Recipient dermal macrophages survive GVHD. (A) Dual immunofluorescence and FISH (insets) of a cytospin of remnant-digested dermis from a female patient with acute GVHD 40 d after transplantation. Donor-derived male DC and lymphocytes can be seen in addition to a donor eosinophil (arrows). A recipient macrophage is clearly visible. Bar, 20 µm. (B) Immunofluorescence staining of skin affected by GVHD showing CD163⁺ macrophages in direct contact with infiltrating CD3⁺ T cells (top left and bottom magnified inset). Eosinophils appear orange because of their bright AF and nonspecific staining (asterisks, a, and b). They are also highly visible in the control with secondary antibodies only (top right) and were confirmed in the section at high magnification (bottom, inset, a and b). Bar, 20 µm. The figure shows the results of one of two patients with GVHD analyzed in the same fashion. (C) Dual immunofluorescence and FISH (insets) of a cytospin of digested dermis from a female patient who had prior acute GVHD taken at 365 d after transplantation. Recipient macrophages still persist and are highly granulated. Engrafting male donor macrophages by comparison are weakly granulated. Bar, 20 µm. Dashed lines in insets indicates stitching of high power fields. (D) DNA ploidy analysis of leukocytes from normal skin. DAPI histograms are shown from a representative experiment on the left, and the means with cumulative data from five independent experiments are shown on the right. The inset shows a binucleate dermal macrophage from a male patient. Bar, 10 µm. *, P < 0.05 by Mann-Whitney U test compared with neighbor (NS, P = 0.55).