Pilot Project: Application of microfluidics to in situ adaptive immunity

试点项目：微流控在原位适应性免疫中的应用

• 批准号: 9920104
• 负责人: Savas Tay
• 金额: $ 12万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9920104
• 关键词: Address; Antigen-Presenting Cells; Antigens; Applications Grants; Autoimmune Diseases; Autoimmunity; B-Lymphocyte Subsets; B-Lymphocytes; Behavior; Biological Assay; Blocking Antibodies; CD4 Positive T Lymphocytes; CD69 antigen; CXCL13 gene; Calcium Signaling; Cell Communication; Cells; Cellular Assay; Chicago; Clinical; Collaborations; Disease; Enzyme-Linked Immunosorbent Assay; Fluo 4; Hour; Human; Humoral Immunities; Image; Immune; In Situ; Individual; Inflammation; Laboratories; Lupus Nephritis; Lymphocyte; MHC Class II Genes; Machine Learning; Measures; Methods; Microfluidic Microchips; Microfluidics; Microscope; Operative Surgical Procedures; Organ; Outcome; Outcome Measure; Pathogenesis; Pathogenicity; Pathologic Processes; Pathology; Patients; Pilot Projects; Population; Predictive Value of Tests; Production; Publishing; Rheumatoid Arthritis; Sampling; Scleroderma; Signal Transduction; Site; Specificity; Stains; Surface; Synovial Membrane; T-Lymphocyte; T-Lymphocyte Subsets; Techniques; Technology; Testing; Time; Tissue Sample; Tissues; Tonsil; Universities; Viola; Work; adaptive immune response; adaptive immunity; antibody conjugate; base; behavioral study; calcium indicator; cell behavior; cell motility; cell type; confocal imaging; cytokine; experimental study; human tissue; in vivo; innovation; insight; new technology; new therapeutic target; novel; peripheral blood; release of sequestered calcium ion into cytoplasm; response; secondary lymphoid organ; transcriptomics

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.

项目概要 抗原限制的细胞：细胞相互作用驱动适应性免疫。虽然对于了解致病性至关重要 机制，可用于研究人体免疫细胞在组织中的相互作用的技术有限。 疾病。正如 UofC ACE 合作项目所示，克拉克实验室开发了新颖的 识别和量化人体组织多色共焦图像中同源相互作用的技术。这 克拉克实验室计算方法的当前版本，细胞距离映射版本 3 (CDM3) 使用深度 机器学习识别潜在抗原呈递细胞 (APC) 和 CD4+ T 之间的同源相互作用 细胞。然而，可用于功能性研究细胞与细胞相互作用的补充技术有限 由CDM3确定。从历史上看，这个问题一直通过检查从外周血中分离出来的细胞亚群来回避。 血。尽管通过这种方法产生了一些有价值的见解，但发现的子集 外周血不一定反映发炎组织中发现的血。充分了解当地情况 细胞与细胞之间的相互作用会导致组织内的病理学，因此有必要开发新的检测方法 适用于从临床可获得的组织样本中分离出的少量细胞。在本次拨款申请中， 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 在已发表的工作和本次拨款申请中证明，我们可以分离单个细胞或细胞对，并执行 用于细胞运动、表面表达、信号传导和细胞因子产生的单细胞功能测定。我们建议 扩展这些技术方法来研究 MHC 类别限制的 B:T 细胞中分离的协作 人体组织。我们假设在不同 B 细胞和 T 细胞之间的相互作用中观察到的特异性 体内亚群反映了内在的细胞差异和能力。该假设将在下面进行检验 具体目标： 目标 1：开发使用微流体研究个体 T 细胞-B 细胞相互作用的方法。 目标 2：使用基于微流体技术检查类风湿性关节炎中 T 细胞-B 细胞的原位相互作用 功能测定。