Allogeneic response-induced upregulation of OTUD1 exacerbates aGVHD progression in HCT patients. (A) A schematic diagram of the mice experiments. (B) Heat map showing the differential thiol-dependent deubiquitinating enzymes. (C) T cells were isolated from syn-HSCT (n = 8) and allo-HSCT mice (n = 8) and the mRNA levels of Otud1, Otud3, Otud4, Otud5, Otud6b and Otud7b were evaluated by reverse transcription quantitative polymerase chain reaction (RT-qPCR). (D) CD4⁺ T cells and CD8⁺ T cells purified from C57BL/6 mice were stimulated with anti-CD3 (2 μg/mL) and anti-CD28 (0.4 μg/mL) antibodies for 48 hours in vitro. Western blot was performed to evaluate the protein levels of OTUD1 in CD4⁺ T and CD8⁺ T cells with or without stimulation. (E) A schematic diagram of the patients experiments. (F) PBMCs were isolated from patients with aGVHD grade 0 to 1 (n = 109) and grade 2 to 4 (n = 57) and the mRNA levels of Otud1, Otud2, Otud4, Otud5, Otud6b, Otud7b were analyzed by RT-qPCR. (G) ROC curve for the Otud1 mRNA level predicts aGVHD, cutoff value = 0.04633. (H) The cumulative incidence of II-IV aGVHD after HCT between Otud1^hi (>0.04633) and Otud1^lo (<0.04633) patients. (I) The OS of patients with Otud1^hi or Otud1^lo after HCT. The cumulative incidence of aGVHD and the survival curve were analyzed by log-rank (Mantel-Cox) test. Data in panels C,F are represented as mean ± standard deviation (SD); ∗P < .05, ∗∗P < .01, ∗∗∗P < .001 (2-tailed unpaired Student t test). Data in panel D are representative of 3 independent experiments and are summarized as mean ± SD of 3 experiments. HCT, hematopoietic cell transplantation; ROC, receiver operating characteristic.