Background: Gliomas are heterogeneous tumors with poor outcomes following current therapies, including immunotherapy. The tumor microenvironment (TME) is a critical determinant of therapeutic response in gliomas. We have classified the immune TME of gliomas by multiplex immunofluorescence (mIF). Methods: Tissue taken at initial resection from 354 patients with newly−diagnosed glioma grades 2-4 were analyzed using three mIF panels of markers for T, B, and myeloid cells. Tumor cores were characterized by the relative abundances of: (i) 15 primary immune phenotypes, (ii) 96 secondary immune phenotypes, and, (iii) 29 biologically meaningful multi-marker immune phenotypes. Results: Using unsupervised cluster analysis of WHO grade 4 gliomas we identified four subtypes α, β, γ, and δ that were internally reproducible. Immune subtype α was characterized by high abundance of antigen-presenting cells (APCs) and low levels of MHC II− monocytes. Subtype β was high in regulatory T cells and myeloid cells, but low in lymphocytes with effector functions. Subtype γ displayed high abundance of immune cell phenotypes, particularly lymphocytes with effector or helper functions. Subtype δ was low in lymphoid and myeloid immune phenotypes and APCs, with poorer outcomes. Grade 3 tumors could also be classified into α, β, γ, and δ subtypes, indicating generalizability of these immune TME subtypes across high grade gliomas. Conclusions: We have identified internally reproducible criteria for classifying gliomas according to the immune microenvironment, findings that could aid our understanding of the natural progression of low- and high-grade gliomas and inform the rational application of immune-oncologic therapeutic interventions.

The authors have declared no competing interest.

This study was partly supported by the National Institutes of Health (P30 CA076292 Quantitative Imaging Core, and R01 NS131912) and a Moffitt Cancer Center Team Science Award.

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