**5. Immunotherapy for malignant gliomas**

Our immune system can be viewed as an intricate balance of opposing functions that lead to either immunity or tolerance. Perturbations that disrupt this stable equilibrium could lead to autoimmune disease or tolerance to malignant cells. In general, the immune system has the ability to recognize and to react to foreign antigens, which leads to their removal as well as to the destruction of cells that express them. Before attempting immunotherapy for cancer, one needs to understand the crucial balancing acts of the immune response that eventually lead to a desired outcome; in addition, the central nervous system has unique features that require special considerations.

In this section, our goals are to introduce the readers to the basics of peripheral immunology focusing on how foreign antigens activate the immune response leading to immunity vs. tolerance. Nevertheless, a detailed discussion of immunity is not within our scope; in some disciplines, we will just be scratching the surface. We will detail antigen processing and presentation, T cell priming, with attention to the synapse between T cells and the antigenpresenting cell (APC) and clonal expansion. Because of our interest in brain tumors, we will compare the systemic immune response to that of the CNS, discussing the historical thoughts of the immune privileges of the CNS and more recent evidence of processing of CNS antigens via the glymphatic pathway. The stage will be set for a discussion of immunotherapy for brain tumors, including priming in the periphery, priming in the CNS, and passive transfer of immunity. The last section lists the clinical trials that employ immunotherapy for brain tumors and their proposed modes of action.

(Joffre, Segura et al. 2012). This cross-priming process has been implicated in immune re‐ sponses not only to infection, but also to cancer and autoimmune disease (Jarry, Jeannin et al.

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T cell priming by DCs induces activation, cytokine secretion, and clonal proliferation (Mempel, Henrickson et al. 2004). For T-cell activation, both the MHC-bound antigen and the MHC itself must be recognized by the T-cell receptor (TCR) and the co-receptor, respectively (Abbas, Lichtman et al. 2014). This process of priming naïve T cells into effector and helper cells occurs in lymphoid organs (Joffre, Segura et al. 2012). DCs prime the T cells during three stages: 1. Contact for exchange of information between the T cell and the dendrite in the lymphocyte pool, 2. The formation of a stable bond followed by secretion of interleukin-2 and interferon-

The synapse between the T cell and the APC requires the interaction of not only the TCR and MHC, but also adhesion molecules and co-receptors to receive signals from the APC (see section 5.1.3 below). Early on during this process various cytokines are released. Certain cytokines lead to clonal expansion of antigen-specific lymphocytes, some of which become differentiated into effector T cells that can remove infected cells. Others differentiate into memory T cells that serve to remain inactive until re-exposed to the same antigen (Abbas, Lichtman et al. 2014). During future encounters, DC-bearing antigens will migrate to the paracortical region in the lymph node to search for a T cell that recognizes its antigen,

To begin the process of priming T cells, the DC must physically contact the naïve T cell. This process tends to occur in lymphoid tissue, specifically in the draining lymph nodes, spleen, and Peyer's patches, after infiltration of antigen-APC complexes from peripheral tissue through lymph vessels (Mempel, Henrickson et al. 2004; Hugues 2010). Mempel et al. showed that naïve T cells re-circulate continually between the blood and lymph nodes searching for antigen. In the absence of antigen, the T cells move randomly in the three dimensions in a stopand-go manner leading to approximately 500-5000 T cells contacting one DC per hour (Mempel, Henrickson et al. 2004; Miller, Hejazi et al. 2004; Hugues 2010). In the absence of antigen, DCs enter the lymph node via the sub-capsular cortex and travel to the paracortex where T cells are localized. Then, the dendrites on DCs scan the T cells which results in transient interactions of up to a few minutes (Hugues 2010). In the presence of antigen, the data supports a three-phase model. First, within a few hours of lymph node entry, contact between naïve T cell-DC with peptide increases in duration, now lasting up to five minutes. Within ten hours of antigen entry into the lymph node, mobility slows dramatically as T cells and DCs form more stable bonds, which will persist from two to twenty four hours. This step also promotes the up-regulation of activation markers. After thirty hours, the bonds separate and this is followed by increased mobility of T cells, corresponding with T cell proliferation (Miller, Wei

γ, and 3. Rapid movement and clonal expansion (Mempel, Henrickson et al. 2004).

ultimately activating clonal expansion (Bousso, 2003).

*5.1.2.1. T lymphocyte — Antigen presenting cell contact*

et al. 2002; Mempel, Henrickson et al. 2004; Hugues 2010).

2013)

*5.1.2. T cell priming and activation*

#### **5.1. Peripheral immunology**

As part of the adaptive immune system, antigens enter the body through epithelium and are immediately met in the infected tissue by APCs, most commonly dendritic cells (DCs), which then process the antigens into protein fragments (Hugues 2010; Joffre, Segura et al. 2012; Abbas, Lichtman et al. 2014). DCs are a type of APC that can induce priming of naïve CD4+ and CD8+ T cells into helper and cytotoxic T cells through a series of steps that include antigen processing, antigen presentation, and interactions with co-stimulatory molecules, in addition to the secretion of various cytokines (Hivroz, Chemin et al. 2012). If not processed locally by an APC, the antigens drain into lymph nodes via lymphatic vessels where an APC will be waiting (Abbas, Lichtman et al. 2014). These antigens are processed internally through degradation in the cytosol, processing in the endoplasmic reticulum, and transportation to the cell surface by the Golgi apparatus (Joffre, Segura et al. 2012). APCs then travel to the lymph nodes, where naïve T cells can recognize displayed protein fragments of antigens (Abbas, Lichtman et al. 2014). DCs serve as the most specialized of the APCs and assist in differentiating naïve T cells into both effector and memory cells. Once activated, effector cells then travel via the blood stream to the site of infection where they can recognize antigens being presented by other types of cells and initiate cytotoxic responses (Abbas, Lichtman et al. 2014).

