Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
Nat Biomed Eng. 2023 Sep;7(9):1170-1187. doi: 10.1038/s41551-023-01085-3. Epub 2023 Aug 31.
The design of chimeric antigen receptor (CAR) T cells would benefit from knowledge of the fate of the cells in vivo. This requires the permanent labelling of CAR T cell products and their pooling in the same microenvironment. Here, we report a cell-barcoding method for the multiplexed longitudinal profiling of cells in vivo using single-cell RNA sequencing (scRNA-seq). The method, which we named shielded-small-nucleotide-based scRNA-seq (SSN-seq), is compatible with both 3' and 5' single-cell profiling, and enables the recording of cell identity, from cell infusion to isolation, by leveraging the ubiquitous Pol III U6 promoters to robustly express small-RNA barcodes modified with direct-capture sequences. By using SSN-seq to track the dynamics of the states of CAR T cells in a tumour-rechallenge mouse model of leukaemia, we found that a combination of cytokines and small-molecule inhibitors that are used in the ex vivo manufacturing of CAR T cells promotes the in vivo expansion of persistent populations of CD4 memory T cells. By facilitating the probing of cell-state dynamics in vivo, SSN-seq may aid the development of adoptive cell therapies.
嵌合抗原受体 (CAR) T 细胞的设计将受益于对体内细胞命运的了解。这需要对 CAR T 细胞产品进行永久标记，并将其汇集在相同的微环境中。在这里，我们报告了一种用于使用单细胞 RNA 测序 (scRNA-seq) 对体内细胞进行多路纵向分析的细胞条形码方法。该方法我们称之为屏蔽的基于小核苷酸的 scRNA-seq (SSN-seq)，与 3' 和 5' 单细胞分析兼容，并通过利用普遍存在的 Pol III U6 启动子来稳健表达带有直接捕获序列修饰的小 RNA 条形码，从而能够记录从细胞输注到分离的细胞身份。通过使用 SSN-seq 来跟踪白血病肿瘤再挑战小鼠模型中 CAR T 细胞状态的动态，我们发现，在体外制造 CAR T 细胞中使用的细胞因子和小分子抑制剂的组合促进了持久的 CD4 记忆 T 细胞群体在体内的扩增。通过促进体内细胞状态动力学的探测，SSN-seq 可能有助于过继细胞疗法的发展。
