The pediatric immune system has a wide variety of efficient mechanisms in place to detect and destroy foreign pathogens and malignancies that pose a threat to the host. Many cancers, including neuroblastoma, have developed unique abilities to suppress and/or evade the host immune system. The type of tumor and the extent of its infiltration into the immune cells has been associated with prognosis [12,30]. High-risk (HR) neuroblastoma tumors are characterized as immunologically “cold” and devoid of antitumor immune cells and/or have been infiltrated by immune-suppressive T-regulatory cell types rather than being “hot” and thus infiltrated by effector immune cells such as cytotoxic T cells and natural killer (NK) cells, which can kill tumors cells, and dendritic cells (DC), which are antigen-presenting and activate T cells [31,32,33]. HR neuroblastoma tumors have less tumor-infiltrating lymphocytes [34] than low-risk (LR) and medium-risk (MR) tumors [9]. These findings are supported by studies that show that HR tumors display lower levels of tumor infiltrating lymphocytes (TIL), NK and DC genetic signatures; higher levels of T-regulatory cells; and worse patient outcomes compared with LR tumors [35]. Importantly, LR patients, characterized by higher TIL density, have demonstrated higher disease-free survival (DFS), event-free survival (EFS) and overall survival (OS) [7].

Neuroblastoma tumors employ a variety of methods in which they suppress TILs and NK and DC cells while increasing the levels of regulatory T cells (Tregs). The immunosuppressive tumor microenvironment (TME) comprises myeloid-derived suppressor cells (MDSCs), stromal cells, tumor-associated macrophages [8] and neuroblastoma cancer cells [36]. MDSCs induce several immunoregulatory mechanisms including disialoganglioside (GD2), transforming growth factor (TGF-B), and the transcriptional coactivator TAZ (transcriptional coactivator with PDZ-binding motif) [1,36].

GD2 suppresses DC antigen presentation and the expression of MHC-I and MHC-II molecules, rendering neuroblastoma tumor cells invisible to cytotoxic CD8+ T lymphocytes, and repressing the ability of CD4+ helper T lymphocytes to mount an immune response via cytokine signaling inhibition [12,36]. Neuroblastoma tumors also express reduced levels of interferon (IFNγ), interleukin (IL-6), IL-8, IL-10, IL-12, and IL-21 [12,36,37]. Table 1 summarizes few of the cytokines and there interactions with the ICIs. Under normal conditions, these cytokines function by amplifying the host’s immune responses against pathogens and cancerous cells [38]. Their reduction in neuroblastoma tumors impedes TIL activation and infiltration, contributing to the low immunogenicity of the neuroblastoma TME [12,36,37]. Treatments that target GD2, such as the anti-GD2 antibody dinutuximab, have been developed and have undergone phase trials in neuroblastoma patients with promising results [7]. This discovery has marked an important breakthrough in pediatric solid tumor research and represents promising potential for further investigation in immunotherapy treatments in oncology.

Transforming growth factor beta (TGF-β) is responsible for cell growth, proliferation, differentiation, and apoptosis [37] in various cancers. Although it has been shown that TGF-β plays an inhibitory, anti-tumor role in early cancer stages, recent studies have revealed that TGF-β also displays cancer-promoting properties, including the impairment of NK function, which in turn enhances tumorigenesis and metastasis while inducing immunosuppression and drug resistance [12,37]. This makes TGF-β a promising target in the development of novel immunotherapies.

Additionally, TAZ has a major impact on the NB TME’s immunosuppressive environment. Increased TAZ expression induces the expression of immune checkpoint programmed death-1 (PD-1) in both T lymphocytes and NK cells. It has been established that the interaction between programmed death-ligand 1 (PD-L1) and PD1 is linked to decreased lymphocyte proliferation, downregulation of NK function, and the survival of cancer cells [36,39]. Importantly, studies have shown that higher expression of PD-1 and PDL-1 is related to poor prognosis [24]. From a therapeutic point of view, it would be beneficial to silence TAZ expression with Food and Drug Administration-approved drugs that target TAZ signaling [36]. Further, inhibition of the PD-1/PD-L1 immunomodulatory checkpoints molecule (ICMS) mechanism has been used to successfully treat some chemotherapy-resistant cancers in adults [36]. Neuroblastoma cells may not be recognized by CTL due to their poor MHC-I expression, but they should be vulnerable to NK cells as a result [40,41]. NK cells can kill neuroblastoma cells, though in some circumstances pre-activation of isolated NK cells is necessary [42]. Nonetheless, neuroblastoma appears to be shielded from NK-mediated death in patients by additional escape mechanisms that adjust the harmony between activating and inhibitory impulses on NK cells. For instance, the ligands (PVR, nectin-2, MICA, MICB, and ULBPs) for the NK cell activating receptors DNAM-1 and NKG2D are not expressed at high levels in neuroblastoma tumors. Thus, NK cell therapies may prove useful in both stand-alone and combination treatments for NB [43].

