Cre-dependent gene expression enables thymic development of autoreactive tumor-associated antigen targeting CAR-T cells.

Chimeric antigen receptor (CAR)-T cells have revolutionized cancer therapy by enabling highly specific adoptive cellular treatments. However, limited in vivo persistence and tumor relapse remain major challenges. To address this, we developed a platform for continuous in vivo generation of CAR-T cells by genetically engineering hematopoietic stem and progenitor cells to express a tumor-specific CAR followed by administration directly into the thymus. We demonstrated in single-cell RNA sequencing and serial transplantation studies that the thymic microenvironment supports short-term hematopoietic stem cells and initiates a transcriptional program that drives T cell lineage differentiation. A key challenge of thymic CAR-T cell development is thymic negative selection, which eliminates developing thymocytes expressing autoreactive antigen receptors such as CD19 CARs. We show that reducing CAR co-stimulation and modifying the endogenous T cell receptor repertoire can enhance the survival of these cells during thymic development. Additionally, inducible CAR gene expression enables sustained thymic production of CD19 CAR-T cells, which can target both normal B cells and CD19+ tumor cells. Our findings highlight mechanisms governing the fate of CAR-T cell precursors in the thymus and support inducible expression systems as a strategy to bypass central tolerance, offering a path toward durable, self-renewing CAR-T cell therapies.