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

Introduction/Rationale: 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.