cJun Overexpression Sensitizes CAR-T Cells to PD-1 Axis Blockade by Preserving an Intratumoral PD-1⁺ Tcf1⁺ Stem-Like Reservoir

Abstract

T cells possess a unique capacity to recognize and kill cells expressing specific antigens,making them an attractive platform for cancer immunotherapy. Decades of iterative advances in immunology, synthetic biology, and genetic engineering have led to the development of adoptive T cell therapies, including chimeric antigen receptor (CAR) T cells, which have transformed how malignancies are treated. Despite the successes of CAR-T cells in hematological cancers, durable efficacy remains limited in solid tumors due to defects in CAR-T cell trafficking, exhaustion, and toxicities that constrain their therapeutic benefit. This thesis examines the immunological principles underlying T cell based therapies, the evolution of cancer immunotherapy, and the mechanisms that govern CAR-T cells response and failure in the solid tumor setting. Additionally, this thesis focuses on the role PD-1⁺ Tcf1⁺ stem-like T cells play in mediating responses to immune checkpoint blockade and examines whether CAR-T cells are capable of forming and maintaining this critical stem-like reservoir in the solid tumor microenvironment (TME). This work demonstrates that ROR1-targeting CAR-T cells rapidly downregulate Tcf1 in- vivo, undergo exhaustion, and fail to respond to PD-1 axis blockade. Overexpression of the AP- 1 transcription factor cJun enables the formation of an intratumoral PD-1⁺ Tcf1⁺ CAR-T cell reservoir, yet cJun overexpression alone is insufficient to overcome CAR-T cell exhaustion in the solid TME due to PD-1 induced post-transcriptional downregulation of cJun. PD-L1 blockade is able to restore cJun overexpression, promotes robust intratumoral CAR-T cell expansion culminating in dramatic tumor clearance. Collectively, these findings identify PD-1 as a negative regulator of cJun and demonstrate that, despite their MHC-independent design, CAR-T cells can be engineered to form intratumoral stem-like reservoirs that overcome resistance to checkpoint blockade. This work provides mechanistic insight into CAR-T cell failure in solid tumors and informs the rational design of next generation immunotherapies with improved durability and efficacy.