Emerging Hallmark Of Gliomas Microenvironment in Evading Immunity: A Basic Concept Part 2

T‑cell anergy

GBM has been shown to deplete T cells and desensitize them to the tumor’s presence [103]. This theory was based on findings in cases of chronic lymphocytic choriomeningitis virus (LCMV) infection [104, 105], but has now been demonstrated to occur in cancer as well [106]. Numerous inhibitory receptors were up-regulated following chronic antigen exposure [107]. Checkpoint inhibitors block inhibitory signals that regulate lymphocytes; among the up-regulated immune checkpoint inhibitory receptors are the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), PD-1, and PD-L1 (Fig. 2), which have been approved by the FDA as T-cell-based treatment for cancer [108].

First, immunity is one of our body's main defense systems against infection and disease. When our immune system is working properly, it can effectively identify and destroy viruses and bacteria, protecting our body from them. However, when our immune system is weakened, we become vulnerable to a variety of diseases, including choriomeningitis.

Therefore, maintaining adequate immunity is very important to prevent choriomeningitis. There are some ways to improve immunity, such as good eating habits, adequate rest, moderate exercise, stress reduction, smoking cessation, and so on. In addition, we can also prevent certain diseases through vaccination, including choriomeningitis.

Of course, if you already have choriomeningitis, early treatment is also very important. Taking timely treatment measures can shorten the duration of the disease and reduce the occurrence of sequelae. At the same time, pay attention to rest, maintain good nutrition, and diet to help the body recover quickly.

In conclusion, choriomeningitis is closely related to immunity. Maintaining adequate immunity and preventive measures are important means of preventing choriomeningitis. At the same time, for patients who are already sick, early treatment is also crucial. Let us pay attention to health, maintain a good lifestyle, prevent and treat diseases, and welcome a bright future. From this point of view, our Xu Ya can improve immunity, and cistanche can significantly improve immunity because meat paste also has anti-virus and anti-cancer effects, which can strengthen the immune system's ability to fight and improve the body's immunity.

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PD-1 is a surface receptor that serves as an immunological checkpoint. This receptor is expressed on the surface of activated T cells, NK cells, B lymphocytes, macrophages, DCs, and monocytes [109]. PD-1 suppresses immune cells’ inflammatory activities when attached to its ligand, the PD-L1 [110]. Nduom and colleagues examined the expression of PD-L1 in 94 patients and discovered that it was a poor predictive factor for GBM [111]. However, Wang and colleagues used transcriptome data to evaluate 976 glioma samples and discovered that PD-L1 expression was positively linked with higher WHO glioma classification (Fig. 3) [112].

The phosphoinositide 3-kinase (PI3K), AKT [113], and mammalian target of rapamycin (mTOR) appear to influence PD-L1 expression (PI3K/AKT/mTOR pathway) [114]. In addition, this pathway is known to modulate various other characteristics of cancer to optimize tumor survival [115]. As indicated by a study on gastrointestinal stromal tumors, PD-1/PD-L1 is thought to promote CD8+ apoptosis [116]. The MAPK signaling pathway is a signaling mechanism that contributes to glioma’s immunosuppressive properties. Recent research has put more attention on the relationship between the PD-1/ PD-L1 axis and the MAPK pathway. Stutvoet and colleagues demonstrated that inhibiting the MAPK pathway reduced the induction of PD-L1 protein in lung cancer cells by epidermal growth factor (EGF) and interferon (IFN) [117]. Indeed, IFN-γ released by tumor-inflating lymphocytes (TIL) is a powerful activator of PD-L1 expression in glioma [108].

Immunosuppressive T‑cell recruitment using chemokines

CXCR2 and CXCL8 are two of the most prevalent chemokines in the glioma microenvironment [118]. The upregulation of both chemokine receptors was found to be associated with a bad outcome [118]. GBMs express high levels of CXCR2 which are known mostly for its role in angiogenesis [119]. CXCL8, on the other hand, leads to local and systemic immunosuppression [120] which enables GBM to evade host immunosurveillance. GBM-associated systemic immunosuppression is connected to the increase of immunosuppressive T cells, such as Tregs and myeloid-derived suppressor cells (MDSCs) [121, 122]. MDSCs exert their effect by suppressing T-cell proliferation and activation. MDSCs regulate inflammatory responses in the normal population, therefore, preventing autoimmune illness [123, 124]. CXCL8 expression by GBM has been shown to regulate the entry of MDSCs into the tumor environment via the CXCR2 receptor [125].

