Abstract
Adoptive cell therapy of donor-derived, antigen-specific T cells expressing native T cell receptors (TCRs) is a powerful strategy to fight viral infections in immunocompromised patients. Determining the fate of T cells following patient infusion hinges on the ability to track them in vivo. While this is possible by genetic labeling of parent cells, the applicability of this approach has been limited by the non-specificity of the edited T cells. Here, we devised a method for CRISPR-targeted genome integration of a barcoded gene into Epstein-Barr virus-antigen-stimulated T cells and demonstrated its use for exclusively identifying expanded virus-specific cell lineages. Our method facilitated the enrichment of antigen-specific T cells, which then mediated improved cytotoxicity against Epstein-Barr virus-transformed target cells. Single-cell and deep sequencing for lineage tracing revealed the expansion profile of specific T cell clones and their corresponding gene expression signature. This approach has the potential to enhance the traceability and the monitoring capabilities during immunotherapeutic T cell regimens.