Abstract Despite remarkable progress in treating B cell malignancies, T cells expressing chimeric antigen receptors (CARs) remain largely ineffective in solid tumors. We demonstrated that unlike conventional T and CAR-T cells, V24-invariant natural killer T cells (NKTs) and CAR-NKTs effectively traffic to tumor sites in xenogeneic models of neuroblastoma (NB), which led to initiation of a first-in-human phase 1 clinical trial evaluating autologous GD2- specific CAR-NKTs in children with NB. Interim results from this trial have shown that the treatment is well- tolerated and produces antitumor activity, underscoring the need for systematic investigation of NKTs as an alternative cellular platform for CAR-redirected immunotherapy. We reported that repeated antigenic stimulation of NKTs and CAR-NKTs leads to their effector-like differentiation, exhaustion, and loss of antitumor activity. Our preliminary data demonstrate that Wnt signaling—specifically, activation of transcription factor LEF1—is required and sufficient for maintenance of the CD62L+ central memory-like NKT subset and is associated with NKT metabolic and functional fitness. These findings provide rationale for examining the role and potential therapeutic targeting of LEF1 in CAR-NKTs for cancer immunotherapy. Pre-clinical in vivo studies of CAR-NKTs have been limited to xenogeneic models, which do not allow for evaluation of the full spectrum of downstream innate and adaptive immune responses. To address this gap, we have developed protocols to generate and expand highly pure murine NKTs that express a murine CAR targeting the GD2 antigen expressed on human and murine NB cells. We have also adapted a syngeneic NB model that faithfully recapitulates the clinical and pathological characteristics of high-risk NB in children. We hypothesize that 1) LEF1 transcriptional activity is required for and can be therapeutically enhanced to maintain CAR-NKT cell metabolic fitness, in vivo persistence, and durable antitumor activity; and 2) CAR-NKT antitumor activity depends on direct targeting of tumor cells, control of tumor-associated macrophages, transactivation of NK cells and induction of tumor-specific T cells. These hypotheses will be tested in the following specific aims: 1) to examine and therapeutically explore the mechanism responsible for maintenance of human central memory-like NKT and CAR-NKT cells, and 2) to explore the mechanism by which CAR-NKTs mediate antitumor activity in a syngeneic NB model. We will use genetic loss-of-function and gain-of-function approaches to study the role of LEF1 in CAR-NKT functional differentiation, metabolism, and antitumor activity using in vitro experimental systems with human cells and in vivo xenogeneic NB models in mice. To study the mechanism by which CAR-NKTs mediate antitumor activity in the syngeneic setting, we will perform a side-by-side comparison of murine NKTs and T cells expressing a GD2- specifc CAR. The proposed experiments will mecha...