As previously mentioned, the combined immunosuppressive mechanisms of neuroblastoma cancer cells create an immunologically “cold” environment devoid of activated T lymphocytes and NK cells, with T-regulatory cells upregulated. Under normal circumstances, NK cells and cytotoxic T cells play a prominent role in preventing tumor progression and metastasis by inducing tumor cell lysis and inflammation [12]. T-regulatory (Tregs) cells function to modulate the immune system by inhibiting other immune effector cells to prevent the over activation of the immune system. The overexpression of Tregs further inhibits the already immunologically inactive TME by suppressing NK and cytotoxic T cell functioning [51,52]. This is especially seen in high-risk neuroblastoma tumors where there are increasingly higher levels of immunosuppressive stromal cancer cells, MDSCs and Tregs [36,52].

Immunotherapy can fall into two different types (Figure 1), one being active immunotherapy and the other being passive. Active immunotherapy relies on directly attacking cancer cells through the stimulation of the immune system [53]. Passive immunotherapy utilizes the acceptance of an organism’s immune system of antibodies, cytokines, and transformed immune cells to act directly on tumor cells [54]. Immunotherapy treatments that can target neuroblastoma cells have been in development over the course of recent decades.

Cancer immune surveillance is a mechanism by which immune cells recognize and eliminate tumor cells. Neuroblastomas produce a highly immunosuppressive environment that has an effect on the body as well as locally in the tumor. Neuroblastomas employ indirect immunoregulatory mechanisms by chartering immune suppressive agents to dampen the activity of immune system. TGF, galectin-1, MIF, soluble GD2 (sGD2), and arginase-2 are some of the soluble mediators that neuroblastoma cells produce, and which have the ability to inhibit lymphocyte activation [55]. Neuroblastoma cancer cells also produce several other immunosuppressive molecules such as, sMICA, sB7-H6, sHLA-E, sHLA-G, IL-10 and HMGB1 [56]. Neuroblastoma tumors are intermixed with myeloid and stromal cell populations with defective activating functions or enhanced suppressive functions, which can prevent TIL from effectuating an anti-tumor response.

One of the areas in which immunotherapies have become focused is that associated with the passive invocation of the immune system to respond to a tumor in ways that would not occur without outside intervention. These therapies target the activation of immune cells such as T-cells to assist in the aim to provide an anti-tumor effect through the infiltration of the immune cells in the tumor. High-risk neuroblastoma with higher T cell infiltration has been associated with improved survival [57]. Increasing the levels of T cell infiltration into the tumor cells must be a goal for the improvement of patient outcomes.

A form of passive treatment for immunotherapy that has shown promising results is the use of monoclonal antibodies (mAbs). One of these immune therapies focuses on the use of mAbs in order to recognize GD2, which is overexpressed relative to a control in neuroblastoma. GD2 is a disialoganglioside that is ubiquitously expressed on the surface of all neuroblastoma cells (source) [29]. This makes GD2 an appealing target due to its specificity for the disease. Adoption of mAbs such as anti-GD2 in both upfront and relapse treatment protocols has dramatically increased survival rates and altered the landscape for children with high-risk neuroblastoma [28]. Even in patients with relapsed disease, dinutuximab may have a treatment value [58]. The success of dinutuximab has sparked investigations into combination therapy with cytotoxic compounds, as well as cellular immunotherapy with (haploidentical) donor NK cells [59,60,61].

Recent technological advancements have made it feasible to analyze the immune response to patient-specific neoantigens that result from tumor-specific mutations. New evidence suggests that clinical immunotherapies may be more effective when these neoantigens are recognized. However, children probably have few, if any, actionable, mutation-generated, immunogenic tumor neoantigens because most juvenile cancers have a very low tumor mutation burden [62,63]. Cancer-testis antigens, as well as other embryonic or differentiation antigens expressed during development and on children’s cancers but not on normal postnatal tissues, may fall under this category [64]. Studies have shown a correlation between the use of these neoantigens and an increase in T cell activity [65]. Recently researchers have identified PHOX2B, a peptide displayed on the surface of neuroblastoma cells, by their MHC molecules [66]. The oncofetal proteins, expressed during embryonic development, though silenced after birth, may be ideal targets for T cell-based immunotherapies in NB due to their restricted expression outside of the tumor. While the comparatively low tumor mutational burden of neuroblastoma may limit neoantigen expression, developmental antigens may be the target of T cell-based immunotherapy. Given the high sensitivity and efficacy of some of these receptors, as has previously been seen with certain modified TCRs, researchers will need to be cautious of the possibility for antigen cross-reactivity. By administering immunomodulating agents that improve the tumor’s immunogenicity, stimulate antigen presentation, increase the patient’s endogenous tumor-reactive T cells, and suppress the tumor’s immunosuppressive microenvironment, it may also be possible to trigger endogenous tumor-reactive T cells in a patient [67,68,69,70]. Tumor-specific neoantigens lead to personalized immunotherapies for patients. Future development of technology involved in the recognition of these neoantigens can help provide an efficient and rapid response.

Cytokines released by cancer cells or cells in the tumor microenvironment support angiogenesis, tumor cell migration and metastasis, and the development of an immunosuppressive microenvironment. These tumor-promoting effects of cytokines also apply to NB, as depicted in Table 2. IL-6 and VEGF have been further characterized as cytokines that stimulate tumor growth and metastasis, while others, such as IFN-γ, can exert anti-NB activity by inducing tumor cell apoptosis and by inhibiting angiogenesis (Figure 2 and Table 2).