High-grade gliomas are rapidly progressing tumors of the central nervous system (CNS) with a very poor prognosis despite extensive resection combined with radiation and/or chemotherapy. Histopathological and flow cytometry analyses of human and rodent experimental gliomas revealed heterogeneity of a tumor and its niche, composed of reactive astrocytes, endothelial cells, and numerous immune cells. Infiltrating immune cells consist of CNS resident (microglia) and peripheral macrophages, granulocytes, myeloid-derived suppressor cells (MDSCs), and T lymphocytes. Intratumoral density of glioma-associated microglia/macrophages (GAMs) and MDSCs is the highest in malignant gliomas and inversely correlates with patient survival. Although GAMs have a few innate immune functions intact, their ability to be stimulated via TLRs, secrete cytokines, and upregulate co-stimulatory molecules is not sufficient to initiate antitumor immune responses. Moreover, tumor-reprogrammed GAMs release immunosuppressive cytokines and chemokines shaping antitumor responses. Both GAMs and MDSCs have ability to attract T regulatory lymphocytes to the tumor, but MDSCs inhibit cytotoxic responses mediated by natural killer cells, and block the activation of tumor-reactive CD4 T helper cells and cytotoxic CD8 T cells. The presence of regulatory T cells may further contribute to the lack of effective immune activation against malignant gliomas. We review the immunological aspects of glioma microenvironment, in particular composition and various roles of the immune cells infiltrating malignant human gliomas and experimental rodent gliomas. We describe tumor-derived signals and mechanisms driving myeloid cell accumulation and reprogramming. Although, understanding the complexity of cell-cell interactions in glioma microenvironment is far from being achieved, recent studies demonstrated several glioma-derived factors that trigger migration, accumulation, and reprogramming of immune cells. Identification of these factors may facilitate development of immunotherapy for gliomas as immunomodulatory and immune evasion mechanisms employed by malignant gliomas pose an appalling challenge to brain tumor immunotherapy.

Microglia are resident myeloid cells in the central nervous system (CNS) that control homeostasis and protect CNS from damage and infections. Microglia and peripheral myeloid cells accumulate and adapt tumor supporting roles in human glioblastomas that show prevalence in men. Cell heterogeneity and functional phenotypes of myeloid subpopulations in gliomas remain elusive. Here we show single-cell RNA sequencing (scRNA-seq) of CD11b+ myeloid cells in naïve and GL261 glioma-bearing mice that reveal distinct profiles of microglia, infiltrating monocytes/macrophages and CNS border-associated macrophages. We demonstrate an unforeseen molecular heterogeneity among myeloid cells in naïve and glioma-bearing brains, validate selected marker proteins and show distinct spatial distribution of identified subsets in experimental gliomas. We find higher expression of MHCII encoding genes in glioma-activated male microglia, which was corroborated in bulk and scRNA-seq data from human diffuse gliomas. Our data suggest that sex-specific gene expression in glioma-activated microglia may be relevant to the incidence and outcomes of glioma patients.

13Brain resident and infiltrating innate immune cells adapt a tumor-supportive phenotype in the 14 glioma microenvironment. Flow cytometry analysis supported by a single-cell RNA 15 sequencing study of human gliomas indicate considerable cell type heterogeneity. It remains 16 disputable whether microglia and infiltrating macrophages have the same or distinct roles in 17 supporting glioma progression. Here, we performed single-cell transcriptomics analyses of 18 CD11b+ cells sorted from murine syngeneic gliomas, indicating distinct activity of microglia, 19 infiltrating monocytes/macrophages and CNS border-associated macrophages. Our results 20 demonstrate a previously immeasurable scale of molecular heterogeneity in the innate 21 immune response in gliomas. We identified genes differentially expressed in activated 22 microglia from glioma-bearing mice of different sex, and profound overexpression of the 23 MHCII genes by male microglial cells, which we also observed in bulk human glioma 24 samples. Sex-specific gene expression in microglia in the glioma microenvironment may be 25 relevant to sex differences in incidence and outcomes of glioblastoma patients. 26 27 3 28 Introduction 29 Infiltrating immune system cells represent an abundant non-malignant component of the 30tumor microenvironment (TME). These cells play a pivotal role in tumor progression and 31 modulation of tumor responses to therapy 1 . A high number of macrophages within TME have 32 been associated with a poor prognosis in many cancers because those tumor-educated cells 33 suppress anti-tumor immunity, stimulate angiogenesis, and promote tumor invasion 2 . 34The central nervous system (CNS) is equipped with resident innate immune cells-microglia 35 and CNS border-associated macrophages (BAMs)-consisting of perivascular, meningeal, 36 and the choroid plexus macrophages. Those cells migrate to the CNS early in the prenatal 37 life and maintain a long-lasting population. In malignant gliomas, besides activation of local 38 microglia, circulating monocytes invade the brain from the periphery and differentiate within 39 the tumor; therefore, microglia and infiltrating monocytes/macrophages are commonly 40 referred to as glioma-associated macrophages (GAMs), due to the shortage 41 of immunocytochemical markers allowing their reliable identification 3 . Transcriptome profiling 42 of bulk CD11b+ cells isolated from human glioblastomas (GBMs) and rodent gliomas showed 43 a mixture of protumorigenic and antitumorigenic phenotypes and did not reveal consistent 44 markers and pathways 4-6 . Recent reports have demonstrated that GAMs consist of diverse 45 cell populations with likely distinct roles in tumor progression 7-10 . Dissecting the TME 46 composition and functional heterogeneity of tumor-infiltrating immune cells would extend the 47 understanding of the glioma immune microenvironment and allow to modulate functions of 48 distinct subpopulations for therapeutic benefits. 49Sex differences in incidence, transcriptomes, and patient outcomes in the adul...