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CONTENTS INTERVIEW: Neuroimmunology and multiple sclerosis: an interiview with Katerina Akassoglou Neurology Central OPINION: Immune-brain relationships: new paradigms in searching for treatments for neurodegenerative disease? Neurology Central REVIEW: Autoantibodies in neurological disorders – a review from Judith Greer Neurology Central SPECIAL REPORT: Elucidating the link between the modifiable risk factors of Alzheimer’s disease and neuroinflammation Neurodegener. Dis. Manag. Vol. 6 Issue 5 REVIEW: The role of the immune system in neurofibromatosis type 1-associated nervous system tumors CNS Oncol. Epub ahead of print www.neurology-central.com/2017/02/01/spotlight-on-neuroimmunology/ INTERVIEW

Neuroimmunology and multiple sclerosis: an interview with Katerina Akassoglou

As part of Neurology Central’s Spotlight on neuroimmunology, Lauren Pulling (Editor, Neurology Central) spoke to Katerina Akassoglou about her pioneering work on the interactions of the immune and nervous systems in neurologic disease. Dr Akassoglou’s work centers on the leakage of the blood–brain barrier in disease and injury, and the mechanisms by which blood proteins subsequently activate immune cells that attack the brain. Of particular note is her work on the role of the blood protein fibrinogen in multiple sclerosis – research that she hopes will one day lead to an effective treatment for patients. In this interview, Dr Akassoglou discusses her research, as well as the key challenges in this exciting area of neuroscience and her hopes for the field.

Dr Akassoglou is primarily a Senior Investigator at the Gladstone Institutes (CA, USA) and a Professor of neurology at the University of California, San Francisco (UCSF; CA, USA). She is also an Associate Adjunct Professor of Pharmacology at the University of California, San Diego (CA, USA), and directs the Gladstone/UCSF Center for In Vivo Imaging Research.

QQ First, could you tell us a little about your myelin, but other mechanisms like activation of background? How did you come to work in brain immune cells appeared to be potent drivers neuroimmunology? of disease. In 1998, I was fortunate to receive I became interested in neuroimmunology early the Women In Neuroimmunology Award by on. I pursued my PhD in an immunology lab at Cedric Raine and the International Society for the Hellenic Pasteur Institute (Athens, Greece), Neuroimmunology for my PhD work. It truly studying the cytokine tumor necrosis factor was an unexpected honor for a graduate student (TNF), under the direction of George Kollias like myself. and Lesley Probert. The lab had made several Neuroimmunology is perhaps one of the most transgenic mice expressing TNF, and all these multidisciplinary fields of study that requires mice developed arthritis. However, there was training in multiple fields. During my PhD one transgenic line that was paralyzed without training, I made the observation that activa- any symptoms of arthritis. My PhD project was tion of brain immune cells strongly correlated to find out why TNF caused paralysis in this with leakage of blood in the brain. I was curi- line. I made the unexpected discovery that, in ous as to whether blood in the brain could be this particular line, TNF was expressed only responsible for activating the brain immune cells in the brain and spinal cord. With the expert and cause damage. To obtain training in blood guidance of Hans Lassmann at the University of proteins, I pursued my postdoctoral studies at Vienna (Austria), we discovered that expression The Rockefeller University (NY, USA) under of TNF in the brain was sufficient to activate the guidance of Sidney Strickland. The blood brain immune cells and cause MS-like symp- protein fibrinogen was already recognized as a toms, such as leakage of blood in the brain and marker of disruption of the blood–brain bar- loss of myelin. It was an eye-opener that auto- rier (BBB), but no one had actually asked about immunity is not the only way to induce loss of its role in brain diseases. Was it a cause of the part of

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disease or a consequence? We made the unan- QQ Could you talk us through your work on ticipated discovery that fibrinogen was required the role of fibrinogen in MS? for regeneration of the peripheral nerve. This As I mentioned, we showed that when the BBB is was the first time that fibrinogen knock-out disrupted, fibrinogen leaks into the brain, where mice had been tested for their neurological it binds to receptors of microglia, astrocytes and functions. After having completed training in neurons to induce inflammation, axonal damage immunology, neurobiology and hematology, I and glial scar formation. As a result, axons are was ready to study in my own lab how fibrinogen damaged. Fibrinogen is abundant in MS and is affects brain functions. Indeed, we showed that, a major culprit of the disease. Indeed, fibrino- when fibrinogen gets into the brain, it activates gen is present in human MS lesions at varying microglia, the brain’s immune cells, to become degrees throughout the course of disease, includ- pro-inflammatory cells, promoting autoimmune ing normal appearing white matter, early lesions, responses and neuronal damage. chronic active and chronic inactive lesions. Our studies have shown that fibrinogen in QQ What are your lab’s current research the brain enables autoimmunity and recruits focuses? macrophages to injury sites. It creates a chemo- We focus on the molecular mechanisms that con- tactic gene signature in microglia to activate and trol communications among the brain, immune recruit myelin-specific T cells and peripheral system and blood vessels. We continue to study macrophages to the CNS, leading to demyeli- fibrinogen and other blood proteins and their nation. We showed that we could increase repair effects on microglia in mouse models of MS to the nervous system and protect axons from that we developed. We want to understand those damage by depleting fibrinogen. We also learned interactions in mouse brains and spinal cords that fibrinogen binds to the complement recep- and learn what happens when the BBB is dis- tor 3 on microglia, causing them to release toxic rupted and as the process of demyelination and reactive oxygen molecules. If we inhibited the neurodegeneration begins. We recently found a binding, the microglia were not activated, and way that we might be able to block the effects of the nerve was not damaged. fibrinogen in the brain. We made other significant discoveries, including a fascinating relation- QQ Do you anticipate that your findings could ship between astrocyte activation and neuronal lead to novel treatments for patients? activity and remodeling of the nuclear pore com- We are committed to developing new therapies plex. We are also looking beyond fibrinogen to for neurological diseases, and our focus has its downstream pathways directly linked to neu- been on targeting fibrinogen in the CNS. We rodegeneration. For example, we are using state- are looking at ways to target the harmful activi- of-the-art genomic and proteomic technologies ties of fibrinogen in the brain without affecting to discover new pathways that damage neurons. its function in the blood. Of course, this is a We have also invested a great deal in imaging, tricky thing to attempt. Fibrinogen is essential particularly high resolution in vivo imaging. We for blood clotting, so we cannot simply eliminate knew the microglia were dynamic cells, but we it completely. However, we hope to find ways to needed the ability to see them in action in liv- separate the two activities. That is, we want to ing animals. So we developed methods to image block the deleterious effects of fibrinogen in the the neurovascular interface in vivo in trans- CNS without interfering with its coagulation genic mice. We use high-resolution two-photon activity. We already showed with genetic tools microscopy and fluorescently labeled microglia, that this separation is feasible, and pharmaco- T cells and fibrinogen. With this procedure, we logic tools to selectively target the inflamma- can watch the whole disease process as it moves tory functions of fibrin in the brain are under from a normal brain to full-blown autoimmune development. disease. We’ve made some surprising discover- Another goal might be to use blood-clotting ies. For example, in MS, microglia change shape proteins as biomarkers for MS, which would and cluster around blood vessels early on in the allow us to predict the disease course of patients disease course. This finding supports the theory to study and treat them more effectively and that disruption of the BBB and leakage of blood to facilitate future development of new thera- into the brain may occur before other symptoms pies. We also developed a molecular probe that of disease. detects lesions early in MS. With this probe, we

2 Neurology Central (March 2017) future science group Neuroimmunology and multiple sclerosis: an interview with Katerina Akassoglou Interview showed that coagulation is activated in the CNS similar translational success yet. Indeed, many in animal models of MS and stroke. The probe recent clinical trials based on good results in can also be developed for magnetic resonance mouse studies have failed in humans, and many imaging, and so it may have clinical applications. scientists now wonder if mouse models are only partially recapitulating features of these diseases. QQ Do you think your findings concerning It is timely to look for therapeutic targets not leakage of the BBB and activation of the only in the brain, but also in the immune system innate immune response in MS could translate and the blood, even for diseases not traditionally across to other neurodegenerative diseases? classified as ‘neuroimmune’. Yes, I do. Fibrinogen is not just found in brains of patients with MS. It is found in any neuro- QQ More broadly, what do you think are the logical disease or trauma with disrupted BBB. key questions to be addressed in the field of For example, it is found in the brains of patients neurological disease in the next 5–10 years? with Alzheimer’s disease, ischemia, spinal cord Traditionally, diseases have been classified as injury, traumatic brain injury, stroke, schizo- neurodegenerative (for example, Alzheimer’s), phrenia, HIV encephalopathy and potentially inflammatory (for example, MS), and vascular normal aging. Further studies might benefit (for example, stroke). But more recently, scien- these patients as well. Thus, we suspect that the tists have begun to look at these diseases in the leakage of blood into the brain and activation context of the complex relationships between the of the innate immune response are common brain, immune and vascular systems. There is threads in neurological diseases. now a search for common threads among these diseases. We will need to re-evaluate our under- QQ What are the key challenges in standing of the causes and signals that regulate investigating the role of the immune system disease onset and progression, and recognize that in neurologic disease? How did/do you neurological disorders cannot be described in overcome these? isolation. One of the challenges was how to actually visual- Tragically, effective therapies are lacking for ize the sequence of events occurring after injury major neurologic diseases. Alzheimer’s disease in living organisms and not rely on dogma for was first described more than 100 years ago, how brain diseases start and progress. We devel- and our society and those of many countries oped methods for in vivo microscopy so that we in the world are aging – the greatest risk factor could follow the movement of immune cells in for neurologic disease. An integrated approach the brain. These involve high-resolution two- is more likely to lead to new insights about photon microscopy and fluorescent labelling of the brain and better ways to treat its diseases. cells and proteins. It is important to know what Neuroimmunology is uniquely positioned to happens in the brain when the disease starts and play a central role for the understanding of fun- progresses and study these early mechanisms damental mechanisms of all brain diseases and together with their more obvious consequences. for the discovery of new therapies. I am extremely The ultimate challenge will be to translate encouraged that the scientific community is these findings from mice into human studies. increasingly recognizing this potential. Targeting the immune system in MS has been one of the biggest successes in drug discovery Disclaimer leading to several FDA-approved drugs in the The views expressed in this interview are those of the author, past 10 years. In contrast, other neurological dis- and do not necessarily reflect the views of Neurology Central eases, such as Alzheimer’s disease, have not had or Future Science Group.

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Immune-brain relationships: new paradigms in searching for treatments for neurodegenerative disease?

Michal Schwartz,

Department of Neurobiology, Weizmann Institute of Science, Rehovot, Israel [email protected]

For decades, it was believed that due to its unique structure, the brain functions best as an autonomous tissue behind barriers, equipped with mechanisms of homeostasis and repair. Since immune cells are the body’s champions of repair, it was assumed that the brain-resident innate immune cells, the microglia, mediate this function. The microglia enter the CNS during early development [1], and their maturation occurs step-wise, in synchrony with the needs of the developing brain [2]. Yet, most of the body’s other tissues, while containing resident immune cells, require additional systemic immune support for their repair. In contrast, it was believed that the brain escapes from such help, relying solely on its resident immune population. This dogma prevailed for decades. Below, I briefly summarize milestones that paved the way to changes in this commonly accepted view.