All nucleated cells in the body display a major histocompatibility complex (MHC) I molecule for presenting processed pathogens or infected cells to T lymphocytes once detected (Joffre, Segura et al. 2012). Only CD8+ T cells bear receptors for MHC I; CD4+ T cells bear receptors for MHC II, typically expressed by dendritic cells, macrophages, and B cells (Abbas, Lichtman et al. 2014). Nucleated cells produce peptide antigens from viruses living in the cell, phago‐ cytosed and endocytosed organisms, and proteins derived from mutated self-genes (Joffre, Segura et al. 2012; Abbas, Lichtman et al. 2014). Traditionally, exogenous antigens are pre‐ sented via MHC II-bearing cells and endogenous antigens by MHC I cells, but cross-presen‐ tation permits MHC I cells to present exogenous antigens (Jarry, Jeannin et al. 2013). Additionally, DCs can ingest virally-infected host cells and present the processed antigens via MHC I to CD8+ naïve T cells through cross-priming (Abbas, Lichtman et al. 2014). Similarly, infected DCs can prime CD8+ T cells via MHC I by directly presenting the processed antigen (Joffre, Segura et al. 2012). This cross-priming process has been implicated in immune re‐ sponses not only to infection, but also to cancer and autoimmune disease (Jarry, Jeannin et al. 2013)

#### *5.1.2. T cell priming and activation*

In this section, our goals are to introduce the readers to the basics of peripheral immunology focusing on how foreign antigens activate the immune response leading to immunity vs. tolerance. Nevertheless, a detailed discussion of immunity is not within our scope; in some disciplines, we will just be scratching the surface. We will detail antigen processing and presentation, T cell priming, with attention to the synapse between T cells and the antigenpresenting cell (APC) and clonal expansion. Because of our interest in brain tumors, we will compare the systemic immune response to that of the CNS, discussing the historical thoughts of the immune privileges of the CNS and more recent evidence of processing of CNS antigens via the glymphatic pathway. The stage will be set for a discussion of immunotherapy for brain tumors, including priming in the periphery, priming in the CNS, and passive transfer of immunity. The last section lists the clinical trials that employ immunotherapy for brain tumors

As part of the adaptive immune system, antigens enter the body through epithelium and are immediately met in the infected tissue by APCs, most commonly dendritic cells (DCs), which then process the antigens into protein fragments (Hugues 2010; Joffre, Segura et al. 2012; Abbas, Lichtman et al. 2014). DCs are a type of APC that can induce priming of naïve CD4+ and CD8+ T cells into helper and cytotoxic T cells through a series of steps that include antigen processing, antigen presentation, and interactions with co-stimulatory molecules, in addition to the secretion of various cytokines (Hivroz, Chemin et al. 2012). If not processed locally by an APC, the antigens drain into lymph nodes via lymphatic vessels where an APC will be waiting (Abbas, Lichtman et al. 2014). These antigens are processed internally through degradation in the cytosol, processing in the endoplasmic reticulum, and transportation to the cell surface by the Golgi apparatus (Joffre, Segura et al. 2012). APCs then travel to the lymph nodes, where naïve T cells can recognize displayed protein fragments of antigens (Abbas, Lichtman et al. 2014). DCs serve as the most specialized of the APCs and assist in differentiating naïve T cells into both effector and memory cells. Once activated, effector cells then travel via the blood stream to the site of infection where they can recognize antigens being presented by other types

All nucleated cells in the body display a major histocompatibility complex (MHC) I molecule for presenting processed pathogens or infected cells to T lymphocytes once detected (Joffre, Segura et al. 2012). Only CD8+ T cells bear receptors for MHC I; CD4+ T cells bear receptors for MHC II, typically expressed by dendritic cells, macrophages, and B cells (Abbas, Lichtman et al. 2014). Nucleated cells produce peptide antigens from viruses living in the cell, phago‐ cytosed and endocytosed organisms, and proteins derived from mutated self-genes (Joffre, Segura et al. 2012; Abbas, Lichtman et al. 2014). Traditionally, exogenous antigens are pre‐ sented via MHC II-bearing cells and endogenous antigens by MHC I cells, but cross-presen‐ tation permits MHC I cells to present exogenous antigens (Jarry, Jeannin et al. 2013). Additionally, DCs can ingest virally-infected host cells and present the processed antigens via MHC I to CD8+ naïve T cells through cross-priming (Abbas, Lichtman et al. 2014). Similarly, infected DCs can prime CD8+ T cells via MHC I by directly presenting the processed antigen

of cells and initiate cytotoxic responses (Abbas, Lichtman et al. 2014).

and their proposed modes of action.

186 Tumors of the Central Nervous System – Primary and Secondary

**5.1. Peripheral immunology**

T cell priming by DCs induces activation, cytokine secretion, and clonal proliferation (Mempel, Henrickson et al. 2004). For T-cell activation, both the MHC-bound antigen and the MHC itself must be recognized by the T-cell receptor (TCR) and the co-receptor, respectively (Abbas, Lichtman et al. 2014). This process of priming naïve T cells into effector and helper cells occurs in lymphoid organs (Joffre, Segura et al. 2012). DCs prime the T cells during three stages: 1. Contact for exchange of information between the T cell and the dendrite in the lymphocyte pool, 2. The formation of a stable bond followed by secretion of interleukin-2 and interferonγ, and 3. Rapid movement and clonal expansion (Mempel, Henrickson et al. 2004).

