恶性肿瘤因基因突变引发细胞无限增殖，严重威胁人类健康。尽管传统治疗方式如手术、放化疗已在临床取得进展，但由于肿瘤细胞具有干性特征以及逐渐形成的获得性耐药，常难实现长效治疗，且相关毒副反应显著。近年来，TCR-T 疗法凭借其特异性识别 MHC 呈递的肿瘤抗原，作为一种精准免疫策略逐渐显示出治疗优势。然而，TCR-T 疗效仍受多重限制，包括 TCR 与 MHC 间亲和力低、肿瘤抗原表达异质性及免疫抑制微环境等因素。目前，PD-1/PD-L1 等免疫检查点阻断剂虽被引入联合治疗体系，但其使用可能导致免疫相关毒性增强，且患者个体响应存在差异，限制了其广泛应用潜力。

基于此，本研究结合高通量药物筛选与代谢干预策略，聚焦肿瘤代谢变化对TCR-T 细胞功能的调节，力图探索免疫联合治疗中的潜在干预靶点与机制，为进一步优化治疗效果提供理论依据。

第一部分 血清饥饿降低胆固醇增强黑素瘤对 TCR-T 杀伤敏感性

背景：热量限制及营养剥夺被证实可通过自噬激活、mTOR 信号抑制等机制干预肿瘤发展，然而其在调节肿瘤免疫方面的作用仍不明晰。最新研究表明，肿瘤细胞代谢状态可显著影响其对免疫细胞攻击的敏感性，尤其是胆固醇及脂类的代谢变化在抗原加工呈递、细胞膜构建以及信号转导调控中发挥关键调节作用。因此，明确胆固醇缺失对 TCR-T 细胞免疫效应的影响，有助于优化代谢-免疫联合治疗策略。

目的：（1）构建血清饥饿模型并评估其对 TCR-T 细胞杀伤效能的影响；（2）揭示血清饥饿作用机制；（3）探讨肿瘤细胞中胆固醇动态变化以及 mTORC1 激活在此过程中的关键调控作用；（4）验证其靶向特异性及脱靶风险；（5）探讨细胞骨架基因的调控效应。

方法：（1）以 α95LY TCR-T 细胞和 OVA-OT-1-T 细胞为研究工具；（2）建立人及小鼠黑素瘤细胞血清饥饿模型；（3）通过 RNA 测序、胆固醇回补实验检测杀伤效应的机制；（4）脱靶检测与骨架蛋白调控研究系统评估机制。

结果：在血清饥饿条件下，肿瘤细胞对 TCR-T 杀伤的易感性显著增强。转录组分析提示该变化与胆固醇代谢以及 mTORC1 抑制相关通路的上调密切相关。外源性胆固醇补充可逆转该增强效应，表明胆固醇减少为核心机制。共培养实验证实该效应具有高度靶向性，未引发非特异性杀伤。值得注意的是，实验发现上调mTORC1 活化相关基因会削弱 TCR-T 细胞的杀伤能力，进一步说明该通路在调控免疫应答中同样发挥关键作用。

结论：本研究发现，血清饥饿可通过下调肿瘤内胆固醇水平以及抑制 mTORC1  激活，有效提升肿瘤细胞对 TCR-T 细胞杀伤的响应性且保持靶向特异性，为发展基于代谢重编程的免疫治疗组合策略提供了新的研究视角与理论依据。

第二部分 胶原脯氨酸羟化酶抑制剂增强黑色素瘤细胞对 TCR-T 细胞杀伤的敏感性

背景：小分子化合物不仅可直接抑制肿瘤细胞生长，还能通过重塑免疫微环境，间接增强 T 细胞介导的抗肿瘤效应。已有研究指出，抗 PD-L1 抑制策略在一定程度上可改善 CD8⁺ T 细胞功能，但关于其他非 PD-1/PD-L1 通路小分子在增强肿瘤细胞对 T 细胞杀伤敏感性方面的具体调节机制仍缺乏系统研究。发掘能提升TCR-T 细胞疗效的调控因子，将为免疫治疗提供更多干预可能。

目的：（1）筛选可增强 TCR-T 细胞杀伤反应的小分子药物；（2）探究其作用机制。

方法：（1）以 α95LY TCR-T 细胞与 94a-14 TCR-T 细胞为研究模型；（2）从 MCE Targeted Diversity Library 中筛选增强肿瘤细胞杀伤响应的化合物；（3）通过杀伤实验与 RNA 测序评估敏感性增强机制；（4）利用 HIF-1A 基因调控验证相关机制。

结果：（1）CPHi、VE-821、Idasanutlin、SP96 等小分子化合物可显著增强肿瘤细胞对 TCR-T 介导杀伤的应答；（2）CPHi 上调 HIF-1A 增强 TCR-T 杀伤效力。

