Post-Radiotherapy GSCs EGR1-Induced GAL3 Upregulation and T Cell Exhaustion in GBM
Background: Glioblastoma (GBM), a highly aggressive brain cancer, inevitably recurs due to therapy-resistant glioblastoma stem cells (GSCs). Lymphocyte activation gene 3 (LAG3) contributes to T cell exhaustion, a key immune escape mechanism. Here, we investigate that the more exhausted state of GBM-infiltrating T cells after recurrence is associated with the LAG3 and its ligand galectin 3 (GAL3).  Methods: The analysis of Single-cell RNA sequencing to characterize the landscape of immune cells in newly diagnosed GBMs and recurrent GBMs respectively. Post-radiotherapy GAL3 in GSCs upregulation was validated via cell biology experiments. In vitro/in vivo loss-of-function assays assessed GAL3's impact on CD4+/CD8+ T cell cytotoxicity against GSCs. Chromatin immunoprecipitation quantitative real-time PCR was leveraged to map the regulation of GAL3 after radiotherapy in GSCs.  Results: The phenomenon that recurrent glioma patients had their T cells in a more exhausted state compared to primary glioma patients, is associated with the expression of LAG3. Elevated EGR1 in post-radiotherapy GSCs upregulates LAG3 ligand GAL3 expression in GSCs, impairing CD4+/CD8+ T-cell cytotoxicity. Small molecule inhibitors targeting EGR1 in combination with anti-LAG3 monoclonal antibody suppressed GBM tumor growth and prolonged survival of orthotopic xenograft-bearing mice.  Conclusions: GAL3 in GSCs plays critical roles in maintaining the exhaustion stage of T cells and inhibits cytotoxic capacity of CD4+/CD8+ T cells, highlighting an interplay between GSCs and GBM-infiltrating T cells. Small molecule inhibitors in combination with anti-LAG3 monoclonal antibody may effectively improve GBM treatment. Overall design: GSCs were preconditioned with 5 Gy irradiation followed by a 72-hour culture period to assess the effects of radiotherapy on cellular responses and gene expression.