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

项目摘要 Ag特异性CD 8 T细胞表达T细胞受体（TCR），其以经加工的抗原形式识别抗原。 与主要组织相容性复合体I类（MHCI）分子结合的肽。在健康人中，CD 8 TCR库包含大约106-108个不同的细胞群。可溶性pMHCI多聚体广泛存在于 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;然而， 具有包括低通量、高剪切应力损伤（特别是对稀有细胞）和低复用的局限性 由于有限数量的荧光团，用于高通量细胞分选（>106个细胞），例如用于 为了制造T细胞疗法，磁激活细胞分选（MACS）是常用的，但只能产生 抗体富集或耗尽的细胞级分，和通过阳性选择分选的细胞保持用珠标记， 阻止了直接的下游测定，例如通过流动分析的表型分析。需要新的方法 用于Ag特异性T细胞的多重、高通量和无标记分离。这项提案将开发DNA- 门控分选（DGS）细胞术用于Ag特异性CD 8 T细胞的多重分离。DGS包含分子 通过DNA杂交将磁珠与pMHCI分子偶联的DNA电路，其功能为 通过支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”。通过 使用正交DNA链置换反应，包被有不同pMHCI抗原的珠的文库 可以同时捕获靶细胞群，然后通过洗脱每个亚群， 顺序链置换与荧光团相反，要设计的可能DNA序列的数量 链置换反应以DNA寡聚物的长度n为指数尺度（即，4 n），提供 因此，我们有可能扩展这项技术，以在目前不可能的深度分离Ag特异性T细胞。这 该提案还将使用pMHCI单体实施DGS，当杂交到珠上时多聚化， 产生T细胞捕获所需的结合亲合力，但在细胞释放后，恢复为单体解离 产生无标记分离物。它还将实施光诱导肽交换， pMHCI文库与多重DGS整合。最后，该提案将展示一个重要的 使用病毒特异性T细胞重定向嵌合抗原受体（CAR）T细胞的应用 使用关键的基准如离体功能测定（扩增，转导效率， 细胞毒性）和体内治疗。

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.