肿瘤细胞治疗研究
英文原题：HVEM costimulatory domain boosts CAR T cell efficacy against solid tumors via enhanced TRAF-mediated TNF signaling.
HVEM 通过招募 TRAF 强效激活 TNF 信号通路，尤其是经由 AVEE 基序，从而增强 CAR T 细胞对实体瘤的疗效。
背景：嵌合抗原受体（CAR）T细胞疗法治疗血液系统恶性肿瘤已取得成功，但对实体瘤的疗效有限。疱疹病毒进入介质（HVEM）是一种共刺激分子，可增强CAR-T细胞活性，但其作用机制尚不清楚。本研究探讨HVEM共刺激结构域CAR-T细胞在实体瘤模型中优于4-1BB CAR-T的机制。方法：构建第二代靶向碳酸酐酶IX（CAIX）的CAR-T细胞，分别采用4-1BB、野生型（WT）HVEM或含AVEE突变（MUT）的HVEM共刺激结构域。通过实时细胞分析、ELISA和Western blot评估体外细胞毒性、细胞因子释放及信号通路；采用RNA测序和基因集富集分析（GSEA）比较转录谱；通过共免疫沉淀和质谱鉴定TNF受体相关因子（TRAF）蛋白互作。在NPG小鼠原位和皮下肾癌模型中评估体内疗效，并通过Seahorse实验测定代谢活性。结果：靶癌细胞刺激后，HVEM-CAR T细胞的细胞毒性、细胞因子释放及增殖均强于4-1BB-CAR T细胞。RNA测序和Western blot显示HVEM-CAR T细胞TNF信号增强，AKT和ERK1/2磷酸化升高，代谢水平也更高。HVEM通过AVEE基序募集TRAF1、TRAF2、TRAF3和TRAF5，而4-1BB仅结合TRAF1–3。AVEE基序突变会破坏TRAF结合、降低信号活化，并损害代谢和抗肿瘤功能。体内实验中，与4-1BB-CAR或AVEE突变HVEM-CAR T细胞相比，HVEM-CAR T细胞肿瘤控制更佳、生存更长、瘤内细胞丰度更高。结论：HVEM通过募集TRAF、尤其经AVEE基序强效激活TNF信号，增强CAR-T细胞对实体瘤的疗效。HVEM是有前景的CAR共刺激结构域，可改善实体瘤CAR-T疗法，其意义涉及代谢重编程和持久抗肿瘤活性。
BACKGROUND: Chimeric antigen receptor (CAR) T cell therapy has shown success in hematological malignancies, but its efficacy against solid tumors remains limited. Herpesvirus entry mediator (HVEM), a co-stimulatory molecule, enhances CAR T cell activity, though the underlying mechanisms are unclear. This study investigates how HVEM-based CAR T cells outperform those using 4-1BB in solid tumor models. METHODS: Second-generation CAR T cells targeting carbonic anhydrase IX (CAIX) were engineered with either 4-1BB, wild type (WT) HVEM, or AVEE mutation (MUT) HVEM co-stimulatory domains. In vitro cytotoxicity, cytokine release, and signaling pathways were assessed using real-time cellular analysis, ELISA, and Western blotting. RNA sequencing and gene set enrichment analysis (GSEA) compared transcriptional profiles. Co-immunoprecipitation and mass spectrometry identified TNF receptor-associated factor (TRAF) protein interactions. In vivo efficacy was evaluated in orthotopic and subcutaneous renal cancer models using NPG mice. Metabolic activity was measured via Seahorse assays. RESULTS: HVEM-CAR T cells exhibited stronger cytotoxicity, cytokine release, and proliferation than 4-1BB-CAR T cells in response to target cancer cell stimulation in vitro. RNA sequencing and Western blotting revealed enhanced TNF signaling in HVEM-CAR T cells, with higher phosphorylation of AKT and ERK1/2, accompanied by elevated metabolic levels. HVEM recruited TRAF1, TRAF2, TRAF3, and TRAF5 via its AVEE motif, whereas 4-1BB only bound TRAF1-3. Mutation of the AVEE motif disrupted TRAF binding, reduced signaling activation, and impaired metabolic and antitumor functions. In vivo, HVEM-CAR T cells showed superior tumor control, prolonged survival, and increased intratumoral abundance compared to 4-1BB-CAR or AVEE-mutated HVEM-CAR T cells. CONCLUSIONS: HVEM enhances CAR T cell efficacy against solid tumors by robustly activating TNF signaling through TRAF recruitment, particularly via the AVEE motif. These findings highlight HVEM as a promising co-stimulatory domain for improving CAR T cell therapy in solid tumors, with implications for metabolic reprogramming and sustained antitumor activity.
MEMBER ACCOUNT
登录成功会直接打开下一页。