Almost all brain pathologies are associated with from acute injuries is highly dependent on cir- local inflammation [3], the etiology of which culating monocytes and T cells [10-12], through has only recently started to be deciphered. a well-orchestrated immune network, which Nevertheless, based on the long-held dogma that is initiated within the brain, continues in the the brain is an immune privileged site, attempts periphery and culminates back in the CNS were made to use anti-inflammatory drugs to [13-15] . Nevertheless, it was not clear how such treat brain pathologies in which local inflam- communication could take place if circulat- mation was observed, almost regardless of the ing immune cells are excluded from the CNS primary disease etiology; this approach mostly parenchyma, and possible aberrations in these failed, resulting in much confusion within the pathways in neurodegenerative diseases and research community [4]. brain aging were not characterized. A turning Studies initiated by my team over almost two point in addressing this question was reached decades have demonstrated that the CNS and when the research focus shifted to understand- the immune system engage in bi-directional ing the immunological properties of the borders communication, and that the brain is dependent between the brain and the circulation [13, 16, 17]. on systemic immunity, and not only on the local The CNS barrier system includes the blood– innate immune-resident cells. The dependence brain barrier (BBB), the meningeal barrier, and of the brain on systemic immune support was the blood–cerebrospinal fluid barrier (BCSFB). shown with respect to formation of new neurons, These three barriers are distinct in their struc- cognitive ability [5-7], coping with stress [8], and ture, and recently were also found to differ with recently, in social behavior [9]. respect to their functions in relation to immune Independent studies, some of which preceded cells [18]. Both the meningeal barrier and the those demonstrating the effect on healthy brain BCSFB barrier are continuously populated by function, revealed that the process of recovery adaptive immune cells and by dendritic cells part of

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[14,16,17]. However, in contrast to the other bar- at inhibitory immune checkpoints, known to riers, the BCSFB, which is an epithelial rather tightly control effector memory T cells. endothelial barrier, created by the choroid plexus Specifically, to release suppression of cells that epithelium (CP), was found to serve as an inter- could potential produce IFN-γ, we tested the face that allows trafficking of immune cells effects of an antibody directed against the inhib- under physiological conditions for surveillance itory immune checkpoint PD-1. We treated and for repair [13,14,19,20]. 5XFAD mice, a mouse model of Alzheimer’s The understanding that adaptive immune disease (AD), which expresses most features of cells populate the CP within the BCSFB and human AD, including the accumulation of intra- can orchestrate trafficking though this barrier cellular and extracellular misfolded amyloid- has led to the hypothesis that its mode of opera- beta, loss of synapses, local inflammation, gliosis tion and its fate under different brain patholo- and neuronal loss [23]. In response to a single sys- gies could be critical to determining its ability to temic treatment, the animals showed reversal of cope with disease conditions. It was found that cognitive loss, and a dramatic decrease in plaque T cells that reside in the CP via local produc- burden and gliosis. The effect was maintained tion of IFN-γ control expression of leukocyte for at least 4 weeks after a single treatment [24]. trafficking molecules, thereby allowing traf- Further studies are needed before translating this ficking of leukocytes into the CNS parenchyma therapy into the clinic, to correspond to the cur- when needed. Moreover, a synergy was found rent understanding of the mechanism of action between IFN-γ and danger signals coming from needed for allowing circulating leukocytes to the injured CNS, which augments trafficking traffic into the brain and display their conse- through this interface upon signaling from the quential local disease-modifying activity in AD. brain [14] . The studies will aim to determine the type of Almost paradoxically, in aging and neuro- the antibody, the dose and the regimen, which degenerative diseases, the CP was shown to be will differ from the current immunotherapy suppressed with respect to its ability to support for cancer. This approach in AD may signal a expression of leukocyte trafficking molecules, major transition in the search for AD-modifying apparently due to limited availability of IFN-γ therapy, as it targets the immune system, rather at the CP [20–22]. Transient reduction of regula- than a specific pathological factor within the tory T cell levels resulted in elevation of IFN-γ diseased brain. availability, activation of the CP, infiltration of monocytes and regulatory T cells to the brain, Acknowledgments and reduced burden of disease pathology, includ- This work was supported by the EU Seventh Framework ing reversal of cognitive loss [20, 21]. Such results Program HEALTH-2011 (grant no. 279017) and the ISF- led us to test a treatment based on de-repressing Legacy Heritage Biomedical Science Partnership-research the immune system by blocking inhibitory path- (grant no. 1354/15). M.S. holds the Maurice and Ilse Katz ways. Specifically, we used antibodies directed Professorial Chair in Neuroimmunology.

References for NSAIDs and novel therapeutics. Expert Rev. Neurother. 17, 17–32 (2017). 1 Ginhoux F, Greter M, Leboeuf M et al. Fate mapping analysis reveals that adult microglia derive 5 Kipnis J, Cohen H, Cardon M, Ziv Y, Schwartz from primitive macrophages. Science 330, 841–845 M. T cell deficiency leads to cognitive (2010). dysfunction: implications for therapeutic vaccination for schizophrenia and other 2 Matcovitch-Natan O, Winter DR, Giladi A et al. psychiatric conditions. Proc. Natl. Acad. Sci. USA Microglia development follows a stepwise program 101, 8180–8185 (2004). to regulate brain homeostasis. Science 353: aad8670 (2016). 6 Ziv Y, Ron N, Butovsky O et al Immune cells contribute to the maintenance of neurogenesis and 3 Frank-Cannon TC, Alto LT, McAlpine FE, Tansey spatial learning abilities in adulthood. Nat. Neurosci. MG. Does neuroinflammation fan the flame in 9, 268–275 (2006). neurodegenerative diseases? Mol. Neurodegener. 4: 47 (2009). 7 Wolf SA, Steiner B, Akpinarli A et al CD4-positive T lymphocytes provide a neuroimmunological link 4 Deardorff WJ, Grossberg GT. Targeting in the control of adult hippocampal neurogenesis. neuroinflammation in Alzheimer’s disease: evidence J. Immunol. 182, 3979–3984 (2009).

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8 Lewitus GM, Cohen H, Schwartz M. Reducing immunity: a key role for IL-4. J. Exp. Med. 207, post-traumatic anxiety by immunization. Brain 1067–1080 (2010). Behav. Immun. 22, 1108–1114 (2008). 18 Shechter R, London A, Schwartz M. Orchestrated 9 Filiano AJ, Xu Y, Tustison NJ et al Unexpected role leukocyte recruitment to immune-privileged sites: of interferon-gamma in regulating neuronal absolute barriers versus educational gates. Nat. Rev. connectivity and social behaviour. Nature 535, Immunol. 13, 206–218 (2013). 425–429 (2016). 19 Baruch K, Deczkowska A, David E et al. Aging- 10 Rapalino O, Lazarov-Spiegler O, Agranov E et al. induced type I interferon response at the choroid Implantation of stimulated homologous plexus negatively affects brain function. Science 346, macrophages results in partial recovery of paraplegic 89–93 (2014). rats. Nat. Med. 4, 814–821 (1998). 20 Kunis G, Baruch K, Miller O, Schwartz M. 11 Moalem G, Leibowitz-Amit R, Yoles E, Mor F, Immunization with a myelin-derived antigen Cohen IR, Schwartz M. Autoimmune T cells protect activates the brain’s choroid plexus for recruitment of neurons from secondary degeneration after central immunoregulatory cells to the CNS and attenuates nervous system axotomy. Nat. Med. 5, 49–55 (1999). disease progression in a mouse model of ALS. 12 Shechter R, London A, Varol C et al Infiltrating J.Neurosci. 35, 6381–6393 (2015). blood-derived macrophages are vital cells playing an 21 Baruch K, Rosenzweig N, Kertser A et al. Breaking anti-inflammatory role in recovery from spinal cord immune tolerance by targeting Foxp3(+) regulatory injury in mice. PLoS Med. 6: e1000113 (2009). T cells mitigates Alzheimer’s disease pathology. Nat. 13 Shechter R, Miller O, Yovel G et al. Recruitment of Commun. 6: 7967 (2015). beneficial M2 macrophages to injured spinal cord is 22 Mesquita SD, Ferreira AC, Gao F et al. The choroid orchestrated by remote brain choroid plexus. plexus transcriptome reveals changes in type I and II Immunity 38, 555–569 (2013). interferon responses in a mouse model of Alzheimer’s 14 Kunis G, Baruch K, Rosenzweig N et al. IFN- disease. Brain Behav. Immun. 49, 280–292 (2015). gamma-dependent activation of the brain’s choroid 23 Oakley H, Cole SL, Logan S et al. Intraneuronal plexus for CNS immune surveillance and repair. beta-amyloid aggregates, neurodegeneration, and Brain 136, 3427–3440 (2013). neuron loss in transgenic mice with five familial 15 Raposo C, Graubardt N, Cohen M et al. CNS repair Alzheimer’s disease mutations: potential factors in requires both effector and regulatory T cells with amyloid plaque formation. J. Neurosci. 26, distinct temporal and spatial profiles.J. Neurosci. 34, 10129–10140 (2006). 10141–10155 (2014). 24 Baruch K, Deczkowska A, Rosenzweig N et al. PD-1 16 Louveau A, Smirnov I, Keyes TJ et al. Structural and immune checkpoint blockade reduces pathology and functional features of central nervous system improves memory in mouse models of Alzheimer’s lymphatic vessels. Nature 523, 337–341 (2015). disease. Nat. Med. 22, 135–137 (2016). 17 Derecki NC, Cardani AN, Yang CH et al. Regulation of learning and memory by meningeal

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Autoantibodies in neurological disorders – a review from Judith Greer

Judith M Greer The University of Queensland Centre for Clinical Research, Brisbane, Australia

Autoantibodies occur in many different nervous system disorders, and are increasingly being found in disorders not traditionally associated with the immune system. Determining if the autoantibodies play a functional or pathogenic role is critical in selecting the most appropriate treatment options.

Association of autoantibodies with Sixty years ago, Ernst Witebsky and colleagues disorders of the nervous system set out some rules to determine if a disease was Some diseases affecting the nervous system have autoimmune in origin [11] . These have been sub- a long history of association with autoantibod- sequently refined [12] to include direct evidence ies. For example, the presence of oligoclonal (i.e. transmissibility by antibody of the char- bands of immunoglobulin in the cerebrospinal acteristic lesions of the disease from human to fluid (CSF) has been used for nearly 50 years human or human to animal, or reproduction of in the diagnosis of multiple sclerosis (MS) [1], the functional defects characteristic of the dis- and the presence of antibodies that bind to ease in vitro), indirect evidence (i.e. reproduc- muscle in serum of patients with myasthenia tion of the autoimmune disease in experimental gravis (MG) was described even earlier [2]. More animals or isolation of autoantibodies from the recently, it has become clear that autoantibodies target organ), and circumstantial evidence (e.g. are also found in various types of encephalitis, arising from clinical improvement in response to and in some patients with movement or psychi- immunotherapy). There are many examples in atric disorders or epilepsy [3–7]. Then there are the literature of the indirect and circumstantial autoantibodies associated with neoplasms (e.g., levels of evidence for autoantibodies as the cause anti-Hu antibodies in small cell lung cancer with of neurological disease; however, only the direct paraneoplastic sensory neuropathy [8]) or with evidence of the pathogenicity of autoantibodies other autoimmune diseases that may cross-react will be discussed further. with neural components to cause CNS disease Human to human transmission of a patho- (e.g., cases of patients with type 1 diabetes devel- genic autoantibody would be likely to occur only oping stiff person syndrome, or vice versa, pre- in two situations: either by accident (through sumably due to immune attack against glutamic transfusion from an affected donor), or by acid decarboxylase (GAD), which is present in transplacental transmission of antibodies from both the pancreas and the nervous system [9]). mother to fetus. In approximately 10% of cases What remains to be elucidated in many cases is of babies born to mothers with MG [13] or the whether these autoantibodies are merely a bio- Lambert-Eaton myasthenic syndrome (LEMS) marker of the disease process, or whether they [14], the baby will have transient signs of disease. are pathogenic [10] . Disease transfer is dependent on the presence of antibodies of the IgG isotype and their abil- Levels of evidence in determining the ity to interact with the neonatal Fc receptor, pathogenicity of an autoantibody FcRn, which facilitates transfer of IgG across the part of

