Xu Y X, Wang X Q, Yang S J, Song Q X, Wei J, Zhang X
Department of Hematology, Affiliated Hospital of North Sichuan Medical College, Nanchong 637002, China Medical Center of Hematology, Xinqiao Hospital of Army Medical University, Blood Ecology and Smart Cell Science Innovation Center, State Key Laboratory of Trauma and Chemical Poisoning, Chongqing Key Laboratory of Hematology and Microenvironment, Chongqing 400037, China Jinfeng Laboratory, Chongqing 401329, China.
Medical Center of Hematology, Xinqiao Hospital of Army Medical University, Blood Ecology and Smart Cell Science Innovation Center, State Key Laboratory of Trauma and Chemical Poisoning, Chongqing Key Laboratory of Hematology and Microenvironment, Chongqing 400037, China.
Zhonghua Xue Ye Xue Za Zhi. 2025 Apr 14;46(4):302-313. doi: 10.3760/cma.j.cn121090-20250107-00011.
To investigate the effects and underlying mechanisms of Stat3 knockout in donor T cells on acute gastrointestinal graft-versus-host disease (GI-aGVHD) . BALB/c mice were exposed to lethal irradiation and transplanted with bone marrow and spleen cells from BALB/c mice (syngeneic control group), C57BL/6 mice (wild-type T cell group, WT group), or C57BL/6J-Stat3(em1cyagen) mice (Stat3 gene knockout T cell group, Stat3-KO group) via tail vein injection to establish the aGVHD model. The survival rate, body weight changes, and clinical scores of mice were monitored. Cytometric bead array (CBA) was used to detect the concentrations of serum cytokines. Lymphocytes were isolated from tissues for flow cytometric analysis. H&E staining was performed to observe intestinal pathological changes. FITC-dextran assay was conducted to assess intestinal permeability. Immunohistochemistry was used to evaluate the expression of Ki67 and Muc2. Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) was employed to analyze the gene expression levels of Olfm4, Lysozyme, and Muc2 in the small intestine. Metabolomics was conducted to detect metabolites in serum and intestinal tissues. An in vitro GI-aGVHD organoid model was established by coculturing intestinal organoids with allogeneic T cells, where the number and area of small intestinal organoids were recorded. The GVL effect was assessed using luciferase-transfected ALL cells (ALL/Luc) and bioluminescent imaging. Compared with the WT group, Stat3 knockout T cells alleviated body weight loss, reduced symptoms-such as hunchback and diarrhea-in mice, improved survival rate (<0.05), and reduced serum interleukin (IL) -2, IL-6, interferon-γ, tumor necrosis factor-α, IL-17A, and IL-10 levels (all <0.05), intestinal inflammatory cell infiltration (<0.05), and intestinal mucosal permeability. Further, Muc2 and Ki67 expression levels in the small intestine of the Stat3 knockout group were markedly increased, and Olfm4, Lysozyme, and Muc2 gene expression levels were significantly increased (all <0.05). In vitro, the Stat3 knockout group demonstrated better organoid development than the WT group. Metabolomic analyses indicated that Stat3 knockout in T cells may affect the pathways associated with bile acid secretion and unsaturated fatty acids. ALL/Luc cells in the GVL mouse model proliferated rapidly in the TCD-BM group; however, 80% of the mice in the Stat3-KO group survived tumor-free for >100 days (<0.05) . Knocking out Stat3 in graft T cells reduces T cell damage to intestinal stem cells, thereby ultimately alleviating GI-aGVHD while maintaining a stable GVL effect.
为研究供体T细胞中Stat3基因敲除对急性胃肠道移植物抗宿主病（GI-aGVHD）的影响及潜在机制。将BALB/c小鼠进行致死性照射，然后通过尾静脉注射来自BALB/c小鼠的骨髓和脾细胞（同基因对照组）、C57BL/6小鼠的骨髓和脾细胞（野生型T细胞组，WT组）或C57BL/6J-Stat3(em1cyagen)小鼠的骨髓和脾细胞（Stat3基因敲除T细胞组，Stat3-KO组），以建立aGVHD模型。监测小鼠的存活率、体重变化和临床评分。采用细胞因子微球阵列（CBA）检测血清细胞因子浓度。从组织中分离淋巴细胞进行流式细胞术分析。进行苏木精-伊红（H&E）染色以观察肠道病理变化。进行异硫氰酸荧光素-葡聚糖（FITC-葡聚糖）检测以评估肠道通透性。采用免疫组织化学评估Ki67和Muc2的表达。运用实时定量逆转录聚合酶链反应（qRT-PCR）分析小肠中Olfm4、溶菌酶和Muc2的基因表达水平。进行代谢组学检测血清和肠道组织中的代谢物。通过将肠道类器官与同种异体T细胞共培养建立体外GI-aGVHD类器官模型，记录小肠类器官的数量和面积。使用荧光素酶转染的急性淋巴细胞白血病（ALL）细胞（ALL/Luc）和生物发光成像评估移植物抗白血病（GVL）效应。与WT组相比，Stat3基因敲除T细胞减轻了小鼠体重减轻，减少了如驼背和腹泻等症状，提高了存活率（<0.05），并降低了血清白细胞介素（IL）-2、IL-6、干扰素-γ、肿瘤坏死因子-α、IL-17A和IL-10水平（均<0.05）、肠道炎性细胞浸润（<0.05）以及肠道黏膜通透性。此外，Stat3基因敲除组小肠中Muc2和Ki67的表达水平显著升高，Olfm4、溶菌酶和Muc2的基因表达水平也显著增加（均<0.05）。在体外，Stat3基因敲除组的类器官发育优于WT组。代谢组学分析表明，T细胞中Stat3基因敲除可能影响与胆汁酸分泌和不饱和脂肪酸相关的途径。GVL小鼠模型中的ALL/Luc细胞在去除T细胞的骨髓（TCD-BM）组中迅速增殖；然而，Stat3-KO组中80%的小鼠无瘤存活>100天（<0.05）。敲除移植物T细胞中的Stat3可减少T细胞对肠道干细胞的损伤，从而最终减轻GI-aGVHD，同时维持稳定的GVL效应。