The synapse between the T cell and the APC requires the interaction of not only the TCR and MHC, but also adhesion molecules and co-receptors to receive signals from the APC (see section 5.1.3 below). Early on during this process various cytokines are released. Certain cytokines lead to clonal expansion of antigen-specific lymphocytes, some of which become differentiated into effector T cells that can remove infected cells. Others differentiate into memory T cells that serve to remain inactive until re-exposed to the same antigen (Abbas, Lichtman et al. 2014). During future encounters, DC-bearing antigens will migrate to the paracortical region in the lymph node to search for a T cell that recognizes its antigen, ultimately activating clonal expansion (Bousso, 2003).

#### *5.1.2.1. T lymphocyte — Antigen presenting cell contact*

To begin the process of priming T cells, the DC must physically contact the naïve T cell. This process tends to occur in lymphoid tissue, specifically in the draining lymph nodes, spleen, and Peyer's patches, after infiltration of antigen-APC complexes from peripheral tissue through lymph vessels (Mempel, Henrickson et al. 2004; Hugues 2010). Mempel et al. showed that naïve T cells re-circulate continually between the blood and lymph nodes searching for antigen. In the absence of antigen, the T cells move randomly in the three dimensions in a stopand-go manner leading to approximately 500-5000 T cells contacting one DC per hour (Mempel, Henrickson et al. 2004; Miller, Hejazi et al. 2004; Hugues 2010). In the absence of antigen, DCs enter the lymph node via the sub-capsular cortex and travel to the paracortex where T cells are localized. Then, the dendrites on DCs scan the T cells which results in transient interactions of up to a few minutes (Hugues 2010). In the presence of antigen, the data supports a three-phase model. First, within a few hours of lymph node entry, contact between naïve T cell-DC with peptide increases in duration, now lasting up to five minutes. Within ten hours of antigen entry into the lymph node, mobility slows dramatically as T cells and DCs form more stable bonds, which will persist from two to twenty four hours. This step also promotes the up-regulation of activation markers. After thirty hours, the bonds separate and this is followed by increased mobility of T cells, corresponding with T cell proliferation (Miller, Wei et al. 2002; Mempel, Henrickson et al. 2004; Hugues 2010).

#### *5.1.2.2. Naïve T lymphocyte — Antigen presenting cell synapse*

Naïve T cells are constantly searching for presented antigen on the MHC-antigen complex of mature DCs, from which the T cell and its receptor will require co-stimulation (Mempel, Henrickson et al. 2004). T cell activation relies on the successful synapse of the T cell receptor (TCR) with the peptide-MHC complex on the APC. Additionally, several signaling complexes must connect between the TCR and the adaptor protein linker for activation of T cells and subsequent filamentous actin (F-actin)-dependent TCR cluster formation (Dustin and Depoil 2011). The role of co-stimulatory and co-inhibitory proteins is to modulate the TCR signal to increase or decrease activation of the T cell or to direct the response of that cell down a particular differentiation pathway (Dustin and Depoil 2011).

receptors. IL-2, by acting on the T cell that secreted it, supports the production of T cells specific to the antigen. IL-2 is also needed to maintain regulatory T cells (Abbas, Lichtman et al. 2014).

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Clonal expansion transpires in 1-2 days, leading to the creation of antigen-specific CD4+ and CD8+ cells. CD8+ cells develop into effector cells that ultimately migrate to the site of infection to interact with the specific antigen to which they were primed. CD4+ cells further develop into T helper I (Th1) and T helper 2 (Th2) lymphocytes. Antigen-exposed macrophages and DCs release IL-12 and natural killer cells secrete interferon-γ, thereby promoting the differ‐ entiation of Th 1 cells (Abbas, Lichtman et al. 2014). Th1 cells secrete IL-2, interferon-γ, and lymphotoxin-a which leads to type 1 immunity with enhanced macrophage activation and phagocytosis (Spellberg and Edwards 2001). Interferon-γ also increases the expression of MHC I and II molecules to amplify antigen presentation (Spellberg and Edwards 2001). Th2 cells, stimulated by IL-4, also release IL-4, IL-5, IL-9, IL-10, and IL-13 promoting production of antibodies and type 2 immunity, which minimizes phagocytosis and decreases inflammation (Spellberg and Edwards 2001). In times of overwhelming systemic response or immunosup‐ pression, a type 2 response can supersede the appropriate type 1 response (Spellberg and

All the aforementioned steps have to be executed flawlessly to achieve immunity against a tumor or a tumor antigen. The body is set up to have low affinity to self-antigens, as would be expressed on tumor cells (Luptrawan, Liu et al. 2008). Along those lines, dendritic cells, which have been discovered in tumors, play a large role in presentation of tumor antigens; however, that does not necessarily predict the nature of the immune response. In particular, any flaw in dendritic cells, from cross presentation to IL-12 production, will lead to tolerance and impaired

The CNS was once thought to be immunologically-privileged because of its unique immuno‐ logical features, including 1) lack of immunological surveillance due to low expression of MHC molecules, 2) lack of distinct lymphatic drainage, and 3) protection by the blood brain barrier (BBB), which limits the movement of naïve T cells into the CNS (Chavarria and Cardenas 2013). Nonetheless, the CNS has more recently been discovered to have a finely tuned immune surveillance managed by APC, believed to be microglia, DC, perivascular macrophages and meningeal dendritic cells (Fathallah-Shaykh, Gao et al. 1998; Yang, Han et al. 2010; D'Agostino, Gottfried-Blackmore et al. 2012; Ousman and Kubes 2012; Romo-Gonzalez, Chavarria et al. 2012; Chavarria and Cardenas 2013). Furthermore, more recent evidence, as can be found in gliomas and multiple sclerosis, suggests that the CNS microglia coordinate with peripheral T cells and APC (Yang, Han et al. 2010). Such evidence describes more active inspection of the BBB in specific regions of the brain most notably the meninges, ventricles, circumventricular

organs, and choroid plexus (D'Agostino, Gottfried-Blackmore et al. 2012).

CD8+ T cell response to tumors (Joffre, Segura et al. 2012).

