NICD is essential for OTUD1-mediated TCR signaling transduction that accelerates aGVHD progression. (A) CD4⁺ T cells isolated from C57BL/6 mice were stimulated with anti-CD3 (2 μg/mL) and anti-CD28 (0.4 μg/mL) antibodies and incubated with control (Ctrl) or DAPT (1, 25 μM) for 24 hours in vitro, and then the protein levels of IFN-γ and IL-2 were monitored by ELISA. (B) Jurkat E6.1 cells stably expressing OTUD1 were incubated with anti-CD3 (0.1 μg/mL) antibody and with or without DAPT (5 μM) for 48 hours, and then Smad7, c-rel, p-p65 and p-Erk were assessed by western blot. (C) Jurkat E6.1 cells transfected with pCDH-OTUD1, shCON or Notch2 shRNA (shNotch2) plasmids were stimulated with anti-CD3 (0.1 μg/mL) antibody for 48 hours, and then Smad7, c-rel, p-LCK, p-p65, p-Erk and NFAT were monitored by western blot. (D) A schematic diagram of the mice experiments. (E-F) 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 injected with 80 μg Flag-NICD or Ctrl plasmids (n = 10 for WT + Ctrl group, n = 8 for OTUD1-KO + Ctrl group, n = 10 for OTUD1-KO + NICD group). The survival of aGVHD mice was observed twice 1 day (E), and the aGVHD scores were observed and recorded every 2 days (F). (G) Representative images of intestine, lung and liver of aGVHD mice by HE staining (scale bar, 100 μm; arrows, infiltrating lymphocytes; triangles, apoptotic epithelial cells). (H) Histological scores of intestine, lungs, and liver of aGVHD mice were evaluated. Log-rank (Mantel-Cox) test was used to analyze the survival curve. Data in panels A,H are represented as mean ± SD; ∗P < .05, ∗∗P < .01, ∗∗∗P < .001 (2-tailed unpaired Student t test). Data in panels B,C are representative of 3 independent experiments and are summarized as mean ± SD of 3 experiments.