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placenta (IgG1 binds better to FcRn than IgG4, the target of the antibody is not a receptor, the which binds better than IgG3; IgG2 binds very potential pathogenic mechanisms considered poorly) [15] . In MG and LEMS, the autoantibody usually include cell lysis (either via antibody- targets (at the neuromuscular junction) could be mediated activation of complement, which easily accessed by the autoantibodies. However, appears to be one mechanism by which myelin it is of interest that there have been two recent might be destroyed in MS [22], or antibody- reports of transplacental transmission of NMDA dependent cell-mediated cytotoxicity mediated receptor antibodies, and development of enceph- via Fc receptors on NK cells, macrophages or alitis in at least one of these cases [16, 17]. In this polymorphonuclear cells) or coating (opsoniza- situation, the antibody would have to navigate tion) of the cell to send an “eat me” message across not only the placenta, but also across the to macrophages (this is thought to underlie the blood–CNS barriers. There are many conflicting abundance of myelin-loaded macrophages in reports regarding the permeability of the these MS). If the target is a receptor, then cell lysis is barriers in neonates compared to adults [18]; but, still an option (e.g., antibody and complement in this patient at least, it would appear that the mediated destruction of muscle morphology at IgG has been able to cross into the CNS. the postsynaptic membrane by antibodies tar- The first example of antibody-mediated geting the skeletal muscle acetylcholine recep- transfer of a human nervous system disease to tor in MG [23]), but other mechanisms could an experimental animal was reported in 1975, include blocking or modulation of receptor sig- using immunoglobulin from a patient with MG nalling (e.g., binding of anti-LGI1 antibodies [19] . Somewhat surprisingly, there have been appears to result in closing of the Kv1 chan- relatively few successful examples of human to nel and a reduction of K+ levels in the synapse experimental animal transmission since then. in limbic encephalitis [24]), internalization of The main reason for the difficulty in induc- receptors (e.g., internalization and break down ing the characteristic lesions of the disease in of NMDA receptors following binding of anti- an animal model is likely to be that the protein NMDAR antibody [25]), or downstream effects sequence of the target molecule differs some- such as modulation of cell architecture (e.g., what at the antigen binding site between the two binding of antibodies specific for MOG from species, and even a single amino acid difference patients with acute disseminated encephalomy- could potentially result in a loss of binding of an elitis leads to changes in microtubule arrange- antibody. However, particularly with diseases ment in oligodendrocytes [26]). Other methods of the CNS, the way in which the antibody is of action could be envisaged, e.g., effects on cell transferred will also affect the outcome of the trafficking or on repair mechanisms, but these transfer, as the adult blood–CNS barriers are remain to be elucidated. largely impermeable to antibodies. Even if high levels of antibodies specific for CNS antigens Location, location! are present in the serum of patients, unless the When considering the antibody-mediated CNS blood–CNS barriers have been compromised disorders, a question that frequently arises prior to transfer of the antibody, the antibody is the site of synthesis of the autoantibodies. will usually need to be injected intracisternally Autoantibodies can often be found both in the or intrathecally into a recipient animal in order CSF and the serum (e.g., in NMDAR encepha- to maximise the likelihood that it reaches the litis [27]), or even sometimes in the serum but not target organ. the CSF (e.g., anti-dopamine receptor 2 (DR2) With recent advances in molecular biology, and anti-voltage-gated potassium channel- the ability to reproduce and to measure func- complex (VGKC) antibodies in non-malignant tional defects characteristic of the disease in related encephalitis [28]). Activated B cells and vitro has improved markedly, and such systems plasma cells expressing the adhesion molecules are now providing the best direct evidence of ICAM-1, VLA-4 and ALCAM can cross freely potential pathogenic effects of autoantibodies into the CNS [29, 30], and would have the poten- [20]. The use of such methods is only limited by tial to synthesize antibody both within and out- our current understanding of how autoantibod- side of the CNS. There have also been reports ies could act [21]. Traditionally, we tend to think of the formation of ectopic lymphoid follicles of antibodies exerting functional/pathogenic containing plasma cells in the meningeal space effects only if they bind to surface antigens. If in MS [31], and antibodies formed within the

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CNS could drain via the interstitial fluid and and it is thought that its beneficial effects may CSF into the bloodstream [32]. Brain-specific be mediated through upregulation of expres- antibodies produced in the CNS/CSF also sion of the inhibitory Fc receptor FcγRIIB on potentially have a large absorptive surface on macrophages, thereby shortening the half-life of which to bind (thereby decreasing the levels of autoreactive antibodies or inhibiting their func- free antibody detectable within the CSF) [33], tional effects [38]. Similarly, plasmapheresis also whereas, in the serum, it is less likely they would appears to be most effective in disorders where encounter their specific antigen, and would the target of the antibodies lies outside of the therefore be present at higher levels. It has been CNS, e.g., in CIDP, Guillain-Barré syndrome argued that if antibodies produced within the (GBS), LEMS and MG [39, 40], or in cases of serum were the sole source of the pathogenic autoimmune encephalitis associated with the antibodies, then it might be expected that there presence of a tumor [41] . would be much higher levels of neurological dis- The use of monoclonal antibody therapies is ease in patients with diseases such as systemic becoming increasingly popular, as their speci- lupus erythematosus (SLE) or type 1 diabetes, ficity enhances the potential to target specific who often have high serum levels of autoanti- molecules or mechanisms. For example, rituxi- bodies against molecules that are present in the mab, which depletes CD20+ B cells, has been CNS. However, it could also be that, in such used successfully in the treatment of a wide cases, the autoantibodies target slightly different range of neurological disorders, including MS, epitopes of the antigen which are not as avail- NMO, CIDP, MMN, IgM-MAG neuropathy, able for antibody binding within the CNS, or MG and paraneoplastic opsoclonus-myoclonus which do not induce the same functional effects [42], and has been reported to be particularly upon antibody binding, as suggested by studies effective in disorders associated with IgG4 of anti-GAD antibodies in type 1 diabetes and antibodies [43]. Other monoclonal antibodies stiff person syndrome [9, 34]. currently in development or in clinical trials, or currently approved only for other (often Treatment options autoimmune) disorders include belimumab to The issues of how an autoantibody acts and deplete BAFF [44] and eculizumab to inhibit where it is produced will determine not only complement activation and the formation of the the choice of therapy, but also the likelihood of membrane attack complex [45]. At present, as success. Autoantibodies targeting surface anti- with most of the other treatment options avail- gens have been reported to be more susceptible able, targeting pathogenic antibody production to therapeutic intervention than autoantibodies within the CNS remains a challenge, unless the targeting intracellular antigens [35]. Furthermore, blood–CNS barriers are disrupted. Other types disorders of the peripheral nervous system or of drugs that inhibit the same target molecules neuromuscular junction, or paraneoplastic syn- are also under development, but to improve dromes where the tumor is located peripherally, the efficacy of therapeutic strategies for CNS are typically more amenable to treatment than disorders, it will be necessary to improve drug disease contained within the CNS. Many of the delivery across the blood–CNS barriers. immunomodulatory and immunosuppressive treatments currently available target both B Conclusions cell/antibody and T cell arms of the immune Autoantibodies occur in many different diseases response, but not always in the same way. For of the nervous system, and have the potential to example, the use of interferon-b, which is fre- be of pathogenic relevance. Current therapeutic quently used successfully in relapsing-remitting strategies are more successful in disorders where MS (primarily T cell driven), induced increased the antibodies are produced and/or act in the levels of the B cell activating factor (BAFF) periphery. There is a need to develop more effec- within the CNS [36], and appeared to increase tive ways of targeting autoantibodies that are the relapse rate in patients with the antibody- produced and/or act within the CNS. mediated neuromyelitis optica (NMO) [37]. Intravenous immunoglobulin (IVIg) has been Disclaimer used successfully in MG, chronic inflamma- The views expressed in this article are those of the author tory demyelinating polyneuropathy (CIDP), and do not necessarily represent those of Neurology Central and multifocal motor neuropathy (MMN), or Future Science Group.

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Elucidating the link between the modifiable risk factors of Alzheimer’s disease and neuroinflammation

Stephanie M Schindler1 & Andis Klegeris*,1

Practice points Modifying the neuroinflammatory environment in Alzheimer’s disease as a therapeutic approach ●● Alzheimer’s disease (AD) is the most common neurodegenerative disease and one of the leading causes of morbidity and mortality worldwide. AD is a public health concern imposing a great economic burden. ●● Emerging evidence for a causal role for neuroinflammation, thereby providing a new potential therapeutic target for AD. ●● Pharmacological attempts to cure or halt disease progression have been unsuccessful. ●● Primary prevention by altering the modifiable risk factors (lifestyle-associated, vascular and psychological) for AD may present an alternative therapeutic strategy. Lifestyle-associated risk factors for AD ●● Dietary components such as fiber, antioxidants, carotenoids, vitamins and fatty acids have all been shown to modulate inflammatory responses, suggesting a possible mechanism behind the link between nutrition and AD risk. ●● The Ω-3 PUFAs, such as docosahexaenoic acid and eicosapentaenoic acid, modulate glial cell activation and achieve an anti-inflammatory effect by decreasing the release of the proinflammatory mediators IL-6, TNF-α and nitric oxide. ●● Epidemiological studies identify physical activity and physical exercise as potential intervention strategies for neurodegenerative disease. Physical activity and physical exercise are neuroprotective, can attenuate neuroinflammation and limit disease progression. Vascular risk factors for AD ●● High BMI and midlife obesity increase the risk of developing AD by up to twofold, while Type 2 diabetes mellitus is associated with a relative risk of 1.54. ●● High adiposity environments are characterized by elevated levels of proinflammatory cytokines (e.g., IL-6, IL-1β and TNF-α), increased infiltration of macrophages into the adipose tissue and microglial activation. ●● Modulating the inflammatory environment in the periphery through regular exercise and a balanced diet may help to reduce the risk of exacerbating the inflammatory environment in the brain, thereby decreasing the risk of developing AD. Psychological risk factors for AD ●● High comorbidity between AD and major depressive disorder (MDD) may be due to the underlying inflammatory mechanisms that are present in both diseases. ●● Patients with MDD have been found to have elevated plasma and cerebrospinal fluid levels of circulating proinflammatory cytokines, including IL-6 and TNF-α, similar to AD patients.

1Department of Biology, University of British Columbia Okanagan Campus, Kelowna, British Columbia, V1V 1V7, Canada *Author for correspondence: Tel.: +1 250 807 9557; Fax: +1 250 807 8830; [email protected] part of

Practice points (cont.) Psychological risk factors for AD (cont.) ●● Mixed results show anti-inflammatory and proinflammatory effects of antidepressants; early MDD treatment may delay AD onset later in life. ●● Increasing evidence suggest that high levels of perceived stress are associated with an increased risk of developing AD. ●● Neuroinflammatory mechanisms induced by stress likely contribute to neuronal dysfunction and impaired neurogenesis, which eventually progress to AD. ●● Meditation- and mindfulness-based stress reduction improve functional connectivity between the cortex and the hippocampus, and decrease atrophy in hippocampus, the region most affected by AD. Conclusion ●● AD is considered an epidemic of the 21st century. ●● Efforts to develop effective pharmacological treatments have not been successful. ●● Modifiable risk factors can facilitate and modify the neuroinflammatory environment found in AD, identifying targets for the development of novel therapeutic and lifestyle interventions for AD. ●● Until more effective medications are developed, controlling and managing the modifiable risk factors presents the best strategy for decreasing the incidence of AD.

Increased worldwide longevity through medical interventions, although beneficial, has allowed the age-related Alzheimer’s disease (AD) to become an epidemic of the 21st century. AD pathology involves adverse activation of microglia, the immune cells of the brain and resulting chronic neuroinflammation. Certain diets, physical inactivity and Type 2 diabetes mellitus have been identified as the risk factors for developing AD, which may increase the risk of AD by neuroimmune mechanisms primarily through the overactivation of microglia. Thus, modifying these risk factors may represent an alternative therapeutic strategy for lowering the incidence of AD. We highlight the link between select modifiable risk factors and neuroimmune mechanisms, and demonstrate that by controlling microglial activation and neuroinflammation the prevalence of AD may be decreased.