**5.2. Central nervous system immunology**

Edwards 2001).

Antigen recognition and adhesion involves simultaneous recognition of many molecules. In the receptor layer, antigen recognition occurs by the TCR to the peptide with its co-receptor CD4 or CD8 binding MHC II or MHC I, respectively. This is the first step in the signal cascade. For signal transduction and co-stimulation, several transmembrane signaling molecules, including CD3 and ζ chain, form part of the TCR complex and bind the MHC/antigen complex. Additionally, CD28 (and CTLA-4) on the T cell binds B7-1 (CD80)/B7-2 (CD86) on the APC (Dustin and Depoil 2011). The B7 proteins are created by APC in response to an antigen to ensure that T cells are not activated by self-antigens. This key bond is essential for signaling and thus activation of naïve T cells. Concurrently, the CD40 Ligand on the T cell and CD40 on the APC unite and promote increased production of B7 and secretion of cytokines in the APC in order to further encourage T cell activation (Abbas, Lichtman et al. 2014). For adhesion, the T-cell integrin LFA-1 (Leukocyte function-associated antigen 1) binds ICAM-1 (Intercellular adhesion molecule) on the APC (Dustin and Depoil 2011; Abbas, Lichtman et al. 2014).

The co-stimulatory signals play a key role in determining immunity or tolerance. Due to the required co-receptors and signal transduction, many mechanisms are set in place to prevent T cells from activating against self-protein. Through early central tolerance mechanisms designed to prevent autoimmune disease, immature T cells that react to self-proteins undergo apoptosis early in development (Luptrawan, Liu et al. 2008). This activation-induced cell death is assisted through the interaction of Fas, which is expressed everywhere and in high concen‐ tration in the thymus, with Fas Ligand on T lymphocytes and NK cells (Maher, Toomey et al. 2002); a similar process results in clonally expanded T cells after they are no longer needed. Similarly, if a T cell encounters an antigen on an APC without the appropriate co-stimulation, it is susceptible to developing tolerance to that antigen such that on future encounters it will ignore it, even if given the appropriate co-stimulation (Luptrawan, Liu et al. 2008; Abbas, Lichtman et al. 2014). On cross-presentation by dendritic cells with MHC I and CD8+ T cells, clonal deletion, functional inactivation (anergy) or programming into a suppressive (regula‐ tory) T cell phenotype can result (Joffre, Segura et al. 2012).

#### *5.1.2.3. Clonal expansion*

To amplify activation, T cells and APCs secrete various cytokines. Initially, T cells secrete interleukin-2 (IL-2), which facilitates the binding of IL-2 by augmenting the presence of IL-2 receptors. IL-2, by acting on the T cell that secreted it, supports the production of T cells specific to the antigen. IL-2 is also needed to maintain regulatory T cells (Abbas, Lichtman et al. 2014).

Clonal expansion transpires in 1-2 days, leading to the creation of antigen-specific CD4+ and CD8+ cells. CD8+ cells develop into effector cells that ultimately migrate to the site of infection to interact with the specific antigen to which they were primed. CD4+ cells further develop into T helper I (Th1) and T helper 2 (Th2) lymphocytes. Antigen-exposed macrophages and DCs release IL-12 and natural killer cells secrete interferon-γ, thereby promoting the differ‐ entiation of Th 1 cells (Abbas, Lichtman et al. 2014). Th1 cells secrete IL-2, interferon-γ, and lymphotoxin-a which leads to type 1 immunity with enhanced macrophage activation and phagocytosis (Spellberg and Edwards 2001). Interferon-γ also increases the expression of MHC I and II molecules to amplify antigen presentation (Spellberg and Edwards 2001). Th2 cells, stimulated by IL-4, also release IL-4, IL-5, IL-9, IL-10, and IL-13 promoting production of antibodies and type 2 immunity, which minimizes phagocytosis and decreases inflammation (Spellberg and Edwards 2001). In times of overwhelming systemic response or immunosup‐ pression, a type 2 response can supersede the appropriate type 1 response (Spellberg and Edwards 2001).

All the aforementioned steps have to be executed flawlessly to achieve immunity against a tumor or a tumor antigen. The body is set up to have low affinity to self-antigens, as would be expressed on tumor cells (Luptrawan, Liu et al. 2008). Along those lines, dendritic cells, which have been discovered in tumors, play a large role in presentation of tumor antigens; however, that does not necessarily predict the nature of the immune response. In particular, any flaw in dendritic cells, from cross presentation to IL-12 production, will lead to tolerance and impaired CD8+ T cell response to tumors (Joffre, Segura et al. 2012).

#### **5.2. Central nervous system immunology**

*5.1.2.2. Naïve T lymphocyte — Antigen presenting cell synapse*

188 Tumors of the Central Nervous System – Primary and Secondary

particular differentiation pathway (Dustin and Depoil 2011).

tory) T cell phenotype can result (Joffre, Segura et al. 2012).

*5.1.2.3. Clonal expansion*

Naïve T cells are constantly searching for presented antigen on the MHC-antigen complex of mature DCs, from which the T cell and its receptor will require co-stimulation (Mempel, Henrickson et al. 2004). T cell activation relies on the successful synapse of the T cell receptor (TCR) with the peptide-MHC complex on the APC. Additionally, several signaling complexes must connect between the TCR and the adaptor protein linker for activation of T cells and subsequent filamentous actin (F-actin)-dependent TCR cluster formation (Dustin and Depoil 2011). The role of co-stimulatory and co-inhibitory proteins is to modulate the TCR signal to increase or decrease activation of the T cell or to direct the response of that cell down a

