OTUD1 deficiency inhibits T-cell activation and proinflammatory cytokines production, alleviates aGVHD pathogenicity after allo-BMT (Bone Marrow Transplantation). (A-B) CD4⁺ T cells (A) or CD8⁺ T cells (B) isolated from C57BL/6 mice were stimulated with anti-CD3 (2 μg/mL) and anti-CD28 (0.4 μg/mL) antibodies for 48 hours, and were analyzed to detect the expression of T-bet, RORγt and Gata3 by flow cytometry. (C) Naive CD4⁺ T cells purified from WT or OTUD1^−/− mice on B6 background were stimulated with anti-CD3 (2 μg/mL) and anti-CD28 (0.4 μg/mL) antibodies for 24 or 48 hours. RT-qPCR was performed to evaluate the mRNA levels of IL-2 and IFN-γ. (D) Protein levels of IL-2 and IFN-γ were monitored by ELISA. (E-G) BM cells (1 × 10⁷ per mouse) and splenocytes (5 × 10⁶ per mouse) isolated from WT or OTUD1^−/− mice on B6 background were transferred into lethally irradiated BALB/c mice (n = 8 for WT, n = 8 for OTUD1-KO). The survival (E) of aGVHD mice was observed twice daily, and body weight (F) and aGVHD scores (G) were observed and recorded every 2 days. (H) Representative images were shown in skin, liver, lung, and intestine of aGVHD mice by hematoxylin and eosin (HE) staining (scale bar, 100 μm; arrows, infiltrating lymphocytes; triangles, apoptotic epithelial cells). (I) Histological scores of skin, liver, lungs, and intestine of aGVHD mice were evaluated. (J-K) BM cells (1 × 10⁷ per mouse) isolated from WT mice and CD4⁺ T cells (2 × 10⁶ per mouse) isolated from WT or OTUD1^−/− mice were transferred into lethally irradiated BALB/c mice (n = 13 for BM, n = 20 for WT, n = 18 for OTUD1-KO). The survival of aGVHD mice was observed twice a day (J), aGVHD scores were observed and recorded every 2 days (K). Log-rank (Mantel-Cox) test was used to analyze the survival curve. Data in panels A-D,I are represented as mean ± SD with biological replicates; ∗P < .05, ∗∗P < .01, ∗∗∗P < .001 (two-tailed unpaired Student t test).