Unsupervised Clustering Reveals Noncanonical Myeloid Cell Subsets in the Brain Tumor Microenvironment

The tumor immune microenvironment (TiME) of human central nervous system (CNS) tumors remains to be comprehensively deciphered. Here, we employed flow cytometry and RNA sequencing analysis via data-driven approaches to dissect a diverse TiME and uncover noncanonical immune cell types in human CNS tumors. Myeloid subsets comprised classical microglia, monocyte-derived macrophages, neutrophils, and two noncanonical myeloid subsets: CD3+ myeloids and CD19+ myeloids. T lymphocyte subsets included double-negative (CD4- CD8-) T cells (DNTs). Noncanonical myeloids and DNTs were validated with an independent dataset, suggesting that our DNT phenotype represents ?d T cells. While the proportions of classical myeloids agreed with reported malignancy type-associated TiMEs, unexpectedly high lymphocyte frequencies were detected in gliosarcoma, which also showed a unique expression pattern of immune-related genes. Our findings highlight the potential of data-driven approaches in resolving CNS TiME and reveal the mosaic of immune cell types constituting TiME. Overall design: To compare the TiME phenotypes characterised by flow cytometry (FCM) and to computationally asses the TiME at transcriptome level, bulk RNA sequencing was performed from the FCM cohort samples for which tumor material was available.