First draft submitted: 2 June 2016; Accepted for publication: 28 July 2016; Published online: 7 September 2016

Keywords Alzheimer’s disease (AD) is the most common disease onset and progression. To date, the exact • Alzheimer’s disease neurodegenerative disease and one of the lead- mechanisms underlying the pathogenesis of AD • microgliosis ing causes of morbidity and mortality world- remain unclear. The involvement of amyloid-beta • modifiable risk factors wide. Over 46 million people were estimated and tau protein deposition in disease progression • neurodegeneration to be affected by AD worldwide, with numbers is well established, and emerging evidence has • neuroinflammation projected to rise to 131.5 million in 2050 [1] . suggested a causal role for neuroinflammation, Advancements in healthcare have contributed to thereby providing a potential therapeutic target increased longevity in the global population, but for the treatment of AD [4–7]. Microglia, the resi- since AD is an age-related disease, this has led to dent immune cells of the brain in particular, have the dramatic increase in the number of patients been studied for their role in AD progression [8– suffering from AD. The AD-associated cogni- 12]. In response to a variety of infections, trauma, tive decline is a public health concern, which toxins and stimuli, microglia become activated imposes a significant and increasing economic mounting an immune response [13,14]. In their burden [2,3]. activated (M1) state, microglia take on a phago- A better understanding of the factors that cytic (amoeboid) phenotype aimed at removing accelerate the neurodegeneration and cognitive the insult. Once the insult has been removed, decline observed in AD will allow for the devel- microglia return to their resting (ramified) or opment of strategies for preventing or delaying M2 alternatively activated state [15,16]. In AD,

376 Neurodegener. Dis. Manag. (2016) 6(5) future science group Elucidating the link between the modifiable risk factors of Alzheimer’s disease & neuroinflammation Special Report however, the predominant microglial phenotype factors during midlife may affect and potentially is M1 resulting in a chronic neuroinflammatory delay the onset of neuroinflammation associated environment accompanied by the increased and with AD. sustained release of proinflammatory mediators, including IL-1β, TNF-α, MCP-1 and reactive Lifestyle-associated risk factors oxygen species, which have all been shown to It is accepted that chronic inflammation in the modify disease progression [4,17–20]. Since a large peripheral tissues can affect neuroinflammation body of evidence supports the involvement of in the brain [26–30]. Therefore, strategies that chronic neuroinflammation in the pathogenesis attenuate neuroinflammation directly or indi- of AD, particularly during the early stages of the rectly by reducing systemic inflammation may disease, any strategies that can attenuate or pos- prove beneficial in the prevention or treatment of itively modify the inflammatory environment neurodegenerative diseases such as AD. One fac- may prove to be successful. tor that has been shown to affect inflammation in Currently, there are few approved pharma- the periphery is the diet that a person consumes. cological treatments for AD, such as donepezil, rivastigmine, galantamine and memantine [21], ●●Diet which only slightly ameliorate the clinical signs Dietary components such as fiber, antioxidants, and their progression during the late stages of carotenoids, vitamins and fatty acids have all the disease [22]. By and large, the pharmacologi- been shown to modulate different inflamma- cal attempts to cure or halt disease progression tory responses, suggesting a link between nutri- have been unsuccessful [23]. Thus, disease pre- tion and risk of developing AD [31–34]. Certain vention during the preclinical stage prior to any fruits and vegetables, as well as wine, tea and onset of symptoms, at this point, is likely the coffee contain polyphenols, which are secondary most effective way to decrease the incidence of plant metabolites that have well-characterized sporadic (late-onset) AD. The risk factors asso- anti-inflammatory and neuroprotective effects. ciated with AD can be separated into two cat- Murine microglial BV-2 cells stimulated with egories: the non-modifiable risk factors such as lipopolysaccharide (LPS) following treatment age [3] and family history (e.g., familial, early- with blueberry homogenate, which is rich in onset AD) [24,25] and the modifiable risk factors. polyphenols, had significantly reduced expres- In this special report we will highlight some of sion of COX-2 and inducible nitric oxide syn- the modifiable midlife risk factors, which we thase, as well as decreased secretion of nitric have grouped into three categories (lifestyle- oxide (NO), IL-1β and TNF-α [35]. Similar associated, vascular and psychological) (Box 1). results were achieved by pretreating BV-2 We will also describe how modifying these risk cells with walnut, strawberry and acai berry

Box 1. Highlight of the proposed risk factors for Alzheimer’s disease. Non-modifiable ●● Age: ūū After the age of 65 years the risk of developing AD doubles every 5 years ●● Family history and genetic background: ūū Gene mutations associated with familial AD are located in genes encoding APP, PSEN1 and PSEN2 ūū Sporadic AD is associated with the genes encoding ApoE Modifiable ●● Lifestyle-associated risk factors: ūū Diet ūū Physical inactivity and exercise ●● Vascular risk factors: ūū Type 2 diabetes mellitus ūū Obesity ●● Psychological risk factors: ūū Major depressive disorder ūū Stress AD: Alzheimer’s disease.

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homogenates [36]. Other studies have explored which are typically composed of high ratios the anti-inflammatory effects of the curry spice (∼16:1) of Ω-6 to Ω-3 PUFAs, promote the curcumin [37] and caffeine [38,39] (reviewed in pathogenesis of cardiovascular disease, cancer more detail in Schindler et al. [40]). and autoimmune and inflammatory diseases [53]. The largest body of evidence linking proper While these results are promising, at present, no nutrition to reduced incidence of AD comes broadly accepted dietary recommendations exist from studies on the role of Ω-3 polyunsaturated on PUFA consumption to delay or prevent the fatty acids (PUFAs). Some examples of com- onset of cognitive decline. Moreover, some stud- mon Ω-3 PUFAs include docosahexaenoic acid ies indicate that PUFA supplementation might (DHA), eicosapentaenoic acid and their precur- be beneficial only in nonapolipoprotein E e4 sor α-linolenic acid [41] . These Ω-3 PUFAs are carriers [54]. In general, the overall quality and not produced by the human body de novo, and composition of the diet, and not a single nutri- as such need to be obtained from food, primarily ent, remain important in contributing to the through the regular consumption of fish, seeds protection against diseases, such as AD. and green leafy vegetables, as they are essential for normal neurodevelopment and brain health [32]. ●●Physical inactivity & exercise DHA levels in the brain of AD patients have Over the past 10 years, epidemiological studies been shown to decrease with increasing age, assessing the effectiveness of physical activity therefore studies have reported health benefits (PA) as an intervention strategy for neurodegen- of using Ω-3 supplementation in early AD [32,42]. erative diseases have demonstrated that PA can A prospective study of 815 elderly individuals, attenuate neuroinflammation and limit disease who were followed up for an average of 3.9 years, progression. A meta-analysis conducted in 2009 demonstrated that weekly consumption of fish concluded that the risk of developing AD can be reduced the incidence of AD by 60% [43]. These reduced by 45% with the introduction of PA [55]. observed health benefits may be, in part, due In total, 12.7% AD cases in the world (20.3% to the ability of Ω-3 PUFAs to modulate glial in Europe) in 2010 could be attributed to a lack cell activation and promote the switch to the of PA [56], which is similar to results reported M2 phenotype, thereby reducing neuroinflam- by Barnes and Yaffe demonstrating that a 25% mation [44–46]. Pretreatment of mice microglia reduction in inactivity could prevent nearly one with DHA and subsequent activation with Toll- million AD cases worldwide [57]. like receptor agonists, resulted in a significant Physical exercise (PE) is defined as repetitive increase in glutathione levels, thus enhancing the and purposeful PA generally used to improve antioxidant response by these cells. Furthermore, physiological, physical and functional capaci- DHA achieved an anti-inflammatory effect by ties [58], and has also been shown to protect decreasing the release of the proinflammatory against cognitive decline [59]. An additional ben- mediators IL-6, TNF-α and NO by micro- efit associated with regular PE is increased car- glia [47]. Similar anti-inflammatory effects were diac output leading to increased cerebral blood observed for eicosapentaenoic acid both in the flow. This, in turn, contributes to increases in periphery and in the CNS [48,49]. angiogenesis, synaptogenesis and neurogenesis However, not all PUFAs induce an anti- in different brain areas involved in cognition and inflammatory effect. The Ω-6 PUFAs are pri- memory [59]. The exact molecular mechanisms marily consumed as linoleic acid or arachidonic by which PE exerts its neuroprotective effects acid [41] . Supplementation with an elevated 7:1 are still not completely understood. However, ratio of arachidonic acid to Ω-3 PUFAs resulted animal studies have proposed the neuroimmune- in a two-fold increase in the production of pros- modifying abilities of PE and accompanying taglandin E2 by rat leukocytes; dysregulation of overall anti-inflammatory effect as a potential this eicosanoid is associated with chronic inflam- explanation for the observed neuroprotective mation and AD [50,51]. Despite the opposing effects [58,60–63]. Kohman et al. [60] reported that effects of Ω-3 and Ω-6 PUFAs, Pischon et al. [52] voluntary wheel running in aged mice reduced determined that when consumed in a balanced microglia proliferation in the dentate gyrus 1:1 ratio by men and women in USA, the low- (∼1.5-fold) and resulted in a morphological shift est levels of inflammatory markers, including to a proneurogenic phenotype characterized by C-reactive protein, and soluble TNF receptors 1 the expression of IGF-1. In addition, significant and 2, were observed. In contrast, western diets, increases in neurogenesis (∼50%) were observed.

378 Neurodegener. Dis. Manag. (2016) 6(5) future science group Elucidating the link between the modifiable risk factors of Alzheimer’s disease & neuroinflammation Special Report

Other studies have shown that wheel running worldwide [68]. High midlife BMI and obesity can attenuate the LPS-induced reductions in increases the risk of developing AD by up to two- neurogenesis, and increase the proportion of fold, while T2DM is associated with a relative risk BDNF-expressing microglia in aged mice [64]. of 1.54 for developing AD [57]. Therefore, it is inev- Leem et al. [62] reported a decrease in microglial itable that as the incidence of these two diseases activation (up to 2.7-fold), as indicated by the increases, the incidence of AD will also rise. reduced expression of the microglia marker Iba-1, One potential explanation for the link in rodent models of AD following exposure to between obesity, T2DM and AD may lie with moderate or intense PA. This coincided with the insulin resistance and dysregulation that can reduced expression of the inflammatory markers lead to vascular and neuronal damage, thus caus- COX-2, IL-1β IL-6, MCP-1 and TNF-α [62]. ing reduced cognitive function [69,70]. However, To date most studies have been conducted in the state of chronic peripheral inflammation mice; however, several novel studies with human that is observed in obesity and T2DM may also subjects have demonstrated that PE can protect provide some insight. High adiposity environ- against the onset of neurodegenerative diseases, ments are typically characterized by elevated such as AD, and can contribute to slowing levels of circulating proinflammatory cytokines, down disease progression [59,65]. A randomized including IL-6, IL-1β and TNF-α, increased controlled trial with older adults demonstrated infiltration of M1 macrophages into the adipose that one year of moderately intense aerobic exer- tissue [71,72]. In addition, decreased endothelial cises, defined as three 40-min sessions per week, function has been observed in T2DM and obe- increased the hippocampal volume by 2%, com- sity, which could lead to a leaky blood brain pared with the adults in the stretching group who barrier, thus allowing for an influx of proin- experienced a 1.4% decline over that same time flammatory mediators from the periphery into period [66]. Moreover, the increase in hippocampal the CNS [73]. Animal studies with obese mice volume was associated with higher levels of serum showed that infiltration of macrophages from BDNF [66]. Although additional human studies the periphery to the CNS increased by 53% are needed to further investigate the effects of compared with controls [74]. This infiltration, exercise on neuroinflammation, the results thus in turn, can initiate and propagate a neuroin- far are promising. Exercise could be a valid thera- flammatory environment, such as the one found peutic strategy for reducing the incidence of neu- in AD. Cognitive impairment due to neuroin- rodegenerative diseases such as AD that possess a flammation associated with increased levels of strong neuroinflammatory component [58–59,62]. IL-6, IL-1β and TNF-α in the hippocampus and enhanced activation of microglia within the Vascular risk factors CNS has been demonstrated in obese rodents The prevalence of lifestyle-associated diseases, and diabetic rats [75,76]. such as obesity and Type 2 diabetes mellitus The common underlying feature linking (T2DM) have been steadily increasing over the obesity and T2DM with a risk for developing past years and have been putting significant AD may be the chronic inflammatory environ- stress on healthcare systems worldwide. Obesity ment present both in the peripheral tissues and and T2DM are well known risk factors for a vari- in the brain. Therefore, patients suffering from ety of other diseases, including AD [57]. Due to T2DM or obesity could attenuate the periph- the similarities and overlapping clinical manifes- eral inflammatory environment by introduc- tations, obesity and T2DM have been grouped ing regular exercise and a more balanced diet. together as vascular risk factors for AD and will This, in turn, could contribute to decreasing the be discussed jointly in this section. proinflammatory environment in the brain and repairing vascular dysfunction, thereby poten- ●●Obesity & diabetes mellitus tially decreasing the risk for developing dementia According to WHO, more than 1.9 billion adults or AD later in life. (over the age of 18 years) and 600 million children (under the age of 18 years) are obese worldwide [67]. Psychological risk factors Obesity, defined as a BMI over 30, is also a risk fac- Increased levels of perceived stress and a history tor for T2DM and cardiovascular disease, which of depression are associated with an increased has contributed to T2DM becoming a grow- risk of developing dementia [57,77]. Moreover, ing epidemic, affecting over 347 million people major depressive disorder (MDD) is often one

