ER-mitochondria contacts control surface glycan expression and sensitivity to killer lymphocytes in glioma stem-like cells.

Glioblastoma is a highly heterogeneous aggressive primary brain tumor, with the glioma stem-like cells (GSC) being more sensitive to cytotoxic lymphocyte-mediated killing than glioma differentiated cells (GDC). However, the mechanism behind this higher sensitivity is unclear. Here, we found that the mitochondrial morphology of GSCs modulates the ER-mitochondria contacts that regulate the surface expression of sialylated glycans and their recognition by cytotoxic T lymphocytes and natural killer cells. GSCs displayed diminished ER-mitochondria contacts compared to GDCs. Forced ER-mitochondria contacts in GSCs increased their cell surface expression of sialylated glycans and reduced their susceptibility to cytotoxic lymphocytes. Therefore, mitochondrial morphology and dynamism dictate the ER-mitochondria contacts in order to regulate the surface expression of certain glycans and thus play a role in GSC recognition and elimination by immune effector cells. Targeting the mitochondrial morphology, dynamism, and contacts with the ER could be an innovative strategy to deplete the cancer stem cell compartment to successfully treat glioblastoma.

Glioma Stemlike Cells Enhance the Killing of Glioma Differentiated Cells by Cytotoxic Lymphocytes.

Glioblastoma multiforme, the most aggressive primary brain tumor, is maintained by a subpopulation of glioma cells with self-renewal properties that are able to recapitulate the entire tumor even after surgical resection or chemo-radiotherapy. This typifies the vast heterogeneity of this tumor with the two extremes represented on one end by the glioma stemlike cells (GSC) and on the other by the glioma differentiated cells (GDC). Interestingly, GSC are more sensitive to immune effector cells than the GDC counterpart. However, how GSC impact on the killing on the GDC and vice versa is not clear. Using a newly developed cytotoxicity assay allowing to simultaneously monitor cytotoxic lymphocytes-mediated killing of GSC and GDC, we found that although GSC were always better killed and that their presence enhanced the killing of GDC. In contrast, an excess of GDC had a mild protective effect on the killing of GSC, depending on the CTL type. Overall, our results suggest that during combination therapy, immunotherapy would be the most effective after prior treatment with conventional therapies.

Decitabine Treatment of Glioma-Initiating Cells Enhances Immune Recognition and Killing.

Malignant gliomas are aggressive brain tumours with very poor prognosis. The majority of glioma cells are differentiated (glioma-differentiated cells: GDCs), whereas the smaller population (glioma-initiating cells, GICs) is undifferentiated and resistant to conventional therapies. Therefore, to better target this pool of heterogeneous cells, a combination of diverse therapeutic approaches is envisaged. Here we investigated whether the immunosensitising properties of the hypomethylating agent decitabine can be extended to GICs. Using the murine GL261 cell line, we demonstrate that decitabine augments the expression of the death receptor FAS both on GDCs and GICs. Interestingly, it had a higher impact on GICs and correlated with an enhanced sensitivity to FASL-mediated cell death. Moreover, the expression of other critical molecules involved in cognate recognition by cytotoxic T lymphocytes, MHCI and ICAM-1, was upregulated by decitabine treatment. Consequently, T-cell mediated killing of both GDCs and GICs was enhanced, as was T cell proliferation after reactivation. Overall, although GICs are described to resist classical therapies, our study shows that hypomethylating agents have the potential to enhance glioma cell recognition and subsequent destruction by immune cells, regardless of their differentiation status. These results support the development of combinatorial treatment modalities including epigenetic modulation together with immunotherapy in order to treat heterogenous malignancies such as glioblastoma.

Synergy between CD8 T cells and Th1 or Th2 polarised CD4 T cells for adoptive immunotherapy of brain tumours.

The feasibility of cancer immunotherapy mediated by T lymphocytes is now a clinical reality. Indeed, many tumour associated antigens have been identified for cytotoxic CD8 T cells, which are believed to be key mediators of tumour rejection. However, for aggressive malignancies in specialised anatomic sites such as the brain, a limiting factor is suboptimal tumour infiltration by CD8 T cells. Here we take advantage of recent advances in T cell biology to differentially polarise CD4 T cells in order to explore their capacity to enhance immunotherapy. We used an adoptive cell therapy approach to work with clonal T cell populations of defined specificity. Th1 CD4 T cells preferentially homed to and accumulated within intracranial tumours compared with Th2 CD4 T cells. Moreover, tumour-antigen specific Th1 CD4 T cells enhanced CD8 T cell recruitment and function within the brain tumour bed. Survival of mice bearing intracranial tumours was significantly prolonged when CD4 and CD8 T cells were co-transferred. These results should encourage further definition of tumour antigens recognised by CD4 T cells, and exploitation of both CD4 and CD8 T cell subsets to optimise T cell therapy of cancer.

Immune infiltration of spontaneous mouse astrocytomas is dominated by immunosuppressive cells from early stages of tumor development.

Immune infiltration of advanced human gliomas has been shown, but it is doubtful whether these immune cells affect tumor progression. It could be hypothesized that this infiltrate reflects recently recruited immune cells that are immediately overwhelmed by a high tumor burden. Alternatively, if there is earlier immune detection and infiltration of the tumor, the question arises as to when antitumor competency is lost. To address these issues, we analyzed a transgenic mouse model of spontaneous astrocytoma (GFAP-V(12)HA-ras mice), which allows the study of immune interactions with developing glioma, even at early asymptomatic stages. T cells, including a significant proportion of Tregs, are already present in the brain before symptoms develop, followed later by macrophages, natural killer cells, and dendritic cells. The effector potential of CD8 T-cells is defective, with the absence of granzyme B expression and low expression of IFN-gamma, tumor necrosis factor, and interleukin 2. Overall, our results show an early defective endogenous immune response to gliomas, and local accumulation of immunosuppressive cells at the tumor site. Thus, the antiglioma response is not simply overwhelmed at advanced stages of tumor growth, but is counterbalanced by an inhibitory microenvironment from the outset. Nevertheless, we determined that effector molecule expression (granzyme B, IFN-gamma) by brain-infiltrating CD8 T-cells could be enhanced, despite this unfavorable milieu, by strong immune stimuli. This potential to modulate the strong imbalance in local antiglioma immunity is encouraging for the development and optimization of future glioma immunotherapies.