Antigen-exposed TCRγδ⁺ T cells are present in the blood and GI tract of patients with severe intestinal GVHD. (A) Violin plots depicting median PD-1 expression levels on TCRγδ⁺ cells and TCRαβ⁺ CD4 or CD8 expressing T cells at the indicated time points in the different patient groups. (B) Hierarchical stochastic neighbor embedding (HSNE) map showing distinct subclusters of TCRγδ⁺ T cells found at all time points in all patients. Note the overlap among CXCR3, CCR4, CCR9, and CCR10 expression in TCRγδ⁺ cells (chemokineR^high). (C) Frequency of chemokineR^high TCRγδ⁺ T cells at time points 1 and 3 in HSCT patients with steroid-refractory aGVHD responsive to MSC-based second-line therapy (GVHD-CR) and HSCT patients with steroid-refractory aGVHD nonresponsive to MSC-based second-line therapy (GVHD-NR) (before and after MSC infusion) and at time points 1 and 2 in HSCT patients with aGVHD responsive to steroids (Steroid-CR). (D) Detection of TCRγδ⁺ T cells in a GI tract biopsy sample obtained from a patient with ongoing aGVHD. A combination of antibodies staining PD-1 (green), TCRδ (blue), and CD3 (red) was used to visualize PD-1-expressing TCRγδ⁺ T cells (marked by white quadrants; original magnification, ×400). Healthy ctr, healthy control participants; HSCT ctr, patients who underwent HSCT without aGVHD. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001.