Antigen recognition and adhesion involves simultaneous recognition of many molecules. In the receptor layer, antigen recognition occurs by the TCR to the peptide with its co-receptor CD4 or CD8 binding MHC II or MHC I, respectively. This is the first step in the signal cascade. For signal transduction and co-stimulation, several transmembrane signaling molecules, including CD3 and ζ chain, form part of the TCR complex and bind the MHC/antigen complex. Additionally, CD28 (and CTLA-4) on the T cell binds B7-1 (CD80)/B7-2 (CD86) on the APC (Dustin and Depoil 2011). The B7 proteins are created by APC in response to an antigen to ensure that T cells are not activated by self-antigens. This key bond is essential for signaling and thus activation of naïve T cells. Concurrently, the CD40 Ligand on the T cell and CD40 on the APC unite and promote increased production of B7 and secretion of cytokines in the APC in order to further encourage T cell activation (Abbas, Lichtman et al. 2014). For adhesion, the T-cell integrin LFA-1 (Leukocyte function-associated antigen 1) binds ICAM-1 (Intercellular adhesion molecule) on the APC (Dustin and Depoil 2011; Abbas, Lichtman et al. 2014).

The co-stimulatory signals play a key role in determining immunity or tolerance. Due to the required co-receptors and signal transduction, many mechanisms are set in place to prevent T cells from activating against self-protein. Through early central tolerance mechanisms designed to prevent autoimmune disease, immature T cells that react to self-proteins undergo apoptosis early in development (Luptrawan, Liu et al. 2008). This activation-induced cell death is assisted through the interaction of Fas, which is expressed everywhere and in high concen‐ tration in the thymus, with Fas Ligand on T lymphocytes and NK cells (Maher, Toomey et al. 2002); a similar process results in clonally expanded T cells after they are no longer needed. Similarly, if a T cell encounters an antigen on an APC without the appropriate co-stimulation, it is susceptible to developing tolerance to that antigen such that on future encounters it will ignore it, even if given the appropriate co-stimulation (Luptrawan, Liu et al. 2008; Abbas, Lichtman et al. 2014). On cross-presentation by dendritic cells with MHC I and CD8+ T cells, clonal deletion, functional inactivation (anergy) or programming into a suppressive (regula‐

To amplify activation, T cells and APCs secrete various cytokines. Initially, T cells secrete interleukin-2 (IL-2), which facilitates the binding of IL-2 by augmenting the presence of IL-2

The CNS was once thought to be immunologically-privileged because of its unique immuno‐ logical features, including 1) lack of immunological surveillance due to low expression of MHC molecules, 2) lack of distinct lymphatic drainage, and 3) protection by the blood brain barrier (BBB), which limits the movement of naïve T cells into the CNS (Chavarria and Cardenas 2013). Nonetheless, the CNS has more recently been discovered to have a finely tuned immune surveillance managed by APC, believed to be microglia, DC, perivascular macrophages and meningeal dendritic cells (Fathallah-Shaykh, Gao et al. 1998; Yang, Han et al. 2010; D'Agostino, Gottfried-Blackmore et al. 2012; Ousman and Kubes 2012; Romo-Gonzalez, Chavarria et al. 2012; Chavarria and Cardenas 2013). Furthermore, more recent evidence, as can be found in gliomas and multiple sclerosis, suggests that the CNS microglia coordinate with peripheral T cells and APC (Yang, Han et al. 2010). Such evidence describes more active inspection of the BBB in specific regions of the brain most notably the meninges, ventricles, circumventricular organs, and choroid plexus (D'Agostino, Gottfried-Blackmore et al. 2012).

#### *5.2.1. Centrally-acting peripheral immune cells*

In addition to resident microglia, the primary immune cell in the CNS, peripheral immune cells including peripherally activated T lymphocytes, macrophages, and DC circulate in small numbers within the CNS. They are predominantly in specialized CNS compartments located outside the parenchyma with ability to gain access to the parenchyma through various mechanisms that include the choroid plexus, perivascular or Virchow-Robin spaces, menin‐ geal vessel branch points into the subarachnoid space, and through post-capillary venules (Ousman and Kubes 2012). As in the periphery, these cells are capable of mounting an activated immune response if they encounter an antigen (Ousman and Kubes 2012; Jarry, Jeannin et al. 2013). Additionally, perivascular macrophages sample CSF and can phagocytose suspected antigens (Ousman and Kubes 2012). There is also separate evidence of drainage of CNS antigens to deep cervical lymph nodes, based on intracranial injection of labeled antigen (D'Agostino, Gottfried-Blackmore et al. 2012; Ousman and Kubes 2012). Despite controversy over poor immune surveillance due to low expression of MHC II, it is thought that preactivated T cells can release IFN- γ and TNF-α to simulate MHC II molecule expression (Romo-Gonzalez, Chavarria et al. 2012). Also of debate is the function of central antigen presentation by central DCs. It is known that the integrity of the BBB is compromised during times of infection, trauma, aging, and autoimmunity due to weakening of the vascular endothelium as a result of cytokine release by astrocytes and microglia (D'Agostino, Gottfried-Blackmore et al. 2012; Romo-Gonzalez, Chavarria et al. 2012).

cell apoptosis. Microglia also express FAS molecules themselves, which induce apoptosis upon binding FASL (Yang, Han et al. 2010). Nitric oxide released by microglia in response to activation can also potentiate effector cell death (Yang, Han et al. 2010). Furthermore, microglia display B7-H1 molecules which also support immunosuppression by stimulating T cell apoptosis (Yang, Han et al. 2010). Additionally, glycoprotein CD200 down regulates activated microglia (via CD200 ligand on microglia) and perivascular macrophages in the CNS, acting as an anti-inflammatory and serving to keep microglia in a quiescent state (Ousman and Kubes

