Abstract
BACKGROUND: Isocitrate dehydrogenase mutant (IDHmt) astrocytoma is considered a T cell excluded tumor, yet little is known regarding the underlying cues that may drive T cell exclusion. To aid better understanding of this mechanism in IDHmt astrocytoma, we systematically characterized the immune microenvironment of this tumor type. METHODS: We performed and analyzed multiplex immunofluorescence stainings for lymphocytes, myeloid cells and IDH1-R132H of 1293 regions from 75 IDHmt astrocytomas. We consequently performed bulk, single nucleus and spatial RNA sequencing of samples from the same cohort. Integrative analysis of data from these platforms allowed for the identification of unique tumor and immune cell subsets that are crucial in the spatial organization of the immune microenvironment and T cell containment in IDHmt astrocytoma. RESULTS: IDHmt astrocytoma showed three spatially distinct distribution patterns of T cells: regions with perivascular accumulation of T cells, regions with T cells in the tumor stroma and T cell absent regions. Perivascular accumulation of T cells, known as T-cell cuffs, was the most abundant pattern in our data. Spatially, there was a significant increase of tumor cells around T cell cuffs that showed a gemistocytic phenotype, a tumor cell type that is histologically characterized by a large eosinophilic cytoplasm and ectopically localized nucleus. Gemistocyte-high tumors (> 20% of all tumor cells) demonstrated enhanced expression of genes related to immune cell migration and activation that were reliably expressed by tumor associated macrophages and T cells. Notably, gemistocytic tumor cells were identified as a distinct sub-cluster of the ‘astrocyte-like’ transcriptomic cell state with a phenotype that is linked to astrogliosis and expressed CD44, TNC and TGFB2. Moreover, gemistocytic tumor cells were spatially associated with a CD83+ subpopulation of tumor-associated macrophages that resemble reactive microglia in astrogliosis and show increased expression of the chemo-attractants CCL3, CCL4 and CCL4L2. CONCLUSION: Using different modalities for spatial and single cell profiling of RNA and protein, we uncovered a previously unknown relationship between the immune system and tumor cells in IDHmt astrocytoma. We found that gemistocytic tumor cells showing an ‘astrocyte-like’ state co-occur with perivascular contained T cells and immune-stimulatory macrophages in IDHmt astrocytomas. The transcriptional profiles of these tumor cell subsets and macrophages point towards a CNS specific defense mechanism of glial cells associated with T cell exclusion.