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Background Diffuse Intrinsic Pontine Glioma (DIPG) is a universally fatal pediatric brain tumor with median survival of less than 12 months. While chimeric antigen receptor (CAR) T-cell therapy shows promise, its efficacy in DIPG is limited by a profoundly immunosuppressive tumor microenvironment (TME). MicroRNAs (miRs), small non-coding RNAs that regulate gene expression, play critical roles in both gliomagenesis and immune regulation. Given their expression in both tumor and immune cells, miRs represent a novel, underexplored strategy to modulate the TME and enhance immunotherapy. We hypothesize that targeting dysregulated miR expression in DIPG can reprogram the TME to support CAR T-cell activation and persistence.Methods To investigate the role of miRs in DIPG, we performed small RNA sequencing on post-mortem DIPG tumors, matched normal brain tissue, and healthy brain controls.Results We identified significant dysregulation of multiple miRs in DIPG, including upregulation of immune-regulatory miRNAs, miR-135b, miR-455, and miR-301b, which have been previously reported to play roles in macrophage polarization and T-cell exhaustion. miRNA target prediction and Gene Ontology Enrichment Analysis confirmed association of upregulated miRs with immune system processes, including T-cell differentiation and myeloid cell regulation. Bulk RNA sequencing of the same samples revealed elevated expression of immunosuppressive genes, such as IL10RA, CD53, CD44, and LGALS9, and downregulation of SIRPA, suggesting impaired immune cell clearance. Notably, fourteen predicted targets of miR-135b were significantly downregulated in DIPG/DMG, and nine of these targets – including MEF2C, STAT6, APC, and KLF4 – are involved in immune-related processes, supporting the role of miR-135b in modulating immune regulatory networks in the TME.To assess therapy-induced changes in miR expression, we treated glioma-bearing mice with B7-H3 CAR T cells, control CARs, or left them untreated. Tumors harvested at defined timepoints were profiled for miRs. Principal Component Analysis revealed distinct clustering in B7-H3 CAR-treated tumors. Differential expression analysis identified therapy-associated miRs (including miR-344, let-7d, miR-155, and miR-30c) implicated macrophage polarization and T-cell dysfunction.Conclusions Together, our data suggests that miRs contribute to DIPG immune suppression and are dynamically regulated by CAR T-cell therapy. Ongoing studies aim to define how targeting specific miRs can enhance CAR T-cell function, offering a new avenue to improve outcomes in DIPG.