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The CNS lacks lymphoid tissue. For appropriate immune surveillance, both antigens and APC must be able to travel to lymphoid tissue, ideally via lymphatic channels, for T cell priming (Romo-Gonzalez, Chavarria et al. 2012). For small and hydrophobic molecules as well as transporter substrates, exit through the BBB is easy. Other substances are cleared from CSF through arachnoid granulations or peripheral lymphatics on cranial nerves. Clearance of large particles and matter deep within the parenchyma is more difficult and is ascribed to a high rate of flow of interstitial fluid (Iliff and Nedergaard 2013). This flow of fluid transports antigens from the brain parenchyma for presentation in cervical lymph nodes (Romo-Gonzalez, Chavarria et al. 2012). Through what has been termed the glio-vascular or glym‐ phatic pathway, interstitial solutes are cleared from the brain to the peripheral lymphatic system via perivascular water channels from the para-arterial CSF influx pathway through the interstitium and along the para-venous clearance route (Iliff and Nedergaard 2013). Once filtered from the CNS, antigens are captured by APCs in the cervical lymph nodes and activate lymphocytes that then migrate to the CNS in search for remaining antigens (Romo-Gonzalez, Chavarria et al. 2012). As noted above, several hypotheses for lymph-like drainage of antigens exist including efferent flow via CSF and interstitial fluid past the optic, trigeminal, and acoustic nerves to the cervical lymph nodes, reabsorption through arachnoid villi into the venous sinuses, and through perivascular APC including macrophages and DC (Romo-

A more recent study by Jarry et al. showed that adult microglia can cross-present antigen to naïve CD8+ T cells for priming if there is appropriate microglial activation (Jarry, Jeannin et al. 2013). Their study involved injecting naïve T cells into the brain, as the natural presence of naïve T cells in the brain is limited, with restriction of entry to activated T cells instead. Their study suggests that if naïve T lymphocytes are given the ability for entry into the brain, typically during stressful inflammatory illnesses, coupled with the appropriate microglial

Glioma-associated microglia/macrophages cannot mount a successful anti-tumor T cell response (Yang, Han et al. 2010). Microglia, along with some T lymphocytes, infiltrate gliomas in a pattern that was initially thought to be an immune response against tumor cells but has been more recently realized to actually encourage tumor growth by promoting immunosup‐

response, cross-priming of naïve T cells is possible (Jarry, Jeannin et al. 2013).

2012; Chavarria and Cardenas 2013).

*5.2.3. The glymphatic pathway*

Gonzalez, Chavarria et al. 2012).

*5.2.4. Glioma-associated microglia*

#### *5.2.2. Microglia*

Microglia play a large role in both innate and adaptive immune responses, in addition to regulatory roles in the CNS (Yang, Han et al. 2010). They comprise 5-12% of all CNS cells and are uniformly distributed throughout the CNS parenchyma (D'Agostino, Gottfried-Blackmore et al. 2012). Similar to the peripheral immune APCs, microglia express MHC I and II molecules and CD 80/86 and CD40 co-stimulatory molecules that once activated, proliferate and phago‐ cytose in response to both CD4+ and CD8+ T cells (Fathallah-Shaykh, Gao et al. 1998; Yang, Han et al. 2010; Ousman and Kubes 2012). In the latent state, microglia survey the microen‐ vironment via pinocytosis. Once they sense infection, neuronal injury, or neurodegenerative disease, they up-regulate the expression of MHC and co-stimulatory molecules and release cytokines including IL-1, IL-6, and TNF-alpha as well as neurotrophic and cytotoxic factors, and chemokines for lymphocyte recruitment (Yang, Han et al. 2010; Jarry, Jeannin et al. 2013). These pro-inflammatory cytokines then make the BBB more soluble for entry of peripheral immune cells and potentially naïve T lymphocytes (Yang, Han et al. 2010). Microglia's phagocytic and cytotoxic features are also up-regulated with the triggering of an immune response (Yang, Han et al. 2010). As in the peripheral immune response, microglia CD80/CD86 and CD40 bind the T cell's CD28 and CD40L, respectively (Yang, Han et al. 2010). IFN- γ release sustains this response and promotes phagocytosis and direct tumor-cell cytotoxicity (Fathal‐ lah-Shaykh, Gao et al. 1998; Yang, Han et al. 2010; D'Agostino, Gottfried-Blackmore et al. 2012).

Similar to peripheral tolerance, if there is insufficient co-stimulatory response, the interaction of Fas ligand (FASL) on microglia and Fas receptor on the T cell leads to activation-induced T cell apoptosis. Microglia also express FAS molecules themselves, which induce apoptosis upon binding FASL (Yang, Han et al. 2010). Nitric oxide released by microglia in response to activation can also potentiate effector cell death (Yang, Han et al. 2010). Furthermore, microglia display B7-H1 molecules which also support immunosuppression by stimulating T cell apoptosis (Yang, Han et al. 2010). Additionally, glycoprotein CD200 down regulates activated microglia (via CD200 ligand on microglia) and perivascular macrophages in the CNS, acting as an anti-inflammatory and serving to keep microglia in a quiescent state (Ousman and Kubes 2012; Chavarria and Cardenas 2013).

#### *5.2.3. The glymphatic pathway*

*5.2.1. Centrally-acting peripheral immune cells*

190 Tumors of the Central Nervous System – Primary and Secondary

al. 2012; Romo-Gonzalez, Chavarria et al. 2012).

