Comprehensive analysis of inhibitory checkpoint ligand expression by glioblastoma cells.

Glioblastoma is a highly aggressive brain malignancy commonly refractory to classical and novel chemo-, radio- and immunotherapies, with median survival times of ~15 months following diagnosis. Poor immunological responses exemplified by the downregulation of T-cell activity, and upregulation of immunosuppressive cells within the tumor microenvironment have limited the effectiveness of immunotherapy in glioblastoma to date. Here we show that glioblastoma cells express a large repertoire of inhibitory checkpoint ligands known to control effector T cell responses. Furthermore, flow cytometry analysis reveals that glioblastoma cells with an enhanced stem cell-like phenotype express several investigated ligands at significant levels on their cell surface. This reveals that glioblastoma stem-like cells express suppressive ligands with the potential of suppressing major T cell checkpoint receptors. With this information, it is now essential that we understand the relevance of this extensive repertoire of immune checkpoint ligands and their functional consequence on immune evasion in glioblastoma. This is necessary to develop effective immunotherapeutics and to be able to match treatment to patient, especially in the light of CheckMate 143.

Isolation of highly enriched primary human microglia for functional studies.

Microglia, the resident macrophages of the central nervous system play vital roles in brain homeostasis through clearance of pathogenic material. Microglia are also implicated in neurological disorders through uncontrolled activation and inflammatory responses. To date, the vast majority of microglial studies have been performed using rodent models. Human microglia differ from rodent counterparts in several aspects including their response to pharmacological substances and their inflammatory secretions. Such differences highlight the need for studies on primary adult human brain microglia and methods to isolate them are therefore required. Our procedure generates microglial cultures of >95% purity from both biopsy and autopsy human brain tissue using a very simple media-based culture procedure that takes advantage of the adherent properties of these cells. Microglia obtained in this manner can be utilised for research within a week. Isolated microglia demonstrate phagocytic ability and respond to inflammatory stimuli and their purity makes them suitable for numerous other forms of in vitro studies, including secretome and transcriptome analysis. Furthermore, this protocol allows for the simultaneous isolation of neural precursor cells during the microglial isolation procedure. As human brain tissue is such a precious and valuable resource the simultaneous isolation of multiple cell types is highly beneficial.