Introduction

Glioblastoma is the most common malignant primary brain tumour in adults.1 Survival is poor, with a median survival of 14.6 months with standard treatment of surgical debulking followed by external-beam radiotherapy with concurrent temozolomide (TMZ) chemotherapy and adjuvant TMZ chemotherapy.2 In the last decade, clinical trials investigating targeted therapies have failed to demonstrate any significant improvement in survival, and innovative new treatments are urgently needed.3 Recently, focus has shifted towards novel strategies that modulate the immune response towards the tumour and the surrounding tumour microenvironment.

Recognition and eradication of malignant cells via immune surveillance of tumour-associated antigens (TAAs) is a key function of the immune system.4 TAAs typically represent peptides which are present within tumour cells but usually absent in the surrounding normal tissue. In glioblastoma, these antigens most commonly fall into three main classes; (i) aberrantly expressed non-mutated self-antigens, (ii) mutated self-antigens and (iii) unique antigens or neo-antigens - novel peptide sequences which are the result of somatic mutations in the cancer genome.5 Tumours manipulate the immune system to avoid detection of TAAs and to facilitate their own growth and survival.6

Immunotherapy aims to harness the immune system against tumours, with breakthroughs observed in a number of cancers, most notably malignant melanoma and haematological malignancies. However, translating these approaches into therapies for primary brain tumours represents a distinct challenge due the unique tumour microenvironment and distinctive immune system within the CNS.

The CNS was traditionally considered immune privileged due to (i) the presence of the blood–brain barrier (BBB) that restricts access to immune cells, (ii) an absence of conventional lymphatic drainage restricting the trafficking of antigens to lymph nodes,7 (iii) a scarcity of specialised antigen-presenting cells,8 and iv) downregulation of the major histocompatibility complex (MHC) expression in normal brain parenchyma, limiting antigen presentation.9 In recent years, this dogma has been eroded with substantial evidence now demonstrating that these interlinked factors tightly regulate a fully functional, innate and adaptive immune system within the CNS (Table 1).

Table 1 Current understanding of the CNS immune system
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In this review, we describe the features of the immune system in the CNS, discuss the immune evasion strategies of glioblastoma, the molecular properties of its microenvironment, and the current clinical evidence supporting a role for immunomodulation in the treatment of glioblastoma.

Glioblastoma tumour microenvironment and mechanisms of immune escape

Glioblastoma arises from glial cells, with surrounding brain parenchyma comprising CNS-specific cells including astrocytes, neurons and microglia, and a distinctive extracellular matrix (ECM) composition.10 Glioblastoma induces a TME characterised by immunosuppressive cytokines secreted by tumour cells, microglia and tumour-associated macrophages (TAMs). These factors, notably interleukin-6, interleukin-10, transforming growth factor-beta (TGF-β), and prostaglandin-E collectively inhibit both the innate and adaptive immune systems with suppression of NK activity and T-cell activation and proliferation, induction of T-cell apoptosis, downregulation of MHC expression, and skew of TAMs towards an M2 (immunosuppressive) phenotype.11 The TME is also characterised by tissue hypoxia provided by irregular vascularity and high-tumour oxygen consumption. Tissue hypoxia activates the immunosuppressive STAT3 pathway, leading to hypoxia-inducible factor-1 alpha (HIF-1α) synthesis, activation of regulatory T cells (T-regs) and production of vascular endothelial growth factor (VEGF),11 which can inhibit the maturation and function of dendritic cells. These mechanisms of immune escape are discussed below in more detail and summarised in Fig. 1.

Fig. 1
figure 1

Immune gateways (left). In addition to the resident microglia, there are three distinct macrophage populations within the CNS present at so-called ‘immune gateways’ that act as ports of entry for activated T cells into the CNS. Perivascular macrophages, derived from the embryonic yolk sac, are located around parenchymal vessels (top). The other two populations, derived from bone marrow, are located in the meningeal spaces (middle) and the choroid plexus (bottom) (adapted from ref. 156). Immune evasion in glioblastoma (right): the immunosuppressive tumour microenvironment (TME) of glioblastoma is the result of complex interactions between tumour cells, microglia, tumour-associated macrophages (TAMs), components of the extracellular matrix and tumour infiltrating lymphocytes (TILs), which are predominantly regulatory in phenotype (T-regs). Hypoxia promotes angiogenesis of abnormal blood vessels, further driving tumour growth (adapted from ref. 10)

