BackgroundGlioblastoma (GBM) is refractory to immune checkpoint inhibitor (ICI) therapy. We sought to determine to what extent this immune evasion is due to intrinsic properties of the tumor cells versus the specialized immune context of the brain, and if it can be reversed.MethodsWe used CyTOF mass cytometry to compare the tumor immune microenvironments (TIME) of human tumors that are generally ICI-refractory (GBM and sarcoma) or ICI-responsive (renal cell carcinoma), as well as mouse models of GBM that are ICI-responsive (GL261) or ICI-refractory (SB28). We further compared SB28 tumors grown intracerebrally versus subcutaneously to determine how tumor site affects TIME and responsiveness to dual CTLA-4/PD-1 blockade. Informed by these data, we explored rational immunotherapeutic combinations.ResultsICI-sensitivity in human and mouse tumors was associated with increased T cells and dendritic cells (DCs), and fewer myeloid cells, in particular PD-L1+ tumor-associated macrophages. The SB28 mouse model of GBM responded to ICI when grown subcutaneously but not intracerebrally, providing a system to explore mechanisms underlying ICI resistance in GBM. The response to ICI in the subcutaneous SB28 model required CD4 T cells and NK cells, but not CD8 T cells. Recombinant FLT3L expanded DCs, improved antigen-specific T cell priming, and prolonged survival of mice with intracerebral SB28 tumors, but at the cost of increased Tregs. Targeting PD-L1 also prolonged survival, especially when combined with stereotactic radiation.ConclusionsOur data suggest that a major obstacle for effective immunotherapy of GBM is poor antigen presentation in the brain, rather than intrinsic immunosuppressive properties of GBM tumor cells. Deep immune profiling identified DCs and PD-L1+ tumor-associated macrophages as promising targetable cell populations, which was confirmed using therapeutic interventions in vivo.

Edited by Ruma Banerjee Lipoxygenases are widespread enzymes found in virtually all eukaryotes, including fungi, and, more recently, in prokaryotes. These enzymes act on long-chain polyunsaturated fatty acid substrates (C18 to C20), raising questions regarding how the substrate threads its way from solvent to the active site. Herein, we report a comparison of the temperature dependence of isotope effects on first-and second-order rate constants among single-site variants of the prototypic plant enzyme soybean lipoxygenase-1 substituted at amino acid residues inferred to impact substrate binding. We created 10 protein variants including four amino acid positions, Val-750, Ile-552, Ile-839, and Trp-500, located within a previously proposed substrate portal. The conversion of these bulky hydrophobic side chains to smaller side chains is concluded to increase the mobility of flanking helices, giving rise to increased off rates for substrate dissociation from the enzyme. In this manner, we identified a specific "binding network" that can regulate movement of the substrate from the solvent to the active site. Taken together with our previous findings on C-H and O 2 activation of soybean lipoxygenase-1, these results support the emergence of multiple complementary networks within a single protein scaffold that modulate different steps along the enzymatic reaction coordinate.

Background Diffuse midline gliomas (DMG) are highly malignant incurable pediatric brain tumors. A lack of effective treatment options highlights the need to investigate novel therapeutic strategies. This includes the use of immunotherapy, which has shown promise in other hard-to-treat tumors. To facilitate preclinical immunotherapeutic research, immunocompetent mouse models that accurately reflect the unique genetic, anatomical, and histological features of DMG patients are warranted. Methods We established cell cultures from primary DMG mouse models (C57BL/6) that were generated by brainstem targeted intra-uterine electroporation (IUE). We subsequently created allograft DMG mouse models by orthotopically implanting these tumor cells into syngeneic mice. Immunohistochemistry and -fluorescence, mass cytometry, and cell-viability assays were then used to verify that these murine tumors recapitulated human DMG. Results We generated three genetically distinct allograft models representing histone 3 wildtype (H3 WT) and K27M-mutant DMG (H3.3 K27M and H3.1 K27M). These allograft models recapitulated the histopathologic phenotype of their human counterparts, including their diffuse infiltrative growth and expression of DMG-associated antigens. These murine pontine tumors also exhibited an immune microenvironment similar to human DMG, characterized by considerable myeloid cell infiltration and a paucity of T-lymphocytes and NK cells. Finally, we show that these murine DMG cells display similar sensitivity to histone deacetylase (HDAC) inhibition as patient-derived DMG cells. Conclusions We created and validated an accessible method to generate immunocompetent allograft models reflecting different subtypes of DMG. These models adequately recapitulated the histopathology, immune microenvironment, and therapeutic response of human DMG, providing useful tools for future preclinical studies.