*2.2. GBM Microenvironment: Local Immunosuppressive Mechanisms*

Glioblastoma arises in the central nervous system (CNS) [36], which is an immunologically distinct site. In the past, the CNS was considered an immune privileged site, due to its unique properties. For instance, the blood brain barrier, which tightly regulates the transportation of the immune cells from the periphery to the CNS; the lack of antigen presenting cells in a non-inflamed state; and more importantly the lack of a classic lymphatic system [37–39]. The concept of CNS being immune privileged has now been revised. Recent studies have shown that antigens derived from the CNS can efficiently elicit an immune response [40]. More importantly, Louveau et al. [41] discovered a functional lymphatic system, parallel to the dural sinuses, a possible route of transportation of antigen-presenting

cells to the deep cervical lymph nodes, where they can present CNS-derived antigens and prime T cells. These recent studies have provided evidence that CNS-derived antigens can mount a vigorous immune response, offering ground to investigate immunotherapy approaches for GBM.

The GBM environment is characterized by the high influx of tumor-associated macrophages (TAMs). In a non-inflamed state, the myeloid composition of the CNS consists of the tissue-resident macrophages that arise from the yolk sac, the microglia [42]. However, in GBM, the microenvironment is comprised mainly of a mixture of microglia and infiltrating monocytes from the periphery. Glioma cells produce a milieu of monocyte chemoattractant proteins along with other factors, leading to disruption of the blood-brain barrier and facilitating recruitment of monocytes from the periphery [43]. When monocytes arrive at the tumor site, glioma cells drive their polarization to an immunosuppressive M2 phenotype [44,45]. These M2-like TAMs promote tumor growth and migration as well as the immune invasion by hampering the adaptive immunity [44,46,47]. TAMs are the most abundant immune cell population in GBM and can consist up to 50% of the GBM tumor mass. Their importance in tumor growth is highlighted by the correlation between increased TAM numbers and worse prognosis in GBM patients; furthermore, TAM infiltration has been associated with the mesenchymal subtype of GBM, being the most aggressive one [48,49].

Another feature that facilitates the local immune suppression in GBM is T cell dysfunction. Severe T cell exhaustion is observed in GBM, which is characterized by upregulation of expression of co-inhibitory molecules like PD-1, LAG-3 and TIM-3 [50]. Furthermore, an increase in numbers of the regulatory T cells (Tregs), which can suppress the antigenspecific T cells, was found in high grade gliomas compared to low grade gliomas [51]. The recruitment of Tregs at the tumor site is mainly facilitated by the production of the attractant indoleamine 2,3 dioxygenase (IDO) by gliomas [52]. Another facet that contributes to the ''cold" tumor microenvironment is the relatively low mutational burden of GBM cells, associated with limited expression of neoantigens [53,54]. Taken together, GBM has all the characteristics of a tumor with low immunogenicity. The M2-like macrophages that are abundant at the tumor site, the dysfunctional T cells and the low neoantigen expression are some of the barriers that we need to overcome to design successful immunotherapies.

Considering all of the above, a therapeutic strategy that is not hindered by specificity for a single molecular target or differentiation state of tumor cells, that is delivered locally in a single surgical intervention, hence bypassing the BBB, that is self-perpetuating in its antitumor activity, and which can overcome the immune-suppressive tumor microenvironment, may offer opportunities for achieving therapeutic responses in glioblastoma patients. Oncolytic viruses offer such a treatment strategy.
