD3⁺CD25⁺Foxp3⁺; NK cells: CD56⁺CD3⁻; NK T cells: CD3⁺CD1d⁺; B cells: CD19⁺; Langerhans cells (LCs): CD207⁺CD1a⁺; plasmacytoid DCs (pDCs): BDCA2⁺CD123⁺; CD1c⁺ DCs: CD1c⁺HLA-DR⁺; macrophages: CD163⁺CD68⁺; neutrophils: CD15⁺HLA-DR⁻; eosinophils: MBP⁺; basophils: CD123⁺CD203c⁺; and mast cells: CD117⁺CD203c⁺. Data are given either as absolute numbers of positive cells ± SD per mm (epidermis) or per mm² (dermis). *P < .05, **P < .01, ***P < .001. A significance in differences between the various groups is shown only for the various GVHD manifestations. (C-E) Representative images of IF multicolor stainings of CD8⁺ T cells (CD3*A568/CD8*A647) (C), macrophages (CD163*A488/CD68*A568/CD11c*A647) (D), and mast cells (CD117*A488/CD203c*A568) (E) merged with 4′,6-diamidino-2-phenylindole (DAPI) in an aGVHD, clGVHD, and csGVHD patient. The framed cells are shown in a larger magnification on the right. The epidermis is, if shown, located on the left in all images. Slides were scanned using a TissueFAXS imaging system (TissueGnostics GmbH) equipped with a Zeiss Axio Imager.Z1 microscope (Carl Zeiss Inc., Jena, Germany) with filters detecting DAPI, green fluorescent protein, Cy3, and Cy5 fluorochromes. Images were taken with Zeiss LD Plan-Neofluar objectives (primary objective ×20/0.4, ocular objective ×10) at room temperature using a PCO PixelFly camera (Zeiss), exported from the TissueQuest software (TissueGnostics GmbH) as tiff images, and processed in Adobe Photoshop CS5 (Adobe Systems, San Jose, CA).