future science group www.futuremedicine.com 379 Special Report Schindler & Klegeris

of the earliest neuropsychiatric abnormalities to effects of SSRIs, specifically sertraline, have also develop in AD patients. The high comorbidity been detected [87]. Moreover some studies have between AD, stress and MDD may be, in part, detected no significant effect of antidepressants due to the underlying inflammatory mechanisms on cognitive abilities of patients suffering from that are present in all three conditions. MDD [88,89]. Due to these mixed results, additional studies are required to determine the long- ●●Major depressive disorder term effects of antidepressants on cognitive abili- MDD is primarily characterized by an imbalance ties. However, the initial research highlighting the in a number of key monoamine neurotransmit- anti-inflammatory effects of some of these antide- ters of the brain, such as serotonin (5-HT), nor- pressants, may encourage using early MDD treat- epinephrine, dopamine and epinephrine. These ment as a novel therapeutic option for potentially neurotransmitters have neuroimmune modi- delaying dementia and AD onset. fying abilities and can contribute to glial cell activation; as a result, their dysregulated secre- ●●Stress tion can have pathological outcomes including Increasing evidence links high levels of per- chronic neuroinflammation [78]. Patients with ceived stress with an increased risk of developing MDD have been found to have elevated plasma AD [77]. The hippocampus, which is one of the and CSF levels of proinflammatory cytokines, main areas of the brain that is damaged during including IL-6 and TNF-α [79]. Similar results the progression of AD, is also one of the first were obtained by Liu et al. [80] using a stress- regions to be targeted by stress hormones includ- induction animal model, showing decreased ing cortisol, sympathetic and parasympathetic 5-HT levels, increased IFN-γ and TNF-α levels neurotransmitters and cytokines [90]. Acute stress in the prefrontal cortex. They also determined accompanied by increases in glucocorticoids that regular swimming exercise could ameliorate can improve cognitive processes, while chronic the depressive symptoms and reverse the levels stress along with persistently elevated levels of of 5-HT, IFN-γ and TNF-α [80]. Others have glucocorticoids is associated with a decrease in demonstrated that the increase in proinflam- hippocampal function and volume, resulting in matory cytokine levels in MDD exacerbated cognitive deficits [91]. The hippocampus is also neurodegeneration caused by hippocampal involved in shutting down the stress response and frontal cortex atrophy [81] . Due to the high initiated by the hypothalamus–pituitary– overlap in brain areas affected and correlation adrenal axis. Therefore, damage and atrophy of between MDD and AD, MDD may be consid- the hippocampus impairs the shut off mecha- ered an early symptom of neurodegeneration as nism, resulting in sustained hypothalamus– concluded by a 14-year study of healthy patients pituitary–adrenal response and ultimately who developed MDD, followed by AD later in an exacerbated neuroinflammatory environ- life [82], thereby highlighting the importance of ment [92]. Moreover, chronic unpredictable treating MDD as early as possible. mild stress promotes microglial proliferation Several randomized controlled trials have and activation, and increased levels of TNF-α found that treating depression in older adults and IFN-γ in the prefrontal cortex of mice [80]. results in improved cognitive function [83,84]. These neuroinflammatory mechanisms induced Moreover, a number of studies have highlighted by stress likely contribute to neuronal dysfunc- the anti-inflammatory effects exerted by some tion and impaired neurogenesis, which eventu- classes of antidepressants. Clomipramine and ally progress to AD. Therefore, it is important imipramine, which are tricyclic antidepressants to find interventions that may decrease stress in decreased the production of NO and TNF-α, order to prevent cognitive decline. as well as the expression of inducible nitric oxide synthase, IL-1β and TNF-α at the mRNA level Conclusion in murine microglial cultures [85]. Similar results AD is now considered an epidemic of the 21st were obtained for fluoxetine and paroxetine, selec- century [1] . This neurodegenerative disease is tive serotonin reuptake inhibitors (SSRIs), which characterized by dysregulated microglial activa- reduced microglial activation in two separate tion leading to the increased and sustained release in vivo models, and decreased the release of IL-1β, of proinflammatory mediators [93]. The resulting IL-6, TNF-α and NO in microglial cell lines acti- neuroinflammatory environment can contribute vated by LPS [86]. In contrast, proinflammatory to the collateral damage of neurons leading to the

380 Neurodegener. Dis. Manag. (2016) 6(5) future science group Elucidating the link between the modifiable risk factors of Alzheimer’s disease & neuroinflammation Special Report neurodegeneration observed in AD. Despite our midlife to delay the onset of cognitive decline increased understanding of some of the molecu- and progression to AD later in life. Although lar mechanisms that contribute to AD progres- the results on the effect of diet, PE and antide- sion and pathology, our efforts to develop effec- pressants on ameliorating the chronic neuroin- tive pharmacological treatments have not been flammation of this neurodegenerative disease very successful. None of the currently available are promising, the evidence remains inconsist- medications can reverse symptoms or slow down ent. Therefore, increased numbers of large, the progression of the disease. Consequently, long-term, high-quality randomized controlled research has shifted to primary prevention of AD trials are needed to outline more specific die- – that is, reducing the incidence of the disease tary food guidelines and exercise regimens that by eliminating or targeting specific risk factors, can be used to delay the progression of cogni- which may decrease or delay the development tive decline associated with AD. Consequently, of the disease [94]. We reviewed several modifi- until effective disease-modifying drugs able risk factors that can facilitate and modulate become available, outlining the above men- the neuroinflammatory environment found in tioned guidelines, lifestyle modification strat- AD, and thereby allow for the development of egies and risk factor management will be at novel therapeutic and lifestyle interventions for the center of a concerted effort to fight the this specific disease. Through modifications of AD epidemic. lifestyle, such as increasing physical activity and reducing stress through meditation, both the Financial & competing interests disclosure individual risk and the overall prevalence of AD This work was supported by grants from the Natural can be significantly reduced. Overall, manag- Sciences and Engineering Research Council of Canada ing the modifiable risk factors currently presents (NSERC) and the Jack Brown and Family Alzheimer’s the best strategy for decreasing the incidence Disease Research Foundation. The authors have no other of AD until more effective disease-modifying relevant affiliations or financial involvement with any pharmacological interventions become available. organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed Future perspective in the manuscript apart from those disclosed. This special report highlighted the number of No writing assistance was utilized in the production of different risk factors that can be modified in this manuscript.

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The role of the immune system in neurofibromatosis type 1-associated nervous system tumors

Souvik Karmakar1 & Karlyne M Reilly*,1

Practice points ●● Neurofibromatosis type 1 (NF1) patients carry mutations in the NF1 gene and are at increased risk for tumors in the PNS and CNS. ●● The product of the NF1 gene, neurofibromin, downregulates RAS signaling and many cell types in NF1 patients show a hyperactive RAS phenotype. ●● Mutation of NF1 causes changes in cytokine levels, mast cells, macrophages, microglia, T cells and B cells, suggesting that immune system activities are altered. ●● Mouse models of NF1-associated tumors have helped to clarify the causal relationship between immune cell alterations and tumorigenesis. ●● The role of mast cells, microglia and cytokines in helping to drive tumor progression is providing therapeutic opportunities in molecularly targeted therapies and immunotherapy.

With the recent development of new anticancer therapies targeting the immune system, it is important to understand which immune cell types and cytokines play critical roles in suppressing or promoting tumorigenesis. The role of mast cells in promoting neurofibroma growth in neurofibromatosis type 1 (NF1) patients was hypothesized decades ago. More recent experiments in mouse models have demonstrated the causal role of mast cells in neurofibroma development and of microglia in optic pathway glioma development. We review here what is known about the role of NF1 mutation in immune cell function and the role of immune cells in promoting tumorigenesis in NF1. We also review the therapies targeting immune cell pathways and their promise in NF1 tumors.

First draft submitted: 31 May 2016; Accepted for publication: 17 August 2016; Published online: 19 December 2016

Neurofibromatosis type 1 Keywords Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic disease in which patients are • B cells • cytokines at risk for tumors in the PNS and CNS (Figure 1). NF1 patients carry mutations in the NF1 gene • immunotherapy encoding the protein neurofibromin, a RasGAP protein that acts to downregulate active Ras sign- • malignant peripheral nerve aling. NF1 affects one in 3500 people and can be inherited or occur sporadically. Although NF1 sheath tumor • mast cells can affect many different organ systems, effects on the nervous system are a defining feature. In • microglia • neurofibroma the peripheral nervous system, NF1 is characterized by different Schwann cell tumors, particularly • neurofibromatosis type 1 dermal neurofibromas and plexiform neurofibromas (PNF) that can progress to malignant periph- • optic pathway glioma eral nerve sheath tumors (MPNST). In the CNS, NF1 children are at an increased risk for optic • T cells

1Rare Tumors Initiative, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 37 Convent Dr, Bethesda, MD 20814, USA *Author for correspondence: [email protected] part of

pathway gliomas (OPG) and adults with NF1 the tumor location. Standard of care for PNF is are at risk for astrocytoma and glioblastoma, all surgery, although surgery may not be feasible, tumors of glial cells. due to the location of the tumor. New therapeu- Neurofibromas are complex tumors involving tic options, particularly for patients for whom many cell types and occur in almost all NF1 surgery is not an option, are desperately needed, patients. The peripheral nerve fiber normally despite an extensive history of clinical trials in consists of Schwann cells, perineurial cells, fibro- NF1 [5]. blasts, and endothelial cells and all of these cells PNFs can transform to MPNST, an aggres- can be found embedded in neurofibromas. The sive sarcoma. The lifetime risk for MPNST in initiating tumor cell has been shown to be NF1- patients with NF1 is 8–16% [6,7]. Recent studies null Schwann cells [1,2] in which the second wild- have shown that the transformation from PNF type copy of NF1 has been mutated or deleted; to MPNST can go through an atypical neu- however, NF1 heterozygous stromal cells are rofibroma stage that frequently has mutation of INK4A also critical to tumorigenesis. Neurofibromas are CDKN2A, encoding p16 [8]. Once MPNSTs divided into groups based on clinical features. have formed, TP53, encoding p53 [9], and/or Dermal (or cutaneous) neurofibromas occur on SUZ12, encoding a subunit of the PRC2 com- the surface of the skin in more than 90% of plex, are found [10,11] . Fifty percent of MPNSTs NF1 patients and can be particularly disfiguring. can also occur sporadically in the absence of a Subcutaneous neurofibromas occur below the preexisting PNF or NF1. NF1-associated and epidermal layer. PNFs are associated with major sporadic MPNST do not significantly differ in nerves and can occur in both superficial and chromosomal alterations [12], gene expression deep tissues. These PNFs occur in up to 50% profiles [13,14] or RAS pathway phosphoprotein of NF1 patients and are highly invasive, often expression [15,16], although there is some indi- spreading along the entire nerve branch [3,4]. cation that BRAF mutations occur in sporadic Although benign, they can have serious mor- MPNST, but not NF1-associated MPNST [17,18]. bidity or mortality for patients depending on NF1-associated MPNSTs have poorer prognosis

Central nervous system Astro/GBM

OPG

PNF

MPNST Peripheral nervous system

Figure 1. Development of nervous system tumors in NF1 patients. GBM: Glioblastoma multiforme; MPNST: Malignant peripheral nerve sheath tumors; OPG: Optic pathway gliomas; PNF: Plexiform neurofibromas.

