Microenvironmental IL13 Rα2 Expression and Enable Extracellular Vesicle-Mediated Antigen Transfer in Glioblastoma

Background/Objectives: Interleukin-13 receptor alpha 2 (IL13Rα2), expressed in more than 75% of glioblastomas (GBM), is a promising chimeric antigen receptor (CAR) T cell target. We investigated how microenvironmental stresses regulate IL13Rα2 expression and whether extracellular vesicle (EV)-mediated antigen transfer modulates CAR T cell killing. Methods: Patient-derived brain tumor and other GBM cell lines were exposed to hypoxia, desferrioxamine (DFO), or reduced fetal bovine serum (FBS). Western blotting assessed IL13Rα2 expression and recovery following reoxygenation. Fluorescence imaging evaluated EV interactions with recipient cells, and cytotoxicity assays assessed CAR T cell killing. Results: Hypoxia and DFO markedly suppressed IL13Rα2 expression. Exposure to 1% oxygen for 48 h reduced protein levels by 40–70%; expression recovered to near-baseline levels within 48 h of reoxygenation. Reduced FBS concentrations for 48 h also decreased IL13Rα2 expression compared with 10% FBS. Findings suggested potential contributions from ubiquitin-dependent degradation and transcriptional mechanisms. IL13Rα2-expressing EVs showed a slight increase under hypoxia. Fluorescence imaging confirmed that IL13Rα2-containing EVs attached to or entered recipient GBM cells within hours. EV-mediated antigen transfer increased CAR T cell killing by 3–5%. Conclusions: Hypoxia and nutrient availability dynamically regulate IL13Rα2 expression, while EVs can transfer the antigen between GBM cells and modestly enhance CAR T cell killing. These findings highlight reversible antigen modulation and EV-mediated antigen redistribution as mechanisms that could inform strategies to overcome antigen escape and improve immune targeting in GBM.