CCR9⁺ MSCs are the major effector cells homing to the thymus and ameliorating cGVHD in mice. (A) Thymus glands of severe-GVHD recipients were harvested 7 days after HCT for RNA isolation and RNA-sequencing microarray analysis. Heatmaps of RNA expression of CCL25, CXCL2, CCL19, CCL21, CCL17, and CCL22 are shown as (mean + 1) centered log₂ expression. CCR9-WT MSCs (MSCs^CCR9WT) from EGFP-transgenic mice were transduced with lentivirus to overexpress CCR9 (MSCs^CCR9+) and lentivirus encoded CCR9-specific short hairpin RNA (MSCs^CCR9−). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) (B) and flow cytometry (C) were used to analyze the messenger RNA (mRNA) or protein expression of CCR9 in MSCs^CCR9WT, MSCs^CCR9+, and MSCs^CCR9−. EGFP-expressing MSCs^CCR9WT, MSCs^CCR9+, and MSCs^CCR9− were intravenously infused into mice with severe-GVHD on day 7 after HCT. (D) The presence and distribution of EGFP-expressing MSCs were examined via in situ immunofluorescence staining on day 7 after infusion. Total number and distribution of EGFP⁺ cells in the cortex and medulla were quantified per microscopic scale of thymus cryosections in triplicate mice, dotted lines trace the border between the cortex and medulla. Data are presented as mean ± SE. Signals: EGFP, (green, MSCs); CK8 (purple, highly expressed in the cortex and lowly expressed in the medulla); CK5, (red, expressed in the medulla). Scale bars, 50 μm. (E) Mice from each group on day 60 after HCT (1, non-GVHD; 2, MSC^CCR9+ GVHD; 3, MSC^CCR9WT GVHD; 4, MSC^CCR9- GVHD; and 5, non–MSC GVHD). (F) Cutaneous cGVHD symptom score (MSC^CCR9WT vs MSC^CCR9+, P < .01; MSC^CCR9+ vs MSC^CCR9−, P < .001; MSC^CCR9WT vs MSC^CCR9−, ns). (G) Survival curve (MSC^CCR9+ vs MSC^CCR9−, P < .05). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001.