10.2217/cns-2016-0024 CNS Oncol. (Epub ahead of print) future science group The role of the immune system in neurofibromatosis type 1-associated nervous system tumors Review than sporadic MPNSTs and this may be due to a role in tumors of the nervous system in NF1, the young age of onset and/or large volume at particularly as it relates to candidate therapeutics. time of diagnosis, given that NF1-associated MPNSTs form within an existing PNF that Method confounds diagnosis [19–22] . The current stand- To identify literature for this review, PubMed [30] ard of care for MPNST is surgery and chemo was searched using the terms Neurofibromatosis therapy [23], such as doxorubicin, etoposide and type 1; immunotherapy of cancer; tumor isofosfomide, as well as radiation therapy in microenvironment; T lymphocytes; mast cell some cases [20], but these treatments are seldom and inflammation; immunology of CNS; as curative, except in some cases of complete surgi- well as combinations between diseases terms cal resection, and better targeted therapies are (neurofibromatosis; plexiform neurofibroma; being actively sought [5]. malignant peripheral nerve sheath tumor or OPG affect 15–20% of children with NF1, MPNST; optic pathway glioma; astrocytoma; typically under 7 years of age. They form along glioblastoma) and mouse model or clinical trial optic nerves, chiasm or postchiasmal optic or immune system terms (lymphocyte; mast tracks. Children with OPG can present with cell; microglia; macrophage; IFN; TNF; SCF; vision defects, although roughly half of OPG are RANTES) or drug terms once they were impli- asymptomatic and never require intervention [24]. cated by other searches (ketotifen; imatinib; These tumors can stop growing as children get sunitinib; sorafenib; sulindac; pexidartinib; older, making it challenging to decide between bevacizumab; GC-1008; fresolimumab; ipilu- intervention and ‘wait-and-see’ approaches in mimab; nivolumab; pembrolizumab; tocili- young children with vision deficits. Due to their zumab; oncolytic virus). The NIH clinical trials location and indolent growth behavior, they are database [31] was searched with the above disease rarely surgically removed or biopsied, so less is terms and drug terms to search for unpublished, known about their molecular biology. As in other ongoing clinical trials. NF1 tumors, OPG are cellularly complex, with hyperproliferative astrocytes, microglia, CD31+ Immune surveillance by peripheral endothelial cells, small clusters of progenitor cells immune cells in the CNS and retinal ganglionic cells identified by immu- OPG, anaplastic astrocytomas and glioblas- nohistochemistry [25]. Chemotherapy is the cur- toma in NF1 patients occur within the immune rent standard treatment for OPG in cases where privileged CNS. The blood–brain barrier (BBB) the patient’s vision is impaired [26]; however, new protects the brain by regulating the passage of treatments are being tested [5]. Radiation therapy macromolecules and immune cells between the is avoided in NF1 patients, due to the risk of periphery and the CNS and by blocking patho- developing radiation-induced moyamoya lesions gens from entering the brain. The cerebrospinal or secondary tumors in the brain. fluid (CSF) surrounds the brain and is impor- Anaplastic astrocytoma and glioblastoma tant for the draining of CNS antigens into the multiforme (GBM) are deadly malignant pri- deep cervical lymph nodes, via the meningeal mary brain tumors that occur rarely in NF1 lymphatics and the cribriform plate [32]. The patients, although there is some evidence that permeability of the BBB and access by immune the incidence in NF1 patients is greater than in cells is regulated by cytokines. TGF-β is nor- the general population [27]. NF1 is commonly mally expressed in the brain and inhibits cell mutated in sporadic GBM [28] and can also be lost entry into the brain by blocking production through increased proteosomal degradation of of chemoattractants by astrocytes in the BBB neurofibromin [29]. The prognosis for anaplastic and downregulating adhesion molecules on the astrocytoma and GBM is very poor, and chemo- surface of BBB endothelial cells that immune therapy has thus far provided little improvement cells use to cross the BBB. TGF-β also inhib- despite many ongoing clinical trials. The cur- its proliferation of T cells in the brain under rent standard of care for anaplastic astrocytoma homeostatic conditions [33]. Conversely, TNF-α and GBM is surgery with radiation therapy and and IFN-γ enhance the adhesion properties of chemotherapy that includes temozolomide. the BBB endothelial cells. TNF-α is not present The aim of this paper is to summarize what is in normal, healthy brain, but can be induced known about alterations to the immune system in by inflammation, either in the brain or systemi- NF1 patients and how the immune system plays cally. It increases the permeability of the BBB,

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allowing increased passage of immune cells into in cytokine signaling [47] and NF1-mutant leuke- the brain and increased release of brain antigens mic myeloid cells are hypersensitive to granulocyte- into the periphery [33]. macrophage colony stimulating factor (GM-CSF) making them hyperproliferative [48]. Bone marrow ●●Microglia/macrophages of NF1 children with juvenile myelomonocytic Brain-resident microglia are established prior to leukemia show loss of the wild-type allele of birth and perform many homeostatic and repair NF1, supporting neurofibromin’s role as a tumor functions in the brain. Pathological changes in suppressor in myeloid malignancies [49,50]. the brain cause microglia to upregulate proteins for antigen presentation. Microglia can ●●Cytokine aberrations both present antigens to T cells and induce NF1 patients have significantly higher serum an inflammation response to pathogens in the concentrations of the inflammatory cytokines brains [34]. Macrophages and perivascular cells IFN-γ, TNF-α, IL-6 [51], stem cell factor (SCF) can also cross the BBB to enter the CNS and and midkine [52] compared with healthy indi- present antigens to T cells. These cells concen- viduals, suggesting that NF1 causes a hyper trate in the subarachnoid space where they acti- inflammatory state that may contribute to vate incoming T cells from the periphery [35]. tumorigenesis. Analysis of intracellular cytokine Both microglia and macrophages are found in staining in stimulated lymphocytes has shown astrocytic gliomas with levels of infiltration cor- that the increase in TNF-α and IFN-γ in NF1 responding to tumor grade, suggesting a tumor patients is specific to CD8+ lymphocytes, and + + supportive role in brain tumors [36–39]. IFN-γ increase is found in the CD8 /CD57 effector T-cell subset [53]. This study also found ●●Lymphocytes increased expression of IL-2 in CD8+/CD57+ Because the normal, healthy brain has low levels effector T cells of NF1 patients. Comparison of adhesion molecules on the BBB endothelial of NF1 patients with and without MPNST has cells, T cells are relatively inhibited from enter- found upregulation of IGFBP1 and RANTES in ing the brain parenchyma, and so the surveil- patients with MPNST [51] . While some of these lance of the brain by T cells is relatively low biomarkers and cytokines have been shown, or compared with the density of T cells in periph- are expected, to act directly on tumor cells to eral organs [33]. T cells that have been recently drive proliferation [52], it is likely that they also stimulated in the periphery cross the BBB, act on immune cells to promote activation and highlighting the importance of the draining chemotaxis and, as described above, may alter the CNS antigens in the deep cervical lymph nodes permeability of the BBB. Alternatively, they may where they can be encountered by T cells in the be markers of excessively activated immune cells periphery [33–35]. Cytotoxic T cells are present in NF1 patients. Additional research is needed to in brain tumors [40,41] and the survival of GBM better understand the complex interplay of these patients was found to be correlated to increased factors with tumor cell growth, angiogenesis and numbers of effector T cells within the tumor, activation of immune functions. suggesting a tumor inhibitory role of cytotoxic T cells in GBM [42]. Cytotoxic T cells in the ●●Tumorigenesis & immune suppression GBM microenvironment are suppressed by regu- Case reports on NF1 patients who have under- latory T cells and myeloid derived suppressor gone organ transplant raise the possibility that cells (MDSCs) [43–46]. immune suppression promotes tumorigenesis in NF1 patients. The development of post Immunopathology of NF1-associated lung transplant lymphoproliferative disorder tumors: evidence in humans for the role of and MPNST was observed in an NF1 patient the immune system in NF1 tumors without any clinical evidence of sarcoma [54]. ●●Myeloid malignancies An astrocytoma was reported in another NF1 The increased incidence of myeloid disorders in patient following a heart transplant [55], rais- NF1 patients, including myeloid dysplastic syn- ing the possibility that normal immune func- drome, juvenile myelomonocytic leukemia and tion suppresses malignancies, but in the context acute myelogenous leukemia highlights the dys- of immune suppression after transplantation function of myeloid immune cells in NF1. The malignant tumors can develop. While these NF1 protein product, neurofibromin, plays a role case reports do not conclusively provide a link

