CD163⁺cells are abundant in PBMC, skin, and GI tract samples of patients with acute GVHD. (A) Hierarchical stochastic neighbor embedding (HSNE)–guided dissection of blood-derived myeloid cells that belong either to the monocyte or DC lineage. (B) Heatmap showing 11 different monocyte subclusters (CD14⁺CD16^– classical, CD14⁺CD16^{dim/intermediate}, or CD14^neg/dimCD16^bright nonclassical monocytes) and 11 DC subclusters (CD11b^–CD11c⁺CD123^dim conventional cDCs and CD11b^–CD11c^–CD123⁺ plasmacytoid pDCs). Cluster annotation numbers displayed in (A) correspond to the numbers shown in (B). (C) Boxplots showing the relative abundance (median and interquartile range) of distinct monocyte subclusters, analyzed at 2 or 3 consecutive time points as indicated on the x-axis, that are significantly more (top row) or less (middle row) prevalent in HSCT patients who developed aGVHD. DC subclusters most prevalent in therapy refractory aGVHD patients (nonresponsive to MSC-based second-line therapy) are indicated in the bottom row. Healthy ctr, healthy control participants; HSCT ctr, patients who underwent HSCT without aGVHD; Steroid-CR, patients who underwent HSCT and demonstrated aGVHD responsive to steroids; GVHD-CR, HSCT patients with steroid-refractory aGVHD responsive to MSC-based second-line therapy; GVHD-NR, HSCT patients with steroid-refractory aGVHD nonresponsive to MSC-based second-line therapy. (D) Representative images of the abundant presence of CD163⁺ cells (stained in brown) in skin and colon biopsy samples from patients with aGVHD. Note the sporadic presence of CD163⁺ cells in colon and skin biopsy samples collected from patients suspected of skin or gut aGVHD after undergoing HSCT. These biopsy samples however displayed no convincing pathologic features of aGVHD. The biopsy sample obtained after MSC therapy initiation (right panel) is derived from one of the patients with refractory aGVHD who did not respond to steroids and MSC. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001.