*5.2.2. Microglia*

In addition to resident microglia, the primary immune cell in the CNS, peripheral immune cells including peripherally activated T lymphocytes, macrophages, and DC circulate in small numbers within the CNS. They are predominantly in specialized CNS compartments located outside the parenchyma with ability to gain access to the parenchyma through various mechanisms that include the choroid plexus, perivascular or Virchow-Robin spaces, menin‐ geal vessel branch points into the subarachnoid space, and through post-capillary venules (Ousman and Kubes 2012). As in the periphery, these cells are capable of mounting an activated immune response if they encounter an antigen (Ousman and Kubes 2012; Jarry, Jeannin et al. 2013). Additionally, perivascular macrophages sample CSF and can phagocytose suspected antigens (Ousman and Kubes 2012). There is also separate evidence of drainage of CNS antigens to deep cervical lymph nodes, based on intracranial injection of labeled antigen (D'Agostino, Gottfried-Blackmore et al. 2012; Ousman and Kubes 2012). Despite controversy over poor immune surveillance due to low expression of MHC II, it is thought that preactivated T cells can release IFN- γ and TNF-α to simulate MHC II molecule expression (Romo-Gonzalez, Chavarria et al. 2012). Also of debate is the function of central antigen presentation by central DCs. It is known that the integrity of the BBB is compromised during times of infection, trauma, aging, and autoimmunity due to weakening of the vascular endothelium as a result of cytokine release by astrocytes and microglia (D'Agostino, Gottfried-Blackmore et

Microglia play a large role in both innate and adaptive immune responses, in addition to regulatory roles in the CNS (Yang, Han et al. 2010). They comprise 5-12% of all CNS cells and are uniformly distributed throughout the CNS parenchyma (D'Agostino, Gottfried-Blackmore et al. 2012). Similar to the peripheral immune APCs, microglia express MHC I and II molecules and CD 80/86 and CD40 co-stimulatory molecules that once activated, proliferate and phago‐ cytose in response to both CD4+ and CD8+ T cells (Fathallah-Shaykh, Gao et al. 1998; Yang, Han et al. 2010; Ousman and Kubes 2012). In the latent state, microglia survey the microen‐ vironment via pinocytosis. Once they sense infection, neuronal injury, or neurodegenerative disease, they up-regulate the expression of MHC and co-stimulatory molecules and release cytokines including IL-1, IL-6, and TNF-alpha as well as neurotrophic and cytotoxic factors, and chemokines for lymphocyte recruitment (Yang, Han et al. 2010; Jarry, Jeannin et al. 2013). These pro-inflammatory cytokines then make the BBB more soluble for entry of peripheral immune cells and potentially naïve T lymphocytes (Yang, Han et al. 2010). Microglia's phagocytic and cytotoxic features are also up-regulated with the triggering of an immune response (Yang, Han et al. 2010). As in the peripheral immune response, microglia CD80/CD86 and CD40 bind the T cell's CD28 and CD40L, respectively (Yang, Han et al. 2010). IFN- γ release sustains this response and promotes phagocytosis and direct tumor-cell cytotoxicity (Fathal‐ lah-Shaykh, Gao et al. 1998; Yang, Han et al. 2010; D'Agostino, Gottfried-Blackmore et al. 2012).

Similar to peripheral tolerance, if there is insufficient co-stimulatory response, the interaction of Fas ligand (FASL) on microglia and Fas receptor on the T cell leads to activation-induced T The CNS lacks lymphoid tissue. For appropriate immune surveillance, both antigens and APC must be able to travel to lymphoid tissue, ideally via lymphatic channels, for T cell priming (Romo-Gonzalez, Chavarria et al. 2012). For small and hydrophobic molecules as well as transporter substrates, exit through the BBB is easy. Other substances are cleared from CSF through arachnoid granulations or peripheral lymphatics on cranial nerves. Clearance of large particles and matter deep within the parenchyma is more difficult and is ascribed to a high rate of flow of interstitial fluid (Iliff and Nedergaard 2013). This flow of fluid transports antigens from the brain parenchyma for presentation in cervical lymph nodes (Romo-Gonzalez, Chavarria et al. 2012). Through what has been termed the glio-vascular or glym‐ phatic pathway, interstitial solutes are cleared from the brain to the peripheral lymphatic system via perivascular water channels from the para-arterial CSF influx pathway through the interstitium and along the para-venous clearance route (Iliff and Nedergaard 2013). Once filtered from the CNS, antigens are captured by APCs in the cervical lymph nodes and activate lymphocytes that then migrate to the CNS in search for remaining antigens (Romo-Gonzalez, Chavarria et al. 2012). As noted above, several hypotheses for lymph-like drainage of antigens exist including efferent flow via CSF and interstitial fluid past the optic, trigeminal, and acoustic nerves to the cervical lymph nodes, reabsorption through arachnoid villi into the venous sinuses, and through perivascular APC including macrophages and DC (Romo-Gonzalez, Chavarria et al. 2012).

A more recent study by Jarry et al. showed that adult microglia can cross-present antigen to naïve CD8+ T cells for priming if there is appropriate microglial activation (Jarry, Jeannin et al. 2013). Their study involved injecting naïve T cells into the brain, as the natural presence of naïve T cells in the brain is limited, with restriction of entry to activated T cells instead. Their study suggests that if naïve T lymphocytes are given the ability for entry into the brain, typically during stressful inflammatory illnesses, coupled with the appropriate microglial response, cross-priming of naïve T cells is possible (Jarry, Jeannin et al. 2013).

#### *5.2.4. Glioma-associated microglia*

Glioma-associated microglia/macrophages cannot mount a successful anti-tumor T cell response (Yang, Han et al. 2010). Microglia, along with some T lymphocytes, infiltrate gliomas in a pattern that was initially thought to be an immune response against tumor cells but has been more recently realized to actually encourage tumor growth by promoting immunosup‐ pression (Yang, Han et al. 2010). Pathological examination typically reveals a large numbers of microglia dispersed within the tumor and not just in necrotic tissue (Yang, Han et al. 2010). The data of Okada et al. suggest that the glioma-infiltrating cells may compose up to 30% of the glioma tumor, correlating in volume with degree of malignancy (Okada, Kohanbash et al. 2009). The lack of phagocytosis by the microglia is thought be related to decreased expression of MHC II and co-stimulatory molecules CD80/86 and CD40, thus prohibiting appropriate T cell activation (Okada, Kohanbash et al. 2009; Yang, Han et al. 2010). Glioma cells appear to attract microglia by secreting chemoattractants and growth factors including Macrophage Chemoattractive Protein-1 (MCP-1), which binds to the microglial MCP-1 receptor, as well as colony stimulating factor-1, Granulocyte-CSF, and hepatocyte growth factor/scatter factor (Yang, Han et al. 2010). Microglial secretion of epidermal growth factor (EGF), VEGF and MCP-1 promote tumor propagation and angiogenesis (Okada, Kohanbash et al. 2009; Yang, Han et al. 2010). Additionally, the release by microglia of MMPs assists in tumor dispersal (Yang, Han et al. 2010). Interestingly, tumors depleted of microglia actually become less invasive (Okada, Kohanbash et al. 2009).