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between immunosuppression and tumorigenesis, similarly show infiltration of mast cells [67]. The they highlight the need for further research into difference in the dependence of PNF formation the role of immunosurveillance in inhibiting on Nf1−/+ stroma in these two models has been tumorigenesis in NF1 patients. attributed to the progenitor cell compartments in which Nf1 is mutated in each model. ●●Infiltrating immune cells Infiltrating immune cells from the myeloid line- ●●Malignant peripheral nerve sheath tumors age are characteristic of many tumors associated MPNSTs have been modeled in mice in a vari- with NF1. Mast cells have long been recognized ety of different ways by breeding mutant mice as a major component of PNFs [56]. Immature together or by injection of viral vectors. The mast cells are found in early stage neurofibroma different MPNST models take advantage of and mature mast cells are found in late stage spontaneous loss of heterozygosity of tumor sup- neurofibroma, and there is some evidence that pressor genes, including Nf1, as mice age and/or mast cell stabillizers may block neurofibroma by targeted loss of tumor suppressor genes in formation [57]. One study of NF1 patients found specific cell types using either the Cre-lox sys- elevated serum IgE in a subset of patients exam- tem or injection of lentiviral vectors expressing ined. PNF patients had a higher incidence of ele- shRNAs. While mice mutants for Nf1 alone vated IgE compared with other NF1 patients [58], do not develop MPNST, additional muta- suggesting alteration in the B-cell compartment tions in the tumors suppressors p53, PTEN, INK4A ARF and a potential interaction with mast cells [59]. and p16 /p19 can drive development of Macrophages have also been found at substan- MPNST. Compound heterozygotes for Nf1 tial levels in PNFs and MPNSTs, with higher and Trp53 (encoding the p53 protein in mice) levels in MPNST [60]. Optic gliomas show accu- mutations develop high-grade MPNST through mulation of microglia, a macrophage-type cell spontaneous loss of the wild-type copies of Nf1 of the brain [61,62], as well as altered microglia and Trp53 [68–70]. In addition to spontaneous morphology [63]. Studies in mouse models (see loss of Nf1 and Trp53, loss of expression can be below) have now established causal relationships targeted using the Cre-lox system or expression between infiltrating myeloid cells and nervous of shRNAs against Nf1 or Trp53 using lentiviral system tumorigenesis in NF1. vectors. Models in which a lentivirus-expressing shRNA against Trp53 is injected into the sci- Mouse models for NF1-associated tumors atic nerve of mice with Cre-induced loss of Nf1 The NF1-associated nervous system tumors have in Gfap-expressing (Gfap+) or Postn-expressing all been successfully modeled in genetically engi- (Postn+) cells develop low-grade MPNST, with neered mice that has allowed the role of immune mast cell infiltration of tumors [71]. Targeted loss system cells in tumorigenesis to be better eluci- of both Nf1 and Pten in Dhh+ cells leads to high- dated. Mouse models of PNF, MPNST, OPG grade MPNST with mast cell infiltration [72]. and astrocytoma/GBM have all been generated Targeted loss of Nf1 and Cdkn2a encoding by driving loss of the mouse Nf1 gene in par- p16INK4A/p19ARF by injection of adenovirus ticular cell types or in combination with other expressing Cre into sciatic nerves leads to high- tumor suppressors or oncogenes. grade MPNST with mast cell infiltration [73]. The majority of reported MPNST genetically ●●Plexiform neurofibromas engineered models recapitulate the mast cell PNFs have been modeled in mice using the Cre- infiltration seen in human tumors. lox system of targeted gene mutation, as Nf1−/− mutant mice are embryonic lethal and Nf1−/+ ●●Optic pathway gliomas mutant mice develop cancers late in life [64]. Loss OPGs have been successfully modeled in mice of Nf1 in Krox20-expressing (Krox20+) cells on a by targeted loss of Nf1 in Gfap+ cells on a hetero Nf1−/+ genetic background leads to the develop- zygous Nf1 genetic background [74]. Nf1−/+ ment of PNF [65]. In this case, heterozygosity in stroma has been shown to be important for the stroma for Nf1 mutation was critical for for- OPG development, and Nf1−/+ microglia pro- mation of PNFs and for mast cell infiltration into mote OPG formation [25,75]. This mouse model the tumors [66]. In a separate model, biallelic loss of OPG recapitulates the microglia infiltration of Nf1 in Dhh-expressing (Dhh+) cells on a wild- seen in human OPG [61] as well as changes in type genetic background gives rise to PNF that microglia morphology [63].

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●●Astrocytoma/glioblastoma effect on the growth factor GM-CSF is consist- Many of the techniques used to model MPNST ent with hyperactivation of RAS signaling down- in mice have also been used to model astrocy- stream of the GM-CSF receptor through loss of toma and GBM. Compound heterozygotes for Nf1. Targeted loss of Nf1 in newborn mouse Nf1 and Trp53 on the C57BL/6J background pups gives rise to spontaneous myeloid prolifera- develop astrocytoma and GBM with spontane- tive disease and higher numbers of leukocytes, ous loss of the wild-type alleles [76]. In these mice monocytes and neutrophils [83]. the tumor stroma is heterozygous for both Nf1 and Trp53. Mice with targeted loss of Nf1 in ●●T cells GFAP+ cells on a Trp53 null background develop A direct role for T cells in NF1 nervous system a range of astrocytoma and GBM [77]. Targeted tumorigenesis has not yet been established, but loss of Nf1, Pten and Trp53 on a Trp53 hete- given the advances in T-cell-based immuno rozygous background develop GBM with high therapy it is important to understand the penetrance [78]. Although there are no published changes in T cells caused by NF1 mutation. NF1 characterizations of immune cell infiltrates in patients have lower levels of circulating lympho- de novo tumors in these models, an intracranial cytes overall. The levels of chronically activated injection model of a cell line derived from the CD8+/CD57+ T cells and CD8+/CD27- effec- Nf1/Trp53 compound heterozygote model shows tor T cells goes up in NF1 patients with low increased infiltrations of activated microglia, PNF tumor volume compared with NF1 patients monocytes/macrophages and lymphocytes in with no PNFs, and then decreases with increas- immunecompetent mice [79]. ing tumor burden [53], suggesting that as tumor burden increases the T-cell response becomes The effect of NF1 haploinsufficiency on muted. In Nf1−/+ mice, the thymus and spleen immune cells are hypercellular, with subtle increases in imma- Experimental data from model systems have estab- ture and mature T-cell subsets [84]. Bone mar- lished the role for NF1 mutation in phenotypes row transplant of Nf1 null cells into immune and functions of immune cells. This suggests that compromised mice results in increased thymic the tumor pathology of patients with germline cellularity. Targeted loss of Nf1 in maturing mutation in NF1 involves complex interactions T cells did not lead to increased thymic cellu- between tumor cells and aberrant immune cells. larity, so this effect may be due to interactions Effects on immune cells are both developmental between different cell types in the thymus [85]. and functional, likely related to aberrant RAS In culture, both Nf1−/− and Nf1−/+ thymocytes signaling due to lower levels of neurofibromin. as well as mature splenic T cells proliferate less after IL-2 and CD3 stimulation than wild- ●●Hematopoeitic progenitor cells type [84,86], whereas unstimulated proliferation Hematopoeitic progenitor cells isolated from Nf1 of Nf1−/+ thymocytes is increased compared with null mouse fetal livers form more colonies and are wild-type. Loss of Nf1 in maturing T cells leads hyper-responsive to growth factors in colony form- to inhibition of positive selection in the con- ing assays when compared with wild-type pro- text of MHC class I restricted T-cell receptor genitors [80,81] . Effects were observed in granulo- transgenic mice and fewer naive T cells in the cyte/macrophage progenitors, as well as primitive spleen [85]. These data suggest that the effect of stem/progenitor cells. Significantly higher num- Nf1 mutation on T cells is very specific to differ- bers of primitive stem/progenitor cells were found ent stages of T-cell maturation and stimulation, in Nf1 null fetal livers and Nf1 null fetal progeni- and may depend on Nf1 mutation in other cells tor cells were better able to repopulate mouse bone of the immune compartment. Because some of marrow after irradiation of mouse hosts compared the effects on T cells can be seen in Nf1−/+ cells, with wild-type progenitor cells. The engraftment these data have implications for NF1 patients of Nf1 null progenitor cells is dependent on the with germline mutations in NF1. growth factor GM-CSF in the microenvironment, such that postengraftment myeloid cell numbers ●●B cells are much lower in Gmcsf−/− mice engrafted with Similar to T cells, a direct role of B cells in NF1 Nf1−/−;Gmcsf−/− progenitors compared with wild- nervous system tumorigenesis has not been type mice engrafted with Nf1−/−;Gmcsf+/+ progeni- established, but B cells play a role in activat- tor cells [82]. This dependence of the Nf1 mutant ing T cells and mast cells and therefore may

10.2217/cns-2016-0024 CNS Oncol. (Epub ahead of print) future science group The role of the immune system in neurofibromatosis type 1-associated nervous system tumors Review indirectly affect NF1 nervous system tumors. at the effects of different immune cell com- Neurofibromin has been found to relocalize in partments on tumorigenesis and whether B cells upon activation of surface Ig [87], sug- haploinsufficiency for Nf1 in the immune cell gesting a role in Ig signaling and the possibility compartments accelerates tumor formation or that mutation of NF1 impairs B-cell activation. growth. These experiments have also begun to Comparison of Nf1−/+ B cells and wild-type B elucidate which signaling molecules mediate the cells showed subtle gene expression changes interactions between immune cells and tumors. with most expressions differences being less than 1.5-fold [88]. Mouse and human cross- ●●Mast cells in PNF species comparison of Nf1−/+ versus wild-type Experiments in mouse models of neurofibromas B-cell gene expression found increases in cell comparing tumorigenesis on Nf1 heterozygous cycle and DNA repair genes with inhibition of versus wild-type genetic backgrounds suggest genes involved in the immune response. Nf1−/− B that Nf1 heterozygosity in the tumor stroma, par- cells proliferate well in response to IL-4 and anti- ticularly in the bone marrow, is critical to drive bodies to CD40, but do not proliferate as well tumorigenesis [65,66]. However, the dependence as Nf1−/+ B cells in response to stimulation by on Nf1 heterozygous stroma may vary with the antibodies to IgM [86]. Signaling downstream of developmental stage at which the neurofibromas IgM was found to be impaired in Nf1−/− B cells. initiate, since tumors initiating through Nf1 loss in Krox20+ cells require Nf1 heterozygous bone ●●Mast cells marrow, whereas Nf1 loss in Dhh+ cells or in post- Nf1−/+ mice have slightly higher levels of mast cells natal Plp+ cells drives neurofibromas on a wild- and haploinsufficiency forNf1 rescues loss of mast type stroma background [67,94]. Alternatively, cells in c-Kit mutant mice [89]. Mast cells from differences in genetic background or pathogen Nf1−/+ mice form more colonies in culture, suggest- status of the mice in these different experiments ing that the role of Nf1 haploinsufficiency is cell- could explain the variation in the requirement autonomous. Increased proliferation of Nf1−/+ mast for Nf1 heterozygous stroma. The evidence that cells is dependent on RAS/MEK/RAC2/PAK1 Nf1 heterozygous stroma contributes to neuro signaling [90,91] consistent with hyperactivation fibroma formation has led to the hypothesis of RAS signaling when neurofibromin levels are that mast cells are a critical factor in promoting reduced. Nf1−/+ mast cells are also hypersensitive neurofibroma growth. Whether or not Nf1−/+ to migratory stimuli and degranulation signals mast cells are necessary for neurofibroma for- secreted by Schwann cells [92,93]. mation, they likely promote tumorigenesis in NF1 patients. Nf1−/+ mast cells have enhanced ●●Microglia functions that can contribute to tumorigenesis Microglia from Nf1−/+ mice proliferate faster than including increased migration and secretion of wild-type microglia in primary cultures [61,75] growth factors that modify fibroblast behavior, and show twofold increased migration both in collagen secretion and remodeling of the tumor culture and in response to brain injury [75]. Nf1−/+ microenvironment [66,95]. microglia also show higher expression of several transcripts including Mgea5, Ptn, Jag1 [61], ●●Microglia in optic pathway glioma Ptgs2, Il6, Nos2 and Tg fa [75]. In contrast to the Similar to neurofibromas, OPG have shown relationship between mast cells and Schwann mixed results on the requirement for Nf1 hetero cells, Nf1−/+ microglia do not appear responsive zygous stroma. Loss of Nf1 in astrocytes and to astrocyte cultured media, although only pro- glial progenitors gives rise to OPG whether or liferation has been examined thus far [61], and not the stroma is Nf1 heterozygous [74]. When other microglia functions may be affected in the Nf1 is lost in astrocytes specifically, and not in interaction of astrocytes and Nf1−/+ microglia. glial progenitors, Nf1 heterozygosity is required in the genetic background for OPG to form [96]. Immunomodulation of NF1-associated Interestingly, injection of Nf1 null astrocytes tumors: evidence in model systems for into the brains of Nf1 heterozygous mice does causal role of the immune system in NF1 not lead to tumors, suggesting the developmen- tumors tal timing and/or location is critical for tumors The development of mouse models for NF1 to form. Several studies have shown that Nf1−/+ tumors has led to rigorous experiments looking microglia can promote OPG. Nf1−/+ microglia

