PO.IM01.17 · 免疫学
一种61参数CyTOF抗体组，用于全面分析人PBMC以表征免疫活化、检查点表达和细胞因子特征，并应用于癌症免疫学
A 61-parameter CyTOF panel for comprehensive profiling of human PBMC to characterize immune activation, checkpoint expression and cytokine signatures with applications in cancer immunology
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摘要 Abstract
中文摘要
理解癌症中免疫应答的复杂性对于开发有效的免疫疗法和预测患者结局至关重要。高参数细胞术分析能够对免疫细胞表型和功能状态进行深度分析，揭示肿瘤免疫逃逸和治疗耐药的机制。CyTOF™技术是一种单细胞分析平台，使用金属标记抗体在单管中解析50余个标志物。与基于荧光的细胞术不同，其无需光谱解混和单染对照。因此，使用CyTOF技术能够独特地快速设计高参数抗体组，从而同时评估谱系、活化、耗竭和胞内信号通路。本研究旨在设计一种针对人PBMC功能性免疫分析优化的61参数CyTOF抗体组，以支持癌症免疫学研究。该抗体组纳入了主要免疫亚群的谱系标志物以及一套广泛的T细胞表型分析靶标，包括活化、分化、免疫检查点（例如PD-1、CTLA-4、TIGIT）以及与肿瘤免疫相关的胞内细胞因子。未处理和刺激的PBMC经条形码标记、混合并染色。样本经冷冻保存，随后使用CyTOF XT PRO系统采集，以最大限度减少技术变异。刺激PBMC的高维分析证明了该抗体组在单细胞水平解析广泛免疫功能多样性的能力。在效应、记忆、细胞毒性、调节性和耗竭免疫细胞亚群中检测到活化标志物、免疫检查点和胞内细胞因子的共表达模式。这些发现凸显了该抗体组在应用于癌症患者样本或肿瘤相关免疫细胞时，揭示与肿瘤免疫和免疫治疗应答相关特征的潜力。CyTOF系统能够在单个抗体组中实现最高数量的同时测量，从而在高分辨率解析胞内靶标的同时进行全面免疫分析，以探究功能潜力。总体而言，使用CyTOF技术研究功能免疫学可阐明免疫应答的复杂本质，从而理解其与疾病和治疗的关系。仅供研究使用。不用于诊断程序。
查看英文原文 English abstract
Understanding the complexity of immune responses in cancer is critical for developing effective immunotherapies and predicting patient outcomes. High-parameter cytometric analysis enables deep profiling of immune cell phenotypes and functional states, revealing mechanisms of tumor immune evasion and therapeutic resistance. CyTOF™ technology is a single-cell analysis platform that uses metal-tagged antibodies to resolve 50-plus markers in a single tube. Unlike fluorescence-based cytometry, spectral unmixing and single-stain controls are not required. Therefore, it is uniquely possible with CyTOF technology to rapidly design high-parameter panels enabling simultaneous assessment of lineage, activation, exhaustion and intracellular signaling pathways. This study aimed to design a 61-parameter CyTOF panel optimized for functional immune profiling of human PBMC to support cancer immunology research. The panel incorporates lineage markers for major immune subsets and an extensive set of T cell phenotyping targets including activation, differentiation, immune checkpoints (for example, PD-1, CTLA-4, TIGIT) and intracellular cytokines relevant to tumor immunity. Untreated and stimulated PBMC were barcoded, pooled and stained. Samples were cryopreserved and later acquired using a CyTOF XT PRO system to minimize technical variation. High-dimensional analysis of stimulated PBMC demonstrated the panel's ability to resolve extensive immuno-functional diversity at the single-cell level. Co-expression patterns of activation markers, immune checkpoints and intracellular cytokines were detected across effector, memory, cytotoxic, regulatory and exhausted immune cell subsets. These findings highlight the panel's potential to uncover signatures relevant to tumor immunity and immunotherapy response when applied to cancer patient samples or tumor-associated immune cells. CyTOF systems enable the highest number of simultaneous measurements in a single panel, allowing for comprehensive immune profiling alongside high resolution of intracellular targets to interrogate functional potential. Overall, studying functional immunology using CyTOF technology can elucidate the complex nature of immune responses to provide an understanding of how they relate to disease and treatments. For Research Use Only. Not for use in diagnostic procedures.
利益披露 Disclosure
L. J. Tracey, None..
K. Thomson, None.