ID #404 Flipping the switch: CAR T cells induce inflammatory cytokines that are quiescent at baseline in cerebrospinal fluid of diffuse midline glioma patients

Introduction Diffuse midline glioma (DMG) is a devastating and highly aggressive tumor, considered immunologically “cold” due to the lack of infiltrating immune cells within the tumor microenvironment. We aimed to use high-throughput proteomics to characterize the baseline immune microenvironment from the cerebrospinal fluid (CSF) of DMG patients. We then used co-culture of DMG cells and chimeric antigen receptor T cells (CART) to identify how these signals change in the context of immune activation. Methods Analysis was completed on the CSF from 13 DMG patients and 30 pediatric controls who received a lumbar puncture for non-malignant and non-infectious conditions. 89 cytokines and immune surveillance proteins were analyzed using the Olink Proximity Extension Assay. Principal component analysis (PCA) was used for dimensionality reduction and visualization of all cytokine concentrations and Welch’s t-test to compare individual cytokine concentrations between groups. In addition, DMG cell lines 7316-6349 and SUDIPG13P* were plated with mRNA GD2-directed CART at E:T ratio 1:1 for 24 hours, bead separated, and analyzed using bulk RNA sequencing. Induced cytokine signals from CART activation were tested using transmigration assays. Results PCA demonstrated strong inter-group variance, separating the DMG and control groups. Five proteins with known tumorigenic properties were significantly increased in the CSF of DMG patients compared to controls: IL32 (p < 0.05), IL1RN (p < 0.05), IL18 (p < 0.05), TREM1 (p < 0.05), and MMP12 (p < 0.05). Twelve immunostimulatory cytokines were significantly decreased in DMG patients CSF compared to controls including CXCL10 (p < 0.001), CXCL11 (p < 0.0001) and CXCL12 (p < 0.0001). Co-culture of DMG cell lines with GD2-directed CART induced expression of 19 inflammatory signals, with elevations in CXCL10 (p < 0.05), CXCL11 (p < 0.01), CCL2 (p < 0.01) and IFNG (p < 0.05). Transwell migration assays confirmed these inflammatory cytokines enhanced CART migration and cytotoxicity (p < 0.0001). Conclusions CSF from DMG patients revealed suppressed inflammatory cytokines compared to pediatric control patients. CART engagement in vitro increased multiple suppressed signals, including CXCL10 and CXCL11, which promote CART migration and cytotoxicity in a positive immune feedback loop. This study provides new insight in the immunosuppressive CSF of DMG patients, and how direct engagement of CART with tumor cells can enhance T cell migration, providing a framework to induce effective cure.