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Extracellular matrix composition

ECM proteins commonly found in abundance in peripheral tissues including collagens, laminins and fibronectin are typically only associated with vascular basement membranes within the CNS. Instead, predominant ECM proteins in the glioma TME include glycoproteins, hyaluronic acid and heparan sulphate proteoglycans (HSPGs), which may be concentrated in cancer stem cell niches.12 HSPGs, in particular, are upregulated in glioblastoma13 and cause retention of heparin-binding angiogenic growth factors such as fibroblast growth factor (FGF) and VEGF, the local release of which promotes tumour angiogenesis and progression.14 Furthermore, glioma vasculature can upregulate the vessel-associated macromolecules periostin and tenascin C (TNC), which can promote tumour survival.15 These macromolecules can also promote immune evasion with TNC shown to block T-cell movement across glioma-associated blood vessels, preventing their migration into brain parenchyma.16 Periostin, when secreted by glioma stem cells, is able to promote recruitment of tumour promoting macrophages from peripheral circulation.17

Macrophages and microglia

Tumour-associated macrophages (TAMs), along with the resident CNS microglia, can constitute up to 30% of the tumour mass.10 Transcriptome analysis of TAMs has found that they may possess markers consistent with both M1 (classically activated or immunopermissive) and M2 (alternatively activated or immunosuppressive) phenotypes, incongruous with the traditional M1/M2 dichotomy.18 TAM populations can be described both functionally and spatially. For example, CNS-resident microglia exist within the TME alongside distinct populations of bone marrow-derived macrophages (outlined in Table 1),19 and recent research has suggested that within the TME, bone marrow-derived macrophages may localise preferentially to the perivascular niche, while resident microglia localise to peritumoural regions.20 Accumulation of TAMs expressing CD163 (haemoglobin scavenger receptor) and CD204 (macrophage scavenger receptor), considered ‘M2′ phenotype markers, increases as tumour grade increases.21 In glioblastoma, higher CD163 TAM expression correlates with poorer outcomes.11 Cancer stem cells in glioblastoma are able to recruit TAMs by overexpression of the macrophage/microglia cytokine colony-stimulating factor-1 (CSF-1).22 This induces a pro-tumourigenic microenvironment via release of immunosuppressive factors such as IL-10 and overexpression of the indoleamine 2,3-dioxygenase (IDO) enzyme.17,23 M2-TAMs also lack expression of key T-cell co-stimulation molecules24 and drive both tumour angiogenesis and resistance to anti-VEGF agents. Angiogenesis is driven through production of pro-angiogenic molecules including VEGF and FGF, with resistance to anti-angiogenics due to upregulation of alternative angiogenic pathways and stimulation of pericytes to proliferate in the perivascular niche. Pericytes are perivascular cells responsible for modulating blood flow, vessel permeability and remodelling, and their proliferation stabilises new vessels.25,26 TAMs can also enhance the invasiveness of glioma stem cells (GSCs) via the TGF-β1 signalling pathway.27 Further evidence, incongruous with the M1/M2 dichotomy, is that blockade of CSF-1R on the macrophage surface does not deplete glioblastoma TAMs in vivo, but leads to reprogramming of TAMs away from immunosuppressive phenotypes.28 Other emerging evidence includes the discovery of a link between tissue hypoxia and macrophage polarisation; with M1 macrophages present within normoxic tumour regions and M2 macrophages present in areas of hypoxia.29 These findings support the view that macrophage polarisation is not simply location dependent but instead dependent on distinct signals present in the local microenvironment.30 TAMs retain both plasticity and the ability to undergo reprogramming;31 characteristics which are potentially exploitable for therapeutic benefit.32

