CXCR3⁺ T cells are of donor origin and release inflammation-promoting compounds on short-term activation. (A) ELISpot wells showing IFN-γ (top row) and granzyme B (GrzB; bottom row) production by flow-sorted CD4⁺TCRαβ⁺ (left; 1600 cells/well), CD8⁺TCRαβ⁺ (center; 1600 cells/well), and TCRγδ⁺ (410 cells/well for CXCR3⁺ and 79 cells/well for CXCR3^–) T cells plated in ELISpot plates (U-CyTech Biosciences) and stimulated overnight by PMA and ionomycin. (B) Supernatants from ELISpot assays using 65 000 or 12 500 cells (CD4⁺CXCR3^– subset only) were harvested before cell lysis for testing by Luminex. CXCL10 (ligand binding to CXCR3), tumor necrosis factor (TNF), and interleukin 2 (IL-2) levels are shown. Note that cytokine values for CD4⁺CXCR3^– subset are corrected based on 5-fold less T-cell yield after sorting. (C) Results of short tandem repeats (STR) analysis performed on 20 000 flow-sorted TCRγδ⁺ T cells, TCRαβ⁺ T-cell subclusters separated according to the level of CXCR3 expression (supplemental Figure 9), and CD14⁺ myeloid subclusters separated according to the level of CD163 expression and CD14^– myeloid cells. Chimerism data are representative for the results obtained from 4 different patients with aGVHD. (D) Detection of CXCL10⁺ cells (left) and CD3⁺ T cells (right), both in brown, in a duodenum biopsy sample obtained from a patient with steroid-refractory aGVHD. Note that CXCL10 is expressed by epithelial cells lining the villi as well as small lymphocytic cells residing in the lamina propria of the villi in the same localization as CD3⁺ T cells stained in a serial section.