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Background Diffuse midline glioma (DMG) is a fatal tumor characterized by H3K27M mutation that is primarily treated with radiation therapy (RT). RT has been reported to increase type I interferon (IFN1) responses, which can enhance CD8 + T cell infiltration and improve responses to immunotherapy. Using single-cell analysis, we previously showed that RT stimulates IFN-I signalling in microglia and other myeloid cells in diffuse midline glioma (DMG); however, this was accompanied by limited cytotoxic immune infiltration. We found that radiation also led to increased CD47 which is a key immunosuppressive modulator for microglia. This study aims to understand the interplay between IFN1 and CD47 in the tumor immune microenvironment (TIME) in DMG after RT.Methods Using a murine DMG cell line with H3K27M mutation (KAPP), we generated a CD47 knockout (CD47 KO-KAPP) cell line via CRISPR-Cas9. Both the wild-type and CD47 KO-KAPP cells were radiated at 10Gy x1, 10Gy x2 and 10Gy x3 fractions using the Small Animal Radiation Research Platform (SARRP). Cells were then co-cultured with microglia and exposed to 3 fractions of 10Gy RT. Apoptosis was evaluated using cleaved caspase 3 and tracked post RT in real-time with live-cell imaging, IFN1 was evaluated with STING via qPCR and IFNb ELISA, cytokine levels were evaluated using multiplex assay panel and RNA-sequencing was performed comparing irradiated to unirradiated groups (SHAM). Both mice groups were then treated with RT at 3Gy x13. Brains were harvested 10 days post-injection (dpi) before, and 24hrs after RT and were evaluated using flow cytometry and immunofluorescence (n=5 per group).Results Before RT, KAPP cells showed high expression of STING transcriptionally. After RT, higher apoptosis was observed in both KAPP and CD47 KO cells compared to SHAM. Interestingly, no IFNb was observed with and without radiation in KAPP cell line despite STING expression. IFNb levels of both cell lines co-cultured with microglia with and without RT are underway. Mice injected with KAPP (n=9) had a median survival of 36 days and CD47 KO-KAPP are being followed. DMG mice with and without CD47 KO-have been treated with RT and are being followed. In vitro RNA-sequencing and flow cytometry of mice with and without CD47 and treated with RT Is underway.Conclusions Studying the effect of CD47 on the TIME of DMG with and without RT will improve the understanding of the immune response of DMG and possible immuno-therapies.