Single-cell profiling of CSF immune dynamics following intracranial B7-H3 CAR T cell therapy for pediatric brain tumors 2254484

Abstract Introduction CAR T cell therapy is a promising approach for pediatric patients with aggressive brain tumors. Understanding immune responses within the central nervous system (CNS) is critical for optimizing CAR T cell therapy against pediatric brain tumors. In the Loc3CAR clinical trial (NTC05835687), B7-H3- CAR T cells were administered intracranially via an Ommaya reservoir. To investigate therapy-induced immune changes in the cerebrospinal fluid (CSF), we performed longitudinal single-cell RNA sequencing to profile immune populations before and after CAR T cell infusions. Methods CSF samples were collected immediately before and 24 hours after serial intracranial infusions from pediatric patients enrolled in Loc3CAR. Samples were processed for single-cell transcriptomic and TCR profiling using 5’ chemistry from 10x Genomics platform. Cell types were annotated by canonical markers and automated classification. Results Despite low baseline CSF cellularity, most samples yielded high-quality single-cell data. Cell counts increased markedly 24 hours post-infusion, revealing robust immune recruitment. Across patients, CAR+ T cells became detectable and increased over successive infusions, suggesting persistence and expansion in the CNS. Notably, regulatory T cells (Tregs) increased post-infusion, paralleling the expansion of CAR+ T cells. In parallel, nonclassical monocytes showed strong cyclical increases after each infusion, coinciding with reduced macrophage and dendritic cell frequencies. Cell-cell interaction analysis indicated strong incoming signaling toward CAR+ T cells and extensive outgoing signaling from various myeloid subsets. Conclusion Our study shows that single-cell profiling of CSF immune cell components is feasible and revealed dynamic immune remodeling following intracranial B7-H3 CAR T therapy. Repeated infusions promoted CAR+ T cell accumulation but also expanded potentially suppressive Tregs and signaling-active myeloid subsets, highlighting targets for future therapeutic refinement. Funding Source SJCRH/ALSAC CeTII / CePIO, NIH NCI Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)