Regulatory T‑cells (Tregs) and T‑cell apoptosis

Numerous studies on many forms of cancer have established that Tregs are involved in immunosuppression [126, 127]. Tregs are a physiological fraction of CD4+ T cells that inhibit the function of T and B cells [128, 129], six different DCs [130–132], monocytes or macrophages [132], and NK cells [133, 134]. Functional Tregs express CD4+, CD25+, and Foxp3 [126]. Within the glioma microenvironment, both the number and function of CD4+ T cells are reduced, with an abnormally high proportion of Tregs [135].

A time-dependent increase of Tregs was seen in brain tumors in an in vivo research [136]. Hussain and colleagues isolated and labeled immune cells from human glioblastoma tissue to determine their phenotypes [26]. They discovered that glioma-specific CTL was phenotypically CD8+ and CD25−, indicating that they were inactive. The majority of T cells in glioma were CD4+, indicating Treg dominance, as demonstrated by positive intracellular staining for Foxp3 [26]. Another study compared GBM and normal brain tissue and discovered that CD4+ CD25+ Foxp3+ Tregs were present only in GBM tissue [137]. The chemokine CXCR2 induces Treg migration into the glioma microenvironment [138].

Tregs have been shown to trigger T-cell death in vitro. T-cells were grown with Tregs for 72  h and apoptosis was demonstrated using transmission electron microscopy [139]. Numerous hypotheses have been advanced to explain how Tregs trigger T-cell death, including inappropriate T-cell activation [140–142] and depriving T cells of cytokines [139]. The former method favors aggressive apoptosis, whereas the latter favors quiet apoptosis. The cytokine deprivation-induced apoptosis was discovered preclinically when pro-survival cytokines shielded T cells against apoptosis. In addition, it was shown that T cells die gradually over 3–4 days rather than instantly as in cytolysis. In addition, an in vitro investigation showed that the concentration of cytokines was lower in cultures containing Tregs than in cultures containing control T cells [139].

Another way for T cells to undergo apoptosis is via the Fas-mediated pathway. GBM expresses the Fas ligand (CD95L) on its surface, which induces T-cell death upon binding to Fas (CD95/APO-1) on T cells [143]. Fas-mediated apoptosis is a well-established concept of cell death. When Fas binds to its ligand, it recruits Fas-associated proteins to DD (FADD). This protein is responsible for the death of cells by recruiting caspase-8 and caspase-10 [144]. Another method of T-cell apoptosis occurs when CD70 on GBM cells interacts with CD27 on T cells. It has been demonstrated that inhibiting this connection partially protects T cells against GBM cell-induced death [145].

Extracellular matrix

Numerous solid tumors contain abundant extracellular matrix (ECM) molecules, including fibrillar collagens, fibronectin, elastin, and laminins [146]. Up to 60% of the mass of many tumors is composed of extracellular matrix [146]. The tumor cells themselves, but to an even greater extent, cancer-associated fibroblasts (CAFs) are the source of these ECM molecules [147]. CAFs support tumor cells via paracrine stromal cell-derived factor-1 (SDF1) and transforming growth factor beta (TGF) signals, contributing not only to a more malignant tumor phenotype by driving epithelial-to-mesenchymal transition (EMT), but also inducing production of collagen and other ECM molecules [148].

The molecular expression profile can subdivide numerous cancers originating from the same tissue [148]. These molecular subtypes provide a great deal of information regarding the tumor’s metabolism, dysregulation of survival and apoptosis pathways, and the presence or absence of specific proteins [148]. In many cancers, the expression profile of ECM-related genes is also a valuable prognostic factor [148]. In addition to immune suppression markers, high expression of Col3a1, Col4a1, and Col5a2 is associated with a poor prognosis in glioblastoma [148].

Inevitably, the occurrence of metastasis impacts treatment options and therapeutic outcomes. EMT is associated with both increased metastasis and chemoresistance. EMT in cancer is associated with the development of stem-cell-like properties [149]. Loss of epithelial polarization, which is linked to the anchorage of epithelial layers on a basement membrane, is characteristic of EMT [149]. On top of that, ECM had a role in glioma invasion. Glycosylated chondroitin sulfate proteoglycans (CSPGs), a major component of ECM in the brain contribute to inducing glioma invasion.

