Macrophages (MΦs)/microglia that constitute the dominant tumor-infiltrating immune cells in glioblastoma are recruited by tumor-secreted factors and are induced to become immunosuppressive and tumor supportive (M2). Glioma cancer stem cells (gCSCs) have been shown to suppress adaptive immunity, but their role in innate immunity with respect to the recruitment and polarization of MΦs/microglia is unknown. The innate immunosuppressive properties of the gCSCs were characterized based on elaborated MΦ inhibitory cytokine-1 (MIC-1), transforming growth factor (TGF-β1), soluble colony-stimulating factor (sCSF), recruitment of monocytes, inhibition of MΦ/microglia phagocytosis, induction of MΦ/microglia cytokine secretion, and the inhibition of T-cell proliferation. The role of the signal transducer and activator of transcription 3 (STAT3) in mediating innate immune suppression was evaluated in the context of the functional assays. The gCSCs produced sCSF-1, TGF-β1, and MIC-1, cytokines known to recruit and polarize the MΦs/microglia to become immunosuppressive. The gCSC-conditioned medium polarized the MΦ/microglia to an M2 phenotype, inhibited MΦ/microglia phagocytosis, induced the secretion of the immunosuppressive cytokines interleukin-10 (IL-10) and TGF-β1 by the MΦs/microglia, and enhanced the capacity of MΦs/microglia to inhibit T-cell proliferation. The inhibition of phagocytosis and the secretion of IL-10 were reversed when the STAT3 pathway was blocked in the gCSCs. The gCSCs modulate innate immunity in glioblastoma by inducing immunosuppressive MΦs/microglia, and this capacity can be reversed by inhibiting phosphorylated STAT3.

Regulatory T (Treg) cells respond to immune and inflammatory signals to mediate immunosuppression, but how functional integrity of Treg cells is maintained under activating environments remains elusive. Here we found that autophagy was active in Treg cells and supported their lineage stability and survival fitness. Treg cell-specific deletion of the essential autophagy gene Atg7 or Atg5 led to loss of Treg cells, increased tumor resistance, and development of inflammatory disorders. Atg7-deficient Treg cells had increased apoptosis and readily lost Foxp3 expression, especially after activation. Mechanistically, autophagy deficiency upregulated mTORC1 and c-Myc function and glycolytic metabolism that contributed to defective Treg function. Therefore, autophagy couples environmental signals and metabolic homeostasis to protect lineage and survival integrity of Treg cells in activating contexts.

The incidence of PD-L1 expression in GBM patients is frequent but is confined to a minority subpopulation, similar to other malignancies that have been profiled for PD-L1 expression. Higher expression of PD-L1 is correlated with worse outcome.

Glioblastomas are highly infiltrated by diverse immune cells, including microglia, macrophages, and myeloid-derived suppressor cells (MDSCs). Understanding the mechanisms by which glioblastoma-associated myeloid cells (GAMs) undergo metamorphosis into tumor-supportive cells, characterizing the heterogeneity of immune cell phenotypes within glioblastoma subtypes, and discovering new targets can help the design of new efficient immunotherapies. In this study, we performed a comprehensive battery of immune phenotyping, whole-genome microarray analysis, and microRNA expression profiling of GAMs with matched blood monocytes, healthy donor monocytes, normal brain microglia, nonpolarized M0 macrophages, and polarized M1, M2a, M2c macrophages. Glioblastoma patients had an elevated number of monocytes relative to healthy donors. Among CD11b+ cells, microglia and MDSCs constituted a higher percentage of GAMs than did macrophages. GAM profiling using flow cytometry studies revealed a continuum between the M1- and M2-like phenotype. Contrary to current dogma, GAMs exhibited distinct immunological functions, with the former aligned close to nonpolarized M0 macrophages.

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