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.

项目摘要 Ag特异性CD8 T细胞表达T细胞接收器（TCRS），以处理的形式识别抗原与主要组织相容性复合物I类（MHCI）分子结合的肽。在健康的个体中，CD8 TCR曲目约为106-108个不同的细胞种群。可溶性PMHCI多聚体广泛用于通过荧光激活细胞分选（FACS）枚举和分离Ag特异性T细胞；但是，FACS 有局限性，包括低通量，高剪切压力损伤（尤其是稀有细胞）和低多重的深度由于荧光团数量有限。用于高通量电池分类（> 106个单元），例如通常使用制造T细胞疗法，磁性细胞分选（MAC），但只能产生富含抗体的或耗尽的细胞级分，由阳性选择排序的细胞仍然标记为珠子，防止立即进行下游测定，例如通过流量分析进行表型。需要新的方法用于多重的，高通量和无标记的Ag特异性T细胞的分离。该建议将发展DNA- 用于Ag特异性CD8 T细胞多路复用分离的门控分选（DGS）细胞仪。 DGS包括分子 DNA电路通过DNA杂交将磁珠耦合到PMHCI分子，并且该作用为分类的“门”，以捕获，释放和恢复特异性T细胞通过toehold介导的链位移。经过使用正交DNA链的位移反应，涂有不同PMHCI抗原的珠子库可以简单地捕获目标细胞群体，然后每个亚群都可以通过顺序链位移。与荧光团相比，设计可能的DNA序列的数量链位移反应通过DNA寡核的长度n（即4N）呈指数缩放，提供了将这项技术扩展到目前不可能的深度分离Ag特异性T细胞的可能性。这提案还将与PMHCI单体实施DGS，当杂交到珠子到珠子到产生所需的T细胞捕获的所需的结合亲和力，但是在细胞释放后，将其恢复为单体以解离来自导致无标记分离株的T细胞。它还将实施光引起的肽交换以产生大型 PMHCI库与多路复用DGS集成。最后，该提议将证明一个重要的使用病毒特异性T细胞重定向，应用嵌合抗原受体（CAR）T细胞生产它们使用诸如离体功能测定等关键基准（扩展，翻译效率，具有CD19+癌细胞的小鼠中的细胞毒性）和体内治疗。