Expression profiling of single cells and patient cohorts identifies multiple immunosuppressive pathways and an altered NK cell phenotype in glioblastoma

Abstract

Glioblastoma (GBM) is an aggressive cancer with a very poor prognosis. Generally viewed as weakly immunogenic, GBM responds poorly to current immunotherapies. To better understand this problem we used a combination of NK cell functional assays together with gene and protein expression profiling to define the NK cell response to GBM and explore immunosuppression in the GBM microenvironment.In addition, we used transcriptome data from patient cohorts to classify GBM according to immunological profiles. We show that glioma stem-like cells, a source of post-treatment tumour recurrence, express multiple immunomodulatory cell surface molecules and are targeted in preference to normal neural progenitor cells by natural killer (NK) cells ex vivo. In contrast, GBM-infiltrating NK cells express reduced levels of activation receptors within the tumour microenvironment, with hallmarks of TGF-b mediated inhibition. This NK cell inhibition is accompanied by expression of mutiple immune checkpoint molecules on T cells. Single cell transcriptomics demonstrated that both tumour and haematopoietic-derived cells in GBM express multiple, diverse mediators of immune evasion. Despite this, immunome analysis across a patient cohort identifies a spectrum of immunological activity in GBM, with active immunity marked by co-expression of immune effector molecules and feedback inhibitory mechanisms. Our data show that GBM is recognised by the immune system but that anti-tumour immunity is restrained by multiple immunosuppressive pathways, some of which operate in the healthy brain. The presence of immune activity in a subset of patients suggests that these patients will more likely benefit from combination immunotherapies directed against multiple immunosuppressive pathways.

Glioma-derived IL-33 orchestrates an inflammatory brain tumor microenvironment that accelerates glioma progression

“…Since the above results are consistent with IL-33-expressing GBM recruiting peripheral immune cells, we extended the characterization of CD45 + immune cells using single-cell RNA sequencing (scRNAseq). To maintain focus on the innate immune cell repertoire, we chose to perform these studies in IL-33 + and IL-33 − xenografts, a model that allows assessment of the differences in activation states within the tumor immune repertoire, as well as the recruited innate immune cells, such as monocytes and NK cells are reported to be enriched within a glioma tumor environment [91][92][93][94][95] . Cerebral leukocytes were isolated as described above from IL-33 + and IL-33 − tumorbearing hemispheres, sorted for CD45, and RNA sequenced using the 10× Chromium platform.…”

Glioblastoma Immune Landscape and the Potential of New Immunotherapies

“…Alternative stratification of GBM based on consensus immunome clusters (CIC) identified two immunologically active GBM clusters ( 32 ). These clusters expressed genes associated with cytotoxic T lymphocyte (CTLs) and NK cell activation, such as granzyme B ( GZMB ) and interferon gamma ( IFNG ), and genes linked to feedback inhibitory mechanisms including FOXP3 , immune checkpoint inhibitors ( CTLA-4, PD-1, TIM3, VISTA ) and their ligands e.g.…”

“…These clusters expressed genes associated with cytotoxic T lymphocyte (CTLs) and NK cell activation, such as granzyme B ( GZMB ) and interferon gamma ( IFNG ), and genes linked to feedback inhibitory mechanisms including FOXP3 , immune checkpoint inhibitors ( CTLA-4, PD-1, TIM3, VISTA ) and their ligands e.g. PD-L1 and galectin-9 ( 32 ). Nevertheless, these CICs did not discriminate patients with respect to survival outcome, potentially due to the low frequencies of CTLs and NKs and the strong immunosuppressive environment mediated by the myeloid compartment.…”

“…Nevertheless, these CICs did not discriminate patients with respect to survival outcome, potentially due to the low frequencies of CTLs and NKs and the strong immunosuppressive environment mediated by the myeloid compartment. Indeed, GBM tumor-infiltrating lymphocytes (TILs) display an exhausted phenotype ( 33 ), and GBM-infiltrating NK cells express reduced levels of activating receptors e.g., NKp30, NKG2D, and DNAX accessory molecule-1 (DNAM-1) ( 32 ).…”

Exploiting the Complexities of Glioblastoma Stem Cells: Insights for Cancer Initiation and Therapeutic Targeting

“…Moreover, GSCs secrete periostin (POSTN), which recruits M2-like tumor-associated macrophages (TAMs) and promotes GBM aggressiveness [ 234 ]. Others reported that GSCs express multiple immunomodulatory cell-surface molecules [ 235 ], including GM-CSF, that induce TAMs development by promoting the survival and differentiation of bone-marrow-derived monocytes [ 236 ]. Furthermore, GSCs are reported to downregulate the expression of histocompatibility complex Class I (MHC-I) molecules and antigen-processing machinery (APM) components when compared to non-GSCs, by overactivating the WNT pathway [ 135 ].…”