Multiplexed transcriptomic profiling of the fate of human CAR T cells in vivo via genetic barcoding with shielded small nucleotides

Nat Biomed Eng. 2023 Sep;7(9):1170-1187. doi: 10.1038/s41551-023-01085-3. Epub 2023 Aug 31.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • CD4-Positive T-Lymphocytes*
  • Cell- and Tissue-Based Therapy
  • Cytokines
  • Humans
  • Mice
  • Nucleotides
  • Transcriptome*

Substances

  • Cytokines
  • Nucleotides