结论：CPHi 通过调控肿瘤内缺氧信号，提高肿瘤细胞对 TCR-T 介导杀伤的敏感性，为实现代谢重塑与免疫激活协同的联合治疗策略提供了新方向。

Malignant tumors continue to pose a major health burden due to mutations that disrupt cellular growth control, leading to unchecked proliferation. While conventional therapies—such as surgery, radiation, and chemotherapy—have yielded clinical benefits, durable responses remain elusive, largely owing to tumor stemness and adaptive resistance mechanisms. In recent years, T-cell receptor-engineered T cell therapy has emerged as a next-generation immunotherapeutic strategy, offering antigen-specific recognition via MHC-dependent pathways. Nevertheless, its therapeutic potential is limited by factors including subpar TCR–MHC affinity, variable tumor antigen profiles, and a profoundly immunosuppressive tumor milieu. The introduction of immune checkpoint blockade agents (e.g., PD-1/PD-L1 inhibitors) has revitalized interest in combination approaches that counteract T cell dysfunction. However, PD-L1 blockade in combination regimens may aggravate immune-related toxicity, and patient-specific variability can significantly hinder treatment efficacy and broad clinical applicability. Building on this foundation, the present study leverages high-throughput small-molecule screening and metabolic perturbation to examine how tumor-intrinsic metabolic cues shape TCR-T functionality, with the goal of identifying actionable targets to enhance therapeutic synergy.

1. Serum Starvation-induced Cholesterol Reduction Increases Melanoma Cell Susceptibility to Cytotoxic T Lymphocyte Killing

Background: Nutritional interventions such as caloric restriction and serum withdrawal have been implicated in tumor suppression through autophagy induction and mTOR inhibition. However, their impact on antitumor immunity-particularly T cell-based responses-remains under-investigated. Recent data highlight the influence of tumor metabolic status, especially lipid and cholesterol homeostasis, on immune recognition, membrane integrity, and signal transduction. Notably, inhibition of the mTORC1 signaling pathway under nutrient-deprived conditions has been shown to enhance TCR-T cell-mediated cytotoxicity, suggesting a pivotal role of mTORC1 in modulating immune responsiveness. Unraveling how cholesterol availability modulates TCR-T responsiveness could inform the design of metabolism-immune co-targeting strategies.

Objective: (1) To develop a serum-starvation model and examine its effect on TCR-T efficacy; (2) To uncover the relevant molecular underpinnings; (3) To evaluate the role of cholesterol reduction in sensitization, including the contribution of mTORC1 suppression under serum starvation; (4) To assess specificity and minimize the risk of off-target toxicity; (5) To investigate the involvement of cytoskeletal regulatory pathways.

Methods: (1) TCR-T effectors including α95LY and OVA-OT-1 cells were utilized; (2) Human and murine melanoma lines were subjected to serum deprivation; (3) RNA sequencing and cholesterol replenishment assays were performed for mechanistic dissection; (4) Off-target analyses and cytoskeletal gene studies were conducted to assess functional specificity; (5) RHEB gene modulation was conducted to verify mechanistic involvement.

Results: Serum starvation significantly improved tumor cell vulnerability to TCR-T-driven lysis. Transcriptome profiling indicated cholesterol biosynthesis downregulation as a key factor. In addition, reduced mTORC1 activation under serum starvation also contributed to enhanced cytotoxicity. Cholesterol repletion reversed the observed enhancement, confirming its mechanistic centrality. Co-culture experiments demonstrated high target fidelity without promoting nonspecific cytotoxicity.

Conclusion: This study reveals that nutrient restriction, through cholesterol depletion and mTORC1 suppression, augments tumor cell sensitivity to TCR-T cell cytotoxicity while maintaining antigen-specific precision, offering a metabolically informed framework for improving immunotherapeutic outcomes.

2. Collagen Proline Hydroxylase Inhibition Sensitizes Melanoma to TCR-T Cell-Mediated Cytotoxicity

Background: Small-molecule compounds are capable of not only impeding tumor progression directly but also of modulating the immune microenvironment to reinforce cytotoxic lymphocyte activity. While PD-L1 antagonists have shown partial efficacy in rejuvenating CD8⁺ T cell responses, the immunomodulatory actions of other targeted agents in the context of TCR-T cytotoxicity remain to be fully elucidated. Exploring molecular interventions that amplify TCR-T efficacy could offer novel strategies for immune enhancement.

Objective: (1) To identify compounds that potentiate TCR-T–driven tumor cell elimination; (2) To characterize the associated signaling cascades and mechanisms.

Methods: (1) α95LY TCR-T cells and 94a-14 TCR-T cells served as effector models; (2) A compound screen was performed using the MCE Targeted Diversity Library; (3) Killing assays and transcriptomic profiling were employed to dissect functional mechanisms; (4) HIF-1A gene modulation was conducted to verify mechanistic involvement.

Results: A panel of compounds-including CPHi, VE-821, Idasanutlin, and SP96, enhanced TCR-T-mediated killing of melanoma cells. Among them, CPHi upregulated HIF-1A, improving immune cytotoxicity.

Conclusion: CPHi promotes tumor susceptibility to TCR-T cells by orchestrating changes in hypoxia-inducible pathways, supporting its potential as a combinatorial immunotherapeutic adjuvant.