Exosomes

Exosomes play a vital role in evading immunity and inducing tumor progression. Exosomes, released by DCs, express tumor or stimulatory antigens to activate cytotoxic T-cell responses [32]. Previous studies have investigated the critical role of tumor-derived exosomes against immunity. Exosomes, released by impaired DCs, tend to have a greater impact under hypoxia. Exosomes released by hypoxic bone-marrow-derived mesenchymal stem cells (BMSCs) in TEM induce cancer cell invasion and epithelial-mesenchymal transition [150]. Exosomes also contribute to the proliferation, invasion, and migration of human umbilical vein endothelial cells in esophageal squamous cell carcinoma under hypoxia [151]. In gliomas, a recent study demonstrated that exosomal connexins 43 (Cx43) contribute to glioma angiogenesis mediated by exosomes under hypoxia [152]. Moreover, hypoxic glioblastoma-derived exosomes disrupt the permeability of blood–brain barrier (BBB) [153].

Discussion

In general, errors in a cell’s genome are the cause of the development and formation of neoplastic cells. The tumor’s microenvironment contains several factors that promote and sustain its growth. In addition, the resistance to applied therapies is also a result of tumor heterogeneity and its constant alterations [154, 155]. Nonetheless, cancer has developed several immune surveillance evasion mechanisms. These include the avoidance of recognition by the down-regulation of MHC, impaired DC function, immunosuppressive TAMs, Natural Killer (NK) cell inhibition, T-cell anergy, immunosuppressive T-Cell recruitment using chemokines, regulatory T-cells (Tregs), T-cell apoptosis and extracellular matrix. Several of these mechanisms are conducive to progression, the creation of their environment for cell development, and cell death in their favorable environment [156–158]. Similar to other types of cancer, gliomas weaken the immune system through various pathways. 

The immunosuppressive ability of glioma plays a vital role in glioma survival. IL-10, IL-6, TGF, and PGE-2 were found to be immunosuppressive factors in the glioma microenvironment. In addition, the presence of GARP, a surface molecule, allows glioma to survive for a longer time by activating Treg cells [53, 112]. On top of that, glioma-induced tumor progression by weakening BBB integrity. This will lead to accelerating vasculogenesis and impaired arteries which result in hypoxia and promote tumor development [153, 159]. Glioma also disrupted EC as a result of VEGF [43, 44]. All of these pathways are frequently interconnected, resulting in a vicious cycle that promotes glioma survival and progression. Understanding what occurs within the microenvironment of glioma and which mechanisms are responsible for glioma development and progression will reveal how glioma could protect itself from the immune system.

The concept of immunotherapy for GBM

Decreased MHC expression in GBM frequently correlates with a worse prognosis. MHC-I downregulation has previously been attributed to epigenetic and transcriptional dysregulations involved in the stabilization of NFkB, interferon regulatory factors (IRFs), and NODlike receptor family CARD domain-containing protein 5 (NLRC5). These dysregulations are possibly reversible, implying the possibility of reversing MHC-I downregulation in cancer. In addition, STAT3 inhibition, STING activation, chemotherapy, and radiation can all stimulate MHC-I expression [160]. However, there are few trials targeting MHC-I in gliomas.

As previously stated, impaired DC proliferation will further impair CTL function [45]. DC vaccines (DCVs) are a type of immunotherapy that aims to enhance DC activities. DCVs comprised immunostimulatory APCs created in vitro utilizing CD14 monocytes cultured with GM-CSF and IL-4. In short, DCVs are DCs loaded with tumor antigens and injected into the patient [161]. Autologous tumor lysate, cultured tumor cells from surgical specimens, irradiated autologous tumor cells, tumor RNA, or tumor-related peptides were utilized as antigens. In a phase II GBM vaccine experiment, Wheeler and colleagues reported that 53% of GBM patients demonstrated a 1.5-fold increase in cytokine response following vaccination. Responders to vaccination have longer median survival than non-responders (642 days and 430 days) [162]. A large phase III clinical trial is needed to confirm DCV’s efficacy and safety in glioma, as results negating its benefits have also been published [162].