response, but also to counteract the active immunological depressive effects by the tumor itself. We will not list an exhaustive search of all immunotherapeutic strategies but will instead discuss an outline of the approaches than can be used. A thorough discussion can be found in Okada et al. (Okada, Kohanbash et al. 2009). Here, we will emphasize the different categories

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Initiating an immune response against tumors is typically difficult due to poor antigen presentation and the active immunosuppressive effects by tumor cells (Luptrawan, Liu et al. 2008). Peripheral vaccination has been performed using purified antigen and irradiated genetically modified tumor cells. Through vaccination with a tumor antigen, one hopes to induce an immune response peripherally, which translates to CNS immunity as activated T cells cross the BBB. This goal may be achieved by processing the antigen via APCs at the subcutaneous injection site, migration to lymph nodes, and priming naïve T cells. Neverthe‐ less, choosing an appropriate antigen is crucial so as to avoid an autoimmune response causing

Peptide-based vaccines (see Table 1) for glioma epitopes are synthetically derived for specific antigens and run less risk of autoimmune encephalitis. This process has the potential to be individually tailored based on assessment of the patient's peripheral blood for positive response to the various antigens (Okada, Kohanbash et al. 2009). Many antigen epitopes exist and will be briefly covered. Il-13Rα2 appears as a membrane protein in more than 80% of gliomas but not in normal brain tissue, making it a target for immunotherapy (Debinski, Gibo et al. 1999). The tyrosine kinase receptor EphA2, which is involved in cell-cell contact in normal cells, contributes to malignant nature of tumor cells (Kinch, Moore et al. 2003). T-cell epitopes of Survivin, an apoptosis inhibitor protein present in several human cancers, have shown promise via vaccination for patients with pancreatic cancer and melanoma (Otto, Andersen et al. 2005; Wobser, Keikavoussi et al. 2006). These proteins are found in 100% of astrocytomas but not in normal brain tissue (Uematsu, Ohsawa et al. 2005; Okada, Kohanbash et al. 2009). Wilm's Tumor 1 gene, a transcription factor oncogene, is also present in many tumor types, including the majority of GBM but not in normal glial cells (Sugiyama 2002). The transcrip‐ tional cofactor family SOX, Sry-Related High-Mobility Group Box, is present in normal tissue development and is upregulated in various tumors, including gliomas. Vaccinations with SOX have been shown to be therapeutic in mice with gliomas (Ueda, Kinoshita et al. 2008; Okada, Kohanbash et al. 2009). HER-2/neu, in the EGFR family, promotes tumor growth by inhibiting apoptosis and stimulating migration, adhesion, and angiogenesis in many tumor-types, most notably breast, ovarian, colorectal, pancreatic, renal-cell, and GBM (Meric-Bernstam and Hung 2006; Okada, Kohanbash et al. 2009). Additional epitopes have been identified involving EGFR variant III, found in 30-50% of GBMs, Squamous Cell Carcinoma Antigen Recognized by T Cells 1 (SART-1), a gene-coding tumor antigen in many cancer types, including glioma but not in normal tissue, and Cytomegalovirus, which infects a large number of gliomas and may contribute to glioma pathogenesis (Cobbs, Harkins et al. 2002; Saikali, Avril et al. 2007; Okada,

and discuss limitations of immunotherapy.

encephalitis (Okada, Kohanbash et al. 2009).

**6.1. Priming in the periphery**

Kohanbash et al. 2009).

In addition to altering the response of microglial cells, gliomas take an active role in downregulating the immune response. Recent data has shown that reduced phagocytic activity by glioma-associated microglia stems from defective antigen presentation for T cell activation due to decreased MHC II expression as well as suppression of pro-inflammatory cytokine (TNFα) release, especially in high-grade gliomas (Yang, Han et al. 2010). Instead, glioma cells favor TGF- β, IL-10, and PGE2 secretion, which inhibits both cytotoxic function of T cells and IFNγ-induced MHC II expression in microglial cells (Luptrawan, Liu et al. 2008; Okada, Kohan‐ bash et al. 2009; Yang, Han et al. 2010). PGE2 specifically inhibits T cell activation, suppresses natural killers cell activity, and favors a Th2 response by increasing cytokines Il-4, Il-10, and Il-6 while suppressing the Th1 cytokines Il-2, IFN-gamma, and TNF-α (Luptrawan, Liu et al. 2008). Additionally, glioma cells do not express adequate co-stimulatory molecules required for appropriate T cell activation, potentiating anergy through tolerance (Luptrawan, Liu et al. 2008). A homologue to the B7 family (B71/2 (CD80/86)), B7-H1 expression on the surface of glioma cells inhibits CD4+ and CD8+ T cell activation. IFN- γ not only enhances antigen processing but also promotes increased B7-H1 expression, ultimately reducing T lymphocyte effectiveness in the presence of gliomas (Okada, Kohanbash et al. 2009). Additionally, some gliomas display Fas-L leading to apoptosis of Fas-labeled T cells contacting the tumor cells (Okada, Kohanbash et al. 2009).
