Pilot Project: Application of microfluidics to in situ adaptive immunity

项目摘要 抗原限制性细胞：细胞相互作用驱动适应性免疫。虽然对于理解致病性 机制，有限的技术可用于研究人类免疫细胞在组织中的相互作用， 疾病正如UofC ACE合作项目所展示的那样，克拉克实验室开发了一种新颖的 用于识别和量化人体组织的共聚焦图像中的同源相互作用的技术。的 克拉克实验室计算方法的当前版本，细胞距离映射版本3（CDM 3）使用深度 机器学习识别潜在抗原呈递细胞（APC）和CD 4 + T细胞之间的同源相互作用 细胞然而，有限的补充技术可用于功能研究细胞：细胞相互作用 由CDM 3确定。历史上，通过检查从外周血中分离的细胞亚群， 血虽然通过这种方法产生了一些有价值的见解，但在 外周血不一定反映在发炎组织中发现的那些。为了充分了解当地 由于细胞-细胞相互作用导致组织内的病理学，因此有必要开发新的测定法， 应用于从临床可获得的组织样品中分离的少量细胞。在这份资助申请中， 我们建议使用我们实验室开发的新型微流体技术来解决这一迫切需求。作为 在已发表的工作和这项资助申请中证明，我们可以分离单个细胞或细胞对，并进行 用于细胞运动性、表面表达、信号传导和细胞因子产生的单细胞功能测定。我们提出 将这些技术方法扩展到研究分离的细胞中MHC类限制性B：T协作， 人体组织我们假设在不同的B和T细胞之间的相互作用中观察到的特异性 体内的亚群反映了内在的细胞差异和能力。这一假设将在下面进行检验 具体目标： 目标1：开发使用微流体询问个体T细胞-B细胞相互作用的方法。 目的2：使用基于微流控技术的免疫组织化学技术检测类风湿关节炎中原位T细胞-B细胞相互作用。 功能测定

PROJECT SUMMARY Antigen-restricted cell:cell interactions drive adaptive immunity. While critical for understanding pathogenic mechanisms, limited techniques are available to study human immune cell interactions in tissue at sites of disease. As demonstrated in the UofC ACE Collaborative Project, the Clark Lab has developed novel technologies to identify and quantify cognate interactions in multicolor confocal images of human tissue. The current version of the Clark Lab computational approach, Cell Distance Mapping version 3 (CDM3) uses deep machine learning to identify cognate interactions between potential antigen presenting cells (APCs) and CD4+ T cells. However, limited complementary technologies are available to functionally study cell:cell interactions identified by CDM3. Historically, this issue has been skirted by examining cell subsets isolated from peripheral blood. Though some valuable insights have been generated through this approach, the subsets found in peripheral blood are not necessarily reflective of those found in inflamed tissue. To fully understand the local cell-cell interactions that contribute to pathology within tissue, it is necessary to develop novel assays that can be applied to small numbers of cells isolated from clinically obtainable tissue samples. In this grant application, we propose to use novel microfluidic techniques developed in our laboratory to address this pressing need. As demonstrated in published work and this grant application, we can isolate single cells, or cell pairs, and perform single cell functional assays for cell motility, surface expression, signaling and cytokine production. We propose to extend these technical approaches to studying MHC class-restricted B:T collaboration in cells isolated from human tissue. We hypothesize that specificities observed in the interactions between different B and T cell subsets in vivo reflect intrinsic cell differences and capacities. This hypothesis will be tested in the following Specific Aims: Aim 1: Develop methods for interrogating individual T cell-B cell interactions using microfluidics. Aim 2: Examine in situ T cell-B cell interactions in rheumatoid arthritis using microfluidics-based functional assays.