In gliomas, TAM infiltration is dominated by tumor-supportive M2 macrophages. Because TAMs require colony-stimulating factor (CSF) for differentiation and survival, BLZ945, a CSF-1 inhibitor, was utilized to target TAMs in mice GBM models. Inhibition of CSF-1 can decrease the quantity of M2 macrophages, resulting in tumor regression [163]. PLX3397 is a CSF-1 inhibitor that can cross the BBB and reduce TAMs, thus resulting in the alleviation of tumor invasiveness in mouse models of GBM [163]. TAMs-targeted immunotherapy may be useful in the treatment of GBM. However, at the moment this therapeutic modality is still limited to in vivo models [162].

NK cells have significant anti-tumor effects, particularly when CTL function is reduced. Although the number of NK cells in GBMs is deemed low, they retained cytotoxic activity [80]. Enhancing NK cells’ oncolytic capacity might be achieved by counteracting their inhibition, that is through cutting the binding between MHC molecules and killer immunoglobin receptors (KIRs) [95]. Ishikawa and colleagues demonstrated tumor volume decrease using autologous NK cells. In addition, they suggested that this response could be enhanced by combining autologous NK cells with an IL-2 dosage or radiation therapy [164]. Another option is to use allogenic NK cells, which originate from an unrelated donor and are equipped with a KIR receptor that is incapable of recognizing MHC class I molecules. In allogeneic NK cells, the KIR receptor does not recognize tumor MHC molecules, resulting in the absence of NK cell inhibition [95].

Anti-CTLA-4 and Anti-PD-1 therapies have primarily been studied in T cells for their direct immunological implications (Fig.  2). Due to their roles as an immune checkpoint, therapies targeting CTLA-4 and PD-1 are hypothesized to be able to “free” T cells from inhibition to fight tumor cells. CTLA-4 (CD152) is an inhibitory receptor that downregulates T-cell function [165, 166]. This receptor is mainly expressed on Tregs but might be upregulated on other subsets of T cells in pathologic conditions, such as cancer. CTLA-4 suppresses the immune system indirectly by inhibiting signals via the co-stimulatory receptor CD28. CTLA-4 reduces immunological responses to weak antigens such as self- and tumor antigens by increasing the activation threshold of T cells [167]. PD-1 binding to PD-L1 is involved predominantly in inhibitory immune signaling. Although the majority of circulating T cells lack PD-1, their expression can be stimulated by exposure to cytokines, such as IL-2, IL-7, IL-15, IL-21, and TGF-β [167].

Neoantigens, which are formed from tumor-specific protein-coding mutations, are immune stimulatory and can operate as bona fde antigens that aid in tumor rejection. T-cell activation and subsequent tumor lysis driven by neoantigen vaccines offer an appealing precision medicine strategy. The process of developing a personalized neoantigen vaccination begins with a comparison of genetic data received from the patient’s peripheral blood mononuclear cells (PBMCs) and excised tumor tissue [168]. Following the administration of customized vaccinations, APCs come into contact with the neoantigens contained in the vaccine, thereby initiating the process of neoantigen MHC presentation [169]. Immune responses mediated by T cells are triggered when a certain T cell receptor recognizes a particular neoantigen. In addition, these neoantigen-specific T lymphocytes expand, move toward the tumor site, and subsequently enter the tumor. Immune responses can be found that are CD4 positive (which enhances the immune response) or CD8 positive (which has a cytotoxic effect). Tumor cells that have been eliminated create an adaptive immunological memory response by releasing neoantigens [170].

Adoptive T cell therapy, which entails the selection and development of antigen-specific T cell clones ex vivo, enables the enhancement of antigen-specific immunity without the in vivo restrictions associated with vaccine-based techniques. While some clinical responses have been found in vaccine trials, the amplitude of the induced T-cell response has often been small or undetectable and has had a poor correlation with clinical responses. In comparison with vaccination methods, adoptive treatment procedures are capable of circumventing the in vivo restrictions that limit the amplitude and avidity of the targeted response. T cells with a given specificity, function, and affinity for a tumor can be selected in vitro and then expanded to achieve in vivo peripheral blood frequencies that are higher than those achieved by current immunization regimens and are consistent with the levels predicted to be required to mediate tumor elimination in murine tumor therapy models [171]. 

