当前在免疫肿瘤学中，生物标志物发现和靶点鉴定的方法仅依赖于肿瘤免疫性的静态快照。为了全面揭示抗肿瘤免疫应答的时间性质，我们开发了一个包含34个参数的光谱流式细胞术面板，并在关键环境中进行了高通量分析。我们利用两个不同的临床前模型，分别重现肿瘤免疫编辑（NPK-C1）和免疫检查点阻断（ICB）应答（MC38），并在与免疫监控和逃逸和/或ICB应答的关键转折点处对多个相关组织进行了剖析。基于机器学习驱动的数据分析揭示出一种KLRG1表达模式，唯一鉴定了肿瘤内抗体效应型CD4T细胞群体，它们与肿瘤负担相关，并且在进行ICB治疗中发生回归的肿瘤中消失。同样，肿瘤浸润调节性T细胞的Helios-KLRG1+亚群与从免疫平衡转变为逃逸的肿瘤进展相关，并且在对ICB作出反应的肿瘤中也会消失。验证研究证实在人类肿瘤浸润CD4 T细胞中KLRG1标记与肾癌疾病进展相关。这些发现将KLRG1+ CD4 T细胞群体提名为进一步探索肿瘤免疫性的子集，并且证明了纵向光谱流式分析作为动态生物标志物发现引擎的实用性。©作者（或其雇主）2023年。根据CC BY-NC许可进行重用。不允许商业再利用。由BMJ出版。

Current methods for biomarker discovery and target identification in immuno-oncology rely on static snapshots of tumor immunity. To thoroughly characterize the temporal nature of antitumor immune responses, we developed a 34-parameter spectral flow cytometry panel and performed high-throughput analyses in critical contexts. We leveraged two distinct preclinical models that recapitulate cancer immunoediting (NPK-C1) and immune checkpoint blockade (ICB) response (MC38), respectively, and profiled multiple relevant tissues at and around key inflection points of immune surveillance and escape and/or ICB response. Machine learning-driven data analysis revealed a pattern of KLRG1 expression that uniquely identified intratumoral effector CD4 T cell populations that constitutively associate with tumor burden across tumor models, and are lost in tumors undergoing regression in response to ICB. Similarly, a Helios-KLRG1+ subset of tumor-infiltrating regulatory T cells was associated with tumor progression from immune equilibrium to escape and was also lost in tumors responding to ICB. Validation studies confirmed KLRG1 signatures in human tumor-infiltrating CD4 T cells associate with disease progression in renal cancer. These findings nominate KLRG1+ CD4 T cell populations as subsets for further investigation in cancer immunity and demonstrate the utility of longitudinal spectral flow profiling as an engine of dynamic biomarker discovery.© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.