PO.IM01.11 · 免疫学
用于免疫调节剂评估的体外抗原特异性T细胞耗竭模型
In vitro antigen-specific T cell exhaustion model for immunomodulators evaluation
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
识别耗竭型CD8+ T细胞(T EX)潜在的新分子机制，是推进癌症及其他疾病免疫治疗策略的一个关键目标。然而，对体外T EX的高通量分析仍具挑战性。我们利用经受慢性抗原刺激的抗原特异性或TCR转基因T细胞，建立了人类CD8+ T细胞耗竭的体外模型。反复暴露于同源肽抗原可诱导标志性的耗竭表型，包括抑制性受体(PD-1、TIM-3和LAG-3)与转录因子TOX的共表达。耗竭T细胞表现出细胞因子分泌减少(如IFN-gamma)、增殖降低(如Ki-67)及细胞毒性受损。这些特征与肿瘤浸润淋巴细胞及慢性感染环境中观察到的耗竭高度吻合。我们评估了检查点阻断的功能挽救作用。抗PD-1恢复了部分增殖和适度的IFN-gamma产生。然而，由于其他抑制通路仍处于活跃状态，任何一种药物均未能完全逆转耗竭。这些发现与单药治疗仅能部分挽救的临床观察相一致。因此，我们的模型能够定量评估耗竭逆转，并支持组合式检查点策略。为生成足量的抗原特异性T细胞用于高通量检测，我们采用了源自表达多种HLA等位基因和共刺激配体的工程化K562细胞系的人工抗原提呈细胞(aAPC)。该平台支持将稀有的内源性T细胞扩增至每位供者>10^8个细胞，同时保留天然TCR表达。功能验证包括CMV特异性和MART-1特异性T细胞，它们展现出抗原特异性细胞毒性和细胞因子反应。这一可扩展且生理相关的检测系统能够精确模拟T细胞耗竭，并有助于系统性评估免疫治疗干预措施。它可作为定量筛选抑制或逆转T细胞耗竭药物的可靠平台，从而支持先进免疫疗法的合理开发。
查看英文原文 English abstract
The identification of novel molecular mechanisms underlying exhausted CD8⁺ T cells (T EX ) represents a critical objective in advancing immunotherapeutic strategies for cancer and other diseases. Nevertheless, high-throughput analysis of in vitro T EX remains challenging. We developed a human in vitro model of CD8⁺ T cell exhaustion using antigen-specific or TCR-transgenic T cells subjected to chronic antigen stimulation. Repeated exposure to cognate peptide antigens induced hallmark exhaustion phenotypes, including co-expression of inhibitory receptors (PD-1, TIM-3 and LAG-3) and transcription factor TOX. Exhausted T cells exhibited diminished cytokine secretion (e.g. IFN-gamma), reduced proliferation (e.g. Ki-67), and impaired cytotoxicity. These features closely mirror exhaustion observed in tumour-infiltrating lymphocytes and chronic infection settings. Checkpoint blockade was evaluated for functional rescue. Anti-PD-1 restored partial proliferation and modest IFN-gamma production. However, neither agent fully reversed exhaustion, as other inhibitory pathways remained active. These findings align with clinical observations where monotherapy yields partial rescue. Our model thus enables quantitative assessment of exhaustion reversal and supports combinatorial checkpoint strategies. To generate sufficient antigen-specific T cells for high-throughput assays, we employed artificial antigen-presenting cells (aAPCs) derived from engineered K562 lines expressing diverse HLA alleles and co-stimulatory ligands. This platform supports expansion of rare endogenous T cells to >10⁸ cells per donor, preserving native TCR expression. Functional validation included CMV-specific and MART-1-specific T cells, which demonstrated antigen-specific cytotoxicity and cytokine responses. This scalable and physiologically relevant assay system permits precise modelling of T cell exhaustion and facilitates systematic evaluation of immunotherapeutic interventions. It serves as a reliable platform for the quantitative screening of agents that inhibit or reverse T cell exhaustion, thereby supporting the rational development of advanced immunotherapies.
利益披露 Disclosure
E. Cosimo, None..
H. Findlay, None..
R. Benson, None..
A. Patakas, None.