In DCs, due to their capability to acquire, process, and present antigens to T cells, they are a critical component of immunization. While immature DCs in peripheral tissues acquire antigens readily, antigen presentation typically results in immunological tolerance due to a lack of costimulatory molecules [172]. Immune tolerance is induced via a variety of methods, including T cell deletion and Treg cell growth [173]. DCs laden with antigens that have been activated (mature) induce the differentiation of antigen-specific T cells into effector T cells with distinct roles and cytokine profiles. DC maturation is associated with a variety of cellular changes, including (1) decreased antigen-capture activity, (2) increased expression of surface MHC class II molecules and costimulatory molecules, (3) acquisition of chemokine receptors such as CCR7 that direct their migration, and (4) the ability to secrete various cytokines that regulate T cell differentiation including IL-12 [174].

The current state of immunotherapy for glioma

DCVax-L® has shown a benign safety profile in the Phase 3 study, as it has consistently been done in prior early-stage trials and a large group of patients. A study by Liau and colleagues showed that only 7 of the 331 Intention-to-treat (ITT) patients experienced any grade 3 or 4 adverse events that were at least possibly related to the treatment. With such a safety profile, DCV looks promising and can potentially be combined with a range of other treatments, including immune checkpoint inhibitors and targeted therapies [175].

A review from Kennedy and colleagues shows that TAMs in glioma are a formidable foe, espousing an altered activation state within the local tumor microenvironment characterized by deficiencies in antitumor effector functions, upregulation of potent immunosuppressive mediators, and participation in tumorigenic loops of paracrine signaling [176]. Given the compelling evidence that TAMs contribute significantly to the creation and maintenance of immunosuppression and tumor progression, it is unlikely that clinically effective immunotherapy against malignant gliomas will be achieved until we gain a better understanding of how to influence TAM function in the local tumor microenvironment [176].

Golan and colleagues conclude that immunotherapy with NK cells seems to be a promising strategy for treating GBM patients. Furthermore, the use of techniques that increase direct cell-to-cell contact between GBM cells and NK cells could potentiate the antitumor effect [177].

Liu and colleagues concluded that there is an association between CTLA-4 expression with clinicopathological findings and IDH mutation status in gliomas. Moreover, CTLA-4 was positively correlated with other immune-related proteins in glioma. Additional studies are needed to further explore the molecular mechanisms mediating CTLA-4 expression in gliomas and responses to anti-CTLA-4 therapy [178].

CAR T-cell therapy has become a revolutionary approach for treating hematological malignancies and it has great potential for brain tumors. Land and colleagues discussed the various targets of CAR T-cell therapy, among which is EGFRvIII [179]. EGFRvIII is the most common EGFR mutation that occurs in about 45% of GBM patients [179]. In vivo, the study showed that CAR T-Cell targeting EGFRvIII improved the survival of the subject animal, as well as reduced the tumor volume. The subject was mice implanted with an EGFRvIII-positive glioblastoma cell line [180].

Limitations and future directions

Multiple therapeutic combination options must be confirmed through clinical research, which would make determining effective therapeutic combinations significantly more difficult and costly as the number of treatments targeting the various aspects of TME increases. To improve high-grade glioma prognosis, novel therapeutics that target multiple TME aspects could be administered alongside standard treatments.

Conclusion

Through a variety of mechanisms, high-grade gliomas are capable of evading immunosurveillance. This extraordinary ability may be one of the reasons behind glioma’s poor prognosis despite regular treatments. Therefore, future efforts to develop novel therapeutics that simultaneously target multiple areas of high-grade glioma-TME interaction may yield better results than the current standard. Novel therapeutics that specifically target glioma’s immune evasion mechanisms are among the most fascinating and promising areas of CNS oncology.

Acknowledgments

Not applicable.

Author contributions

Author contributions to the study and the manuscript preparation. Conception and design, all authors; writing—original draft, MRA, RM, YH, and AF; writing, review, and editing, all authors; supervision, IBIH, RIS, JW, and AF. Funding acquisition, AF. All authors had full access to the data in the study and take responsibility for the integrity of the data. All authors read and approved the final manuscript.

Funding

AF received the Universitas Padjadjaran Academic Leadership Grant, Bandung, Indonesia. RM is an awardee of the Indonesia Endowment Fund for Education (Lembaga Pengelola Dana Pendidikan Republik Indonesia) and, therefore, might receive financial rewards for publishing papers in Scopus-indexed journals.

Availability of data and materials

All data generated or analyzed during this study are included in this published article (and its supplementary information files).

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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