DNA-gated cytometry for multiplexed sorting of antigen-specific CD8 T cells

用于抗原特异性 CD8 T 细胞多重分选的 DNA 门控细胞术

基本信息

批准号：
10503181
负责人：
Gabriel A Kwong
金额：
$ 39.55万
依托单位：
GEORGIA INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-06-20 至 2027-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10503181
关键词：
Affinity Antibodies Antigens Avidity Benchmarking Binding Biological Assay CAR T cell therapy CD19 gene CD8-Positive T-Lymphocytes Cell Death Cell Fraction Cell Separation Cell Therapy Cell physiology Cells Couples Cytomegalovirus Cytometry DNA DNA Sequence Engineering Fluorescence Fluorescence-Activated Cell Sorting Generations HLA-A2.1 Human Human Herpesvirus 4 Hybrids Individual Infection Influenza K562 Cells Label Length Libraries Light Lymphocytic choriomeningitis virus Magnetism Major Histocompatibility Complex Mediating Modeling Molecular Mus Oligonucleotides Peptide/MHC Complex Peptides Phenotype Population Process Production Raji Cell Reaction Receptor Activation Receptor Cell Sampling Sorting - Cell Movement Splenocyte T cell therapy T-Cell Receptor T-Lymphocyte T-Lymphocyte Epitopes T-cell receptor repertoire Technology Testing Transgenic Mice Ultraviolet Rays Viral Virus Virus Diseases Work adaptive immunity antigen-specific T cells base cancer cell chimeric antigen receptor T cells cytotoxicity design fluorophore in vivo magnetic beads mitochondrial dysfunction monomer mouse model neoplastic cell novel strategies prevent receptor shear stress success transduction efficiency translational potential

项目摘要

PROJECT SUMMARY Ag-specific CD8 T cells express T cell receptors (TCRs) that recognize antigens in the form of processed peptides bound to major histocompatibility complex class I (MHCI) molecules. In healthy individuals, the CD8 TCR repertoire comprises approximately 106–108 different cell populations. Soluble pMHCI multimers are widely used to enumerate and isolate Ag-specific T cells by fluorescence activated cell sorting (FACS); however, FACS has limitations including low-throughput, high shear-stress damage (especially to rare cells), and low multiplexing depth due to limited number of fluorophores. For high-throughput cell sorting (>106 cells) such as for manufacturing T cell therapies, magnetic activated cell sorting (MACS) is commonly used but can only produce antibody-enriched or depleted cell fractions, and cells sorted by positive selection remain labeled with beads, preventing immediate downstream assays such as phenotyping by flow analysis. New approaches are needed for multiplexed, high-throughput and label-free isolation of Ag-specific T cells. This proposal will develop DNA- gated sorting (DGS) cytometry for multiplexed isolation of Ag-specific CD8 T cells. DGS comprise a molecular DNA circuit that couples a magnetic bead to pMHCI molecules through DNA hybridization, and that functions as a sorting ‘gate’ to capture, release, and recover Ag-specific T cells by toehold-mediated strand displacement. By using orthogonal DNA strand displacement reactions, a library of beads coated with different pMHCI antigens can simultaneously capture target cell populations en masse and each subpopulation can then be eluted by sequential strand displacement. In contrast to fluorophores, the number of possible DNA sequences to design strand displacement reactions scales exponentially by the length n of the DNA oligo (i.e., 4n), providing the possibility to extend this technology to isolate Ag-specific T cells at depths that is currently not possible. This proposal will also implement DGS with pMHCI monomers that multimerize when hybridized onto the bead to produce the required binding avidity for T cell capture, but after cell release, revert into monomers to dissociate from T cells resulting in label-free isolates. It will also implement light-induced peptide exchange to produce large pMHCI libraries to integrate with multiplexed DGS. Finally, this proposal will demonstrate an important application for the manufacturing of chimeric antigen receptor (CAR) T cells using virus-specific T cells to redirect them to tumor cells, using key benchmarks such as ex vivo functional assays (expansion, transduction efficiency, cytotoxicity) and in vivo therapy in mice bearing CD19+ cancer cells.

项目概要 Ag 特异性 CD8 T 细胞表达 T 细胞受体 (TCR)，可识别经过加工的形式的抗原。在健康个体中，CD8 与主要组织相容性复合物 I 类 (MHCI) 分子结合。 TCR 库包含大约 106-108 个不同的细胞群，广泛存在于可溶性 pMHCI 多聚体中。然而，FACS 用于通过荧光激活细胞分选 (FACS) 计数和分离 Ag 特异性 T 细胞；具有局限性，包括低通量、高剪切应力损伤（特别是对稀有细胞）和低复用由于荧光团数量有限，因此深度较深，适用于高通量细胞分选（>106 个细胞），例如制造 T 细胞疗法，磁激活细胞分选 (MACS) 很常用，但只能生产富含抗体或耗尽抗体的细胞级分，以及通过正选分选的细胞仍用珠子标记，需要新的方法来防止直接下游分析，例如通过流式分析进行表型分析。用于 Ag 特异性 T 细胞的多重、高通量和无标记分离。用于多重分离 Ag 特异性 CD8 T 细胞的门控分选 (DGS) 细胞术包含分子 DGS。 DNA 电路通过 DNA 杂交将磁珠与 pMHCI 分子耦合，其功能如下：通过立足点介导的链置换来捕获、释放和回收 Ag 特异性 T 细胞的分选“门”。使用正交 DNA 链置换反应，形成涂有不同 pMHCI 抗原的珠子文库可以同时捕获大量目标细胞群，然后可以通过以下方法洗脱每个亚群与荧光团相反，需要设计的可能的 DNA 序列的数量。链置换反应按 DNA 寡核苷酸的长度 n（即 4n）呈指数缩放，提供扩展该技术以在目前不可能的深度分离 Ag 特异性 T 细胞的可能性。该提案还将使用 pMHCI 单体实施 DGS，这些单体在杂交到珠子上时会发生多聚化产生 T 细胞捕获所需的结合亲合力，但在细胞释放后，恢复为单体以解离它也将实施光诱导的肽交换以产生大量的T细胞。最后，该提案将展示一个重要的与多重 DGS 集成的 pMHCI 库。使用病毒特异性 T 细胞重定向来制造嵌合抗原受体 (CAR) T 细胞的申请使用关键基准，例如离体功能测定（扩增、转导效率、细胞毒性）和携带 CD19+ 癌细胞的小鼠体内治疗。