MICROFLUIDIC SYSTEM FOR HIGH-THROUGHPUT EVALUATION OF T CELL FUNCTIONALITY WITH H

描述(申请人提供)：T细胞过继转移是一种很有前途的临床癌症治疗方法，它依赖于增强体内对靶肿瘤细胞的适应性免疫反应。然而，这种疗法的广泛应用受到了每个患者大量T细胞的必要扩张(通常是因为肿瘤抗原特异性而被选择)和移植后T细胞功能丧失的阻碍。我们的长期目标是了解肿瘤环境如何抑制体内T细胞的激活，并能够准确地评估体外扩增的T细胞对癌症治疗的反应性。微流控芯片是高通量并行实验和自动化的理想选择。此外，微流体还提供了相关的长度尺度(~微米)和独特的物理现象(例如层流)来处理细胞。我们可以从这项技术中获得的多重数据类型将使T细胞激活的定量建模以及更好地理解和表征无能。这个R21项目的目标是设计一种多重微流控分析，以高时间分辨率量化T细胞在一小群细胞上的激活。这个假设是，捕捉到体外扩增克隆的T细胞激活的早期动力学将在目前T细胞功能测量的基础上有所改善。该项目的第一个组成部分是开发用于多时间点刺激和细胞裂解的高通量微流控系统；同时，我们将开发生化分析来表征系统的性能和细胞状态。第二个部分是对体外扩增的T细胞进行体外鉴定，以区分无能和反应性行为。这种方法是创新的，因为这里开发的技术极大地提高了现有检测方法在评估采用转移的T细胞方面的能力和吞吐量。此外，这项工作提出并测试了一种使用多重定量方法进行T细胞评估的新范式。这项拟议的研究意义重大，因为它有望扩大癌症治疗的工具箱，并可能扩大其他相关的定量生物科学和医疗技术。

DESCRIPTION (provided by applicant): Adoptive transfer of T cells is a promising clinical cancer therapy that relies on enhancing the adaptive immune response to target tumor cells in vivo. Widespread application of this therapy, however, has been hindered by the necessary expansion of large populations of T cells for each patient (often selected for tumor antigen specificity) and loss of functionality of the T cells post-transfer. Our long-term objective is to understand how T cell activation is dampened in vivo by the tumor milieu and to be able to evaluate the responsiveness ex vivo-expanded T cells accurately for cancer therapy. Microfluidic chips are ideal for high-throughput parallel experimentation and automation. In addition, microfluidics also provides the relevant length scales (~microns) and unique physical phenomena (e.g. laminar flow) to handle cells. The type of multiplex data that we can obtain from this technology will enable quantitative modeling of T cell activation and better understanding and characterization of anergy. The objective of this R21 project is to engineer a multiplex microfluidic assay to quantify T cell activation on a small population of cells with high temporal resolution. The hypothesis is that capturing the early dynamics of T cell activation of ex vivo expanded clones would improve upon current measures of T cell functionality. The first component of this project is to develop the high-throughput microfluidic system for multiple time-point stimulation and lysis of cells; in parallel, we are to develop biochemical assays to characterize the performance of the system and the cell state. The second component is to perform in vitro characterization of ex vivo expanded T cells for distinguishing anergic versus responsive behavior. The approach is innovative because the technology developed here dramatically increases the capabilities and throughput of existing assays in evaluating T cells for adoptive transfer. Furthermore, this work proposes and tests a new paradigm in T cell evaluation using multiplex quantitative means. The proposed research is significant because it is expected to expand the toolbox of cancer therapy and possibly other related quantitative biosciences and medical technologies.