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Background In contrast to the success of adoptive cell therapy with chimeric antigen receptor (CAR) T cells in pediatric hematologic malignancies, the application of these cells against childhood central nervous system (CNS) tumors such as medulloblastoma, has been limited. This limitation likely stems from suboptimal trafficking and the immune suppressive microenvironment. Invariant natural killer T cells (iNKT) are innate-like T cells that may address these critical challenges. iNKT cells can overcome immune cell trafficking limitations into CNS tumors as they naturally migrate towards CCL2, which is overexpressed in medulloblastoma. Additionally, they can potentially neutralize the immune suppressive microenvironment via selective lysis of tumor associated macrophages. We hypothesize that iNKT cells expressing CAR will enhance CAR cell trafficking and overcome the microenvironment, which together constitute the key barriers of CAR T therapy for pediatric CNS tumors like medulloblastoma.Methods Expansion of Human iNK-T Cells: Peripheral blood mononuclear cells (PBMCs) were isolated and iNKT cells were enriched using magnetic selection. Cells were co-cultured with irradiated feeder cells along with α-GalCer and IL-2 for 15 days. On day 5, cells were transduced with the B7-H3 CAR. Expansion of Human T Cells: PBMCs were stimulated with immobilized CD3/CD28 and cultured for 2 days prior to CAR transduction. Cytotoxicity was performed by luciferase or chromium-release method. In Vivo Antitumor Efficacy: Luciferase-labeled tumor cells were implanted orthotopically to monitor tumor progression via bioluminescent imaging. CAR iNKT cell-treated groups were evaluated for tumor burden, animal activity, and survival, and compared with CAR T cell-treated mice.Results We successfully expanded and transduced human iNK-T and T cells with the B7-H3 CAR construct. The transduction efficiency of B7H3 CAR-T cells (67.7±14.8%, n=9) and B7H3 CAR-iNK-T cells (65.1 ± 13%; n=9). Cytotoxicity shows an increased target lysis (>50%) of B7-H3 medulloblastoma cells at 20:1 (E:T) by both B7-H3 CAR T and NKT cells compared to the control. In orthotopic mouse models, treatment with B7-H3 CAR-iNK-T and B7H3-CAR-T cells significantly reduced tumor burden and improved survival compared to controls. Median overall survival was significantly longer in B7H3-CAR-T cells compared to non-transduced T cells (**p = 0.0046). Similarly, median overall survival was significantly higher in B7H3 CAR-iNK-T cells compared to non-transduced iNK-T cells (*p=0.0318).Conclusions B7-H3 CAR iNK-T cells offer a promising immunotherapeutic approach for high-risk medulloblastoma. Their ability to target tumors and reshape the TME supports their use alone or in combination with CAR T cells to enhance durability and efficacy in pediatric CNS tumors.Acknowledgements We would like to thank the National Pediatric Cancer Foundation for supporting this work.