Time-lapse Flow Cytometry for Kinetic Profiling of T-Cell Function

摘要 尽管免疫疗法取得了进展，但仍有重大挑战需要克服 更广泛地适用于不同的癌症，对患者更有效。但是，当前 努力改进过继细胞疗法和免疫检查点封锁以及癌症 疫苗由于缺乏表征功能和表型的方法而受到阻碍。 高通量下不同T细胞亚群随时间的变化。这项SBIR的目标是 第二阶段的项目是开发一种新的流式细胞仪方法，可以解决这一技术 瓶颈。延时流式细胞术使用激光粒子(LP)条形码来跟踪每个细胞 在不同时间点进行的横流测量。建立在已演示的概念验证基础上 数据，这个项目的重点是建立一套新的检测方法来表征T细胞 以时间分辨的方式对不同的刺激作出反应。第一个具体目标是证明 短期时间推移分析细胞因子分泌(t-SEC)。分泌肿瘤坏死因子、干扰素γ和 来自数百万个相同细胞的IL10对不同类型的刺激的反应在 24小时内有多个时间点。第二个具体目标是证明长期的延时 检测T细胞表型(t-pho)，包括10天内T细胞耗竭情况。第三 具体目的是用患者样本验证t-SEC和t-Pheno分析。时间- 分辨率高、高通量、高参数的分析有望加速 更有效、更持久的癌症免疫疗法(全球市场价值1000亿美元)。超越 免疫治疗，延时流式细胞术也有望在基础 免疫学研究和疫苗开发。

Abstract Despite progress in immunotherapies, there are significant challenges to overcome to make them more broadly applicable to different cancers and more effective for patients. However, current efforts to improve adoptive cell therapies and immune checkpoint blockade as well as cancer vaccines are hampered by the lack of a method to characterize the functional and phenotypical changes of different subpopulations of T cells over time at high throughput. The goal of this SBIR Phase II project is to develop a novel flow-cytometry method that can solve this technological bottleneck. The time-lapse flow cytometry uses laser particle (LP) barcodes to track each cell across flow measurements taken at different time points. Built on demonstrated proof-of-concept data, this project is focused on establishing a set of novel assays for characterizing T cells in response to different stimuli in a time-resolved manner. The first specific aim is to demonstrate short-term time-lapse assays to profile cytokine secretion (t-SEC). Secretion of TNF, IFNγ and IL10 from millions of the same cells in response to different types of stimulation are measured at multiple timepoints over 24 hours. The second specific aim is to demonstrate long-term time-lapse assay to monitor phenotype (t-PHENO), including T-cell exhaustion over 10 days. The third specific aim is to validate the t-SEC and t-PHENO assays with patient samples. The time- resolved, high-throughput, high-parameter assays are expected to accelerate the development of more effective and durable immunotherapies for cancer (>$100 B global market). Beyond immunotherapy, time-lapse flow cytometry is also expected to be useful in fundamental immunological research and vaccine development.