Downregulation of MHC

An effective T-cell immune response requires antigen presentation and subsequent recognition, which is dependent on the co-expression of the human MHC proteins. Comparative analyses of gene expression profiles suggest that invading glioblastoma cells are able to escape immune recognition by downregulating expression of MHC molecules.33 The role of antigen presentation within the CNS is thought to fall primarily to the resident microglia, with supporting evidence in vivo demonstrating that microglia are able to cross-present tumour antigens to CD8+ T cells via MHC Class I.34 However, the presence of immunosuppressive cytokines such as IL-10 and TGF-ß within the glioblastoma TME cause microglia to lose MHC expression.11,35 Pericytes may also play a role in antigen presentation within the CNS, and pericyte MHC Class II expression is shown to increase in response to inflammatory cytokine release.36 In the TME, pericytes in contact with glioblastoma cells possess immunosuppressive functions evidenced by changes including a reduction in MHC expression and T-cell co-stimulatory signals.37 Low levels of MHC Class I molecules are also found on glioblastoma cancer stem cells, rendering them resistant to T-cell-mediated killing, and thus contributing towards tumour initiation, progression and resistance to therapy.38

T lymphocytes

In patients with glioblastoma, increased T-cell infiltration is found in both tumour (tumour-infiltrating lymphocyte; TIL) and brain parenchyma.39 Studies of human tumour samples have shown that this influx is counteracted with a number of events that evade the immune response including further downregulating MHC to prevent antigen presentation,40 increasing expression of the inhibitory protein programmed death ligand-1 (PD-L1),41 and increased recruitment of immunosuppressive regulatory T cells (T-regs) which express co-inhibitory molecules including cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and programmed death receptor 1 (PD-1).42,43 Fourfold more T-regs are found in human glioblastoma samples than benign pituitary adenomas and meningiomas, with CTLA-4 expression on these T-regs threefold that on T-regs in peripheral blood.42 PD-L1 expression on circulating monocytes is significantly higher in patients with glioblastoma compared to healthy controls, while expression in tumour infiltrating monocytes is, on average, twice that of circulating monocytes from the same patient.41

Immunological effects of standard therapy

Standard therapy for patients diagnosed with glioblastoma consists of maximal safe debulking surgery, followed by radiotherapy and temozolomide chemotherapy (RT-TMZ).2 In addition, high doses of glucocorticoids are frequently administered to reduce tumour-associated and radiotherapy-induced cerebral oedema. These therapies may potentially both augment or diminish immune responses.

Radiotherapy, chemotherapy and glucocorticoid therapy are independently immunosuppressive. Approximately 70% of patients experience clinically significant reductions in their circulating CD4+ lymphocyte counts following standard treatment, persisting for up to a year and associated with early tumour progression.44 TMZ-induced lymphopenia is considered a significant limitation to clinical translation of immunotherapies.44 Radiation induces M2 macrophages and activation of TGF-β.45,46,47 RT-TMZ has been shown to increase PD-L1 expression in preclinical glioblastoma models, and acquired resistance to radiotherapy has been overcome by PD-L1 blockade in cancer models.48,49 Further, as well as depleting systemic lymphocyte counts, RT-TMZ also tilts the balance between regulatory and effector peripheral blood lymphocytes towards an immunosuppressive state.50,147,148

Tumour heterogeneity is well characterised in glioblastoma, and may contribute towards immune cell heterogeneity within tumours.149,150 Glioblastoma heterogeneity is at least partly maintained by extracellular vesicles (EVs) that contain molecules including DNA, RNA and proteins. The shedding and uptake of these vesicles from tumour cells form an additional communication network capable of modulating the TME. Recent evidence has also shown that PD-L1 is expressed on glioblastoma EVs.151,152 Further, vascular heterogeneity exists, with vessel co-option (glioblastoma migration along existing blood vessels) occurring alongside angiogenesis, increasing the complexity of heterogeneity within glioblastoma.153 Further research into these factors, including utility of techniques such as single-cell RNA sequencing to characterise both tumour, and immune cell heterogeneity154,155 may facilitate a future shift towards more personalised treatment.

In summary, the CNS has an active immune system and glioblastoma employs a number of strategies to evade immune-mediated death. To design therapies that effectively harness the immune system against glioblastoma, a deeper understanding of the complex interactions between tumours and the tightly regulated immune system in the CNS is needed. Future therapies will need to address multi-dimensional challenges including identifying truly tumour-specific antigens to target, increasing tumour immunogenicity, targeting the immune microenvironment and controlling toxicity of therapy.