IMMU-06. A Single-Cell Atlas of Alternative Isoform Usage in Glioma-Infiltrating Macrophages

Abstract Glioma is the most aggressive adult brain tumor, with limited therapeutic options and minimal success from immunotherapies. A major challenge is its immunosuppressive microenvironment, dominated by tumor-associated macrophages (TAMs), which include both tissue-resident microglia and monocyte-derived macrophages. Over the past decade, advances in single-cell RNA sequencing have expanded our understanding of the cellular heterogeneity in glioma TAMs and their roles in immunosuppression. However, most studies have focused on gene-level expression, overlooking isoform-level alterations—despite the fact that over 90% of human multi-exon genes undergo alternative splicing (AS) to generate multiple protein isoforms. To bridge this knowledge gap, we analyzed the isoform-level transcriptomic landscape in glioma TAMs using both short-read and long-read single-cell RNA sequencing. These analyses revealed distinct AS patterns between microglia and monocyte-derived macrophages, including an exon skipping (SE) event in CD74, a key regulator of MHC-II antigen presentation, as well as widespread alternative first exon usage (AFE) as a common regulatory mechanism in immune cells. In addition, an SE event in MS4A7 correlated with clinical responses to anti-PD-1 therapy in both glioma and melanoma patients. These findings collectively underscore the importance of integrating both gene-centric and isoform-centric approaches to better understand the complexity of glioma immune microenvironment and to inform the development of more effective immunotherapeutic strategies.