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

项目总结 抗原特异的CD8 T细胞表达T细胞受体(TCR)，识别经加工的抗原 与主要组织相容性复合体I类(MHCI)分子结合的多肽。在健康的个体中，CD8 TCR谱系包括大约106-108个不同的细胞群。可溶性pMHCI多聚体广泛存在 用于通过荧光激活细胞分选(FACS)计数和分离抗原特异性T细胞；然而，FACS 其局限性包括低吞吐量、高剪应力损伤(特别是对稀有细胞)和低复用率 由于荧光团的数量有限，导致了深度的增加。用于高通量细胞分选(&gt；106细胞)，例如 制造T细胞疗法，磁激活细胞分选(MACs)是常用的，但只能产生 抗体浓缩或耗尽的细胞部分以及通过阳性选择分选的细胞仍然用珠子标记， 防止直接的下游分析，如通过流动分析进行表型分析。需要新的方法 用于多路、高通量和无标记分离抗原特异性T细胞。这项提议将发展DNA- 门控分选(DGS)细胞术用于多重分离抗原特异性CD8T细胞。DGS由一个分子组成 通过DNA杂交将磁珠连接到pMHCI分子的DNA回路，其功能是 通过脚趾介导的链置换来捕获、释放和恢复抗原特异性T细胞的分选‘门’。通过 利用正交DNA链置换反应，建立了包被不同pMHCI抗原的珠库 可以同时捕获目标细胞群体，然后每个亚群可以被洗脱 顺序链位移。与荧光团相比，要设计的可能DNA序列的数量 链置换反应按DNA寡聚的长度n(即4n)指数级扩展，提供 将这项技术扩展到在目前不可能的深度分离抗原特异性T细胞的可能性。这 Proposal还将实施带有pMHCI单体的DGS，当将其杂交到珠子上时，这些单体会发生多聚体聚合 产生捕获T细胞所需的结合亲和力，但在细胞释放后，恢复为单体以解离 从T细胞中分离出无标签的分离物。它还将实施光诱导的多肽交换，以产生 PMHCI库与多路传输的DG集成。最后，这项提议将展示一个重要的 病毒特异性T细胞重定向在制造嵌合抗原受体(CAR)T细胞中的应用 使用关键基准，例如体外功能分析(扩增，转导效率， 对携带CD19癌细胞的小鼠进行体内治疗。

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.