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secrete factors that promote growth of Nf1 null microglia [75] and has been suggested to play a astrocytes [61,63]. Inhibition of microglia, either growth promoting role in OPG. It is upregu- pharmacologically [61,75] or genetically [97], lated in Schwann cells in which Nf1 is knocked inhibits the growth of OPG in vivo. down [99] and promotes mast cell development and activation that may in turn favor PNF ●●Macrophages tumorigenesis. In combination with TGF-β it The role of macrophages in NF1-associated nerv- induces MDSCs to accumulate in the tumor ous system tumors has not been thoroughly stud- microenvironment and upregulates their abil- ied. A study using a c-KIT/FMS kinase inhibitor, ity to suppress effector T cells [100], potentially Pexidartinib, in the Dhh-Cre; Nf1flox/flox mouse blocking an antitumor immune response. model of PNF provides intriguing evidence for a different role for macrophages in early and late ●●IFN-γ neurofibroma tumorigenesis [60]. Macrophage IFN-γ plays a crucial role in inducing Th1 numbers are very high in plexiform tumors and immune response through CD4+ T cells, leading numbers of macrophages go up with increased to CTL recruitment in the tumor microenviron- + malignancy, from normal nerve to PNF to ment [101] . CD8 T cells express IFN-γ within MPNST. Early inhibition of the c-KIT signal- tumor tissue that upregulates the immune ing pathway, important for mast cells, and the checkpoint proteins PD-L1 on tumor cells and FMS kinase signaling pathway, important for PD-1 on CD8+ T cells, shutting down a cyto- macrophages, led to increased tumor growth [60]. toxic antitumor T-cell response [101–103]. IFN-γ Paradoxically, inhibition of these pathways once may also affect the immune response in the the tumors are established led to a subset of brain by increasing the expression of proteins tumors shrinking, with the shrinkage correlat- on BBB endothelial cells necessary for T-cell ing better to numbers of macrophages than to transmigration into the brain [33]. numbers of mast cells. The conflicting role of macrophages in tumor initiation and progression ●●TNF-α needs to be better understood before pharmaco- Mast cells secrete TNF-α and trigger the Th1 logical intervention can be considered. Careful immune response [104] . TNF-α acts together with mouse genetic experiments are needed to dissect IFN-γ to upregulate expression of PD-L1 in tumor out the individual roles of macrophages and mast cells, blocking cytotoxic T-cell effects [101,103]. cells in the initiation and progression of PNF. Chronic dysregulation of TNF-α synthesis in the tumor microenvironment activates fibroblasts Implications of cytokine aberrations for and macrophages and leads to increased angio- immunomodulation in NF1 genesis and tissue remodeling [105] . It promotes Although it has not been thoroughly studied in MDSC development in the bone marrow and NF1, the high concentrations of cytokines found in recruitment to the tumor microenvironment, the serum of NF1 patients may alter immune func- augmenting immunosuppression [106] . It can also tion in favor of immune suppression and tumori- increase permeability of the BBB, increasing the genesis. IL-6, IFN-γ and TNF-α are upregulated number of immune cells in the brain [33]. in NF1 and all play immune suppressive roles. SCF is upregulated in NF1 patient serum and plays a ●●SCF tumor supporting role through its activity on mast SCF regulates mast cell differentiation, prolifera- cells. The chemokine RANTES/CCL5 is found tion, degranulation and cytokine secretion [107] . at higher levels in the serum of MPNST patients As described above, many of the cytokines and is overexpressed by optic pathway glioma asso- secreted by mast cells promote immunosup- ciated microglia. The effect of RANTES on the pression. Increased SCF release by Nf1-mutant immune system could be both immunosuppressive Schwann cells activates the c-KIT receptor on and immune activating. Nf1+/− mast cells leading to mast cell proliferation and their accumulation in the neurofibroma ●●IL-6 microenvironment. IL-6 is produced by tumor-infiltrating myeloid cells [98]. It promotes cancer development ●●RANTES and protects cancer cells from apoptosis. It is RANTES is expressed by immune cells and pro- more highly expressed in Nf1 heterozygous motes the migration of effector T cells, memory

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T cells, neutrophils, monocytes, eosinophils, mast cell infiltration, and increase apoptosis in dendritic cells and macrophages. This could a mouse model. In a compassionate use case, increase the level of immune cells in the tumor imatinib treatment resulted in a 70% reduction microenvironment for both antitumor effects in a PNF [66]. In a Phase II study of imatinib and immunosuppressive effects. RANTES is part in NF1 patients with PNF, roughly a quarter of a cytokine cluster on chromosome 17q11.2 of patients showed reduction in tumor volume, that includes MIP, CCL14, and CCL16, all of although all of the tumors that shrunk were very which encode chemoattractants for immune small when enrolled in the study [110] . effector cells. Because these genes are linked to the NF1 gene, it will be interesting to com- ●●Sunitinib pare the immune response to tumorigenesis in Sunitinib is a multityrosine kinase inhibitor, patients with large deletions on chromosome 17, with activity against VEGFR, PDGFR and compare to patients with mutations limited to c-KIT, and similar to imatinib is predicted to the NF1 gene. It is possible that RANTES plays inhibit PNF growth by multiple mechanisms. different roles at different stages of tumorigen- In preclinical models, sunitinib reduced PNF esis. As a macrophage chemoattractant, it may be number and volume in mice, and altered the cell tumor suppressive at early stages of tumorigen- ular and biochemical properties of Nf1 hetero esis and tumor promoting at later stages, consist- zygous mast cells and fibroblasts in culture [111]. ent with the effects of inhibiting macrophages at Sunitinib is approved for use in renal cell carci- different times during tumor development [60]. noma and gastrointestinal stromal tumors, however, a Phase II clinical trial (NCT01402817) of Molecularly targeted therapy for NF1- sunitinib in PNF patients has been suspended associated tumors directed against the at the time of this writing, and so it remains to immune system be seen whether sunitinib will show benefit in Several therapies expected to modulate immune NF1 patients. cell function have been tested in patients or in animal models. These molecularly targeted ther- ●●Sorafenib apies are predicted to inhibit immune cells in the Sorafenib is a multikinase inhibitor with inhi- tumor microenvironment, with or without inhibition of VEGFR, PDGFR, RAF, and to a bition of tumor cell growth, thereby blocking lesser extent c-KIT. It has been shown to inhibit supportive effects of immune cells on the tumor. tumor growth in a mouse plexiform model [112] . Unfortunately, it was not tolerated by pediat- ●●Ketotifen ric patients in a Phase I study [113] and either Ketotifen is a mast cell stabilizer that is expected no tumor response or acceleration of tumor to block secretion of many of the cytokines that progression was observed in studies on OPG affect immune cell function in the tumor and (NCT01338857) [114], PNF (NCT00727233) [113] promote tumor growth. Case reports on ketotifen and MPNST (NCT00245102). show promising results for neurofibroma when used early as a prevention method [108] . An open- ●●Sulindac label double-blind clinical trial looking at pain Sulindac is a nonsteroidal anti-inflammatory and itching found favorable effects of continu- drug that inhibits multiple biological functions ous ketotifen administration [109] . Additional through COX1/2-dependent and independent independent studies are needed to assess any mechanisms [115,116]. Sulindac inhibits the bio- potential benefit of ketotifen on PNF growth. activity of IL6 [117], as well as inhibiting TGF-α and TGF-β and reduces the number of macro ●●Imatinib phages in the tumor microenvironment [118]. Imatinib is a multityrosine kinase inhibitor, Sulindac has been shown to inhibit MPNST with activity against c-KIT, PDGFR and ABL. cells in culture by inducing apoptosis [119], and so It has been predicted to inhibit PNF growth could inhibit MPNSTs at multiple key pathways. at several key points. The inhibition of c-KIT It has not been tested in NF1 patients yet. blocks mast cell proliferation and the inhibition of PDGFR blocks tumor cell proliferation, as ●●Pexidartinib well as angiogenesis and fibroblast proliferation. As described above, pexidartinib inhibits FMS Imatinib was found to shrink PNFs, reduce kinase, the receptor for M-CSF, and c-KIT, the

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receptor for SCF and important in mast cell pro- diseases, such as Crohn’s disease [127] and rheuma- liferation and migration. Treatment of mouse toid arthritis [128], as well as cancer [129] . Inhibition tumors after tumor formation was shown to of TNF-α is expected to inhibit mast cell activ- shrink tumors [60]. Phase I clinical trials have ity, reduce the expression of immune check- recently been opened to test pexidartinib in point proteins on Treg cells and tumor cells, and PNF (NCT02390752) and in combination inhibit MSDC activity, all of which could lead to with the mTOR inhibitor sirolimus in MPNST increased immune response against NF1 tumors. (NCT02584647). ●●Bevacizumab Potential immunotherapy for NF1- Bevacizumab is a humanized anti-VEGF anti- associated tumors body. VEGF is a potent angiogenesis inducer. The goal of immunotherapy is to restore or aug- The therapeutic effect of bevacizumab is most ment antitumor immune responses by stimulat- often attributed to a normalization of tumor ing the immune system to eliminate cancer cells blood vessels that causes a decrease in tumor through immune-mediated cell killing processes. volume. In addition, VEGF contributes to Immunotherapeutic approaches include cytokine recruitment of macrophages to tumors, is therapies [120], tumor vaccines that elicit strong secreted by mast cells to inhibit differentiation specific immune responses to tumor antigens [121], of dendritic cells [130,131], thereby downregulat- adoptive cell transfer of genetically engineered ing the antitumor immune response, and is a T cells expressing tumor-specific chimeric anti- proliferation, survival and chemotactic factor gen receptors (CAR T cells) [122], monoclonal for Schwann cells in the peripheral nervous sys- antibodies that target and block cell surface tem [132,133]. Bevacizumab has shown to delay molecules on tumor cells [123], and approaches tumor growth for a variety of tumors, includ- that block immunosuppressive molecules such as ing glioblastoma [134–136] and is showing prom- CTLA-4, PD-1 or Treg cells that dampen effec- ise for vestibular schwannomas associated with tive antitumor immune responses [124]. Aside neurofibromatosis type 2 [137–139]. A trial of from pegylated-interferon-α2b (pegIFN-α2b), bevacizumab in combination with the mTOR most potential immunotherapies have not yet inhibitor everolimus in MPNST is ongoing been tested in NF1-associated tumors. Several (NCT01661283). Bevacizumab has the poten- immunotherapies that have already shown prom- tial to target tumor cells, tumor blood vessels ise in NF1 preclinical models or other tumor and the immune response in NF1 tumors. types may be useful in NF1. ●●Fresolimumab ●●PegIFN-α2b Fresolimumab is a humanized anti-TGF-β anti- IFN-α is secreted by fibroblasts and many types body. TGF-β is secreted by Nf1−/+ mast cells and of lymphoid cells, and activates both the innate induces Nf1−/+ fibroblasts to proliferate and and adaptive immune response. It acts on NK synthesize collagen, a major component of the cells and cytotoxic T cells to upregulate their stroma in PNFs [95]. The activation of Nf1−/+ cytotoxicity and survival [120,125]. It also can fibroblasts occurs through hyperactivated RAS act directly on tumor cells to promote apop- and c-ABL signaling downstream of TGF-β and tosis and can block angiogenesis. Pegylation of inhibition of c-ABL with imatinib was shown to IFNs extend their half-life in patient plasma and inhibit fibroblast proliferation and collagen pro- pegIFN-α2b has been used in a clinical trial of duction. In a mouse model of melanoma, TGF-β PNFs [126] . In the Phase I clinical trial it was was shown to be required for tumorigenesis, and observed that just under 30% of patients had promotes migration of MDSCs into the tumor a 15–22% decrease in their tumor volume and and the conversion of CD4+ naive T cells into many more had a decrease in volume of <10%. Tregs, which play an important role in tumors In addition, close to 70% of patients reported a escaping immune surveillance [140] . Inhibition of decrease in pain symptoms. A Phase II trial of TGF-β has the potential to inhibit PNFs through pegIFN-α2b is ongoing (NCT00396019). both increasing immune surveillance and reducing stromal accumulation. Fresolimumab is ●●Anti-TNF-α antibodies under development for metastatic kidney can- Several humanized antibodies have been devel- cer and malignant melanoma [141] and may show oped to target TNF-α for use in inflammatory benefit in neurofibromas.

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●●Ipilimumab xenograft model in immune compromised Ipilimumab is an anti-CTLA4 antibody that mice [147] . Oncolytic herpes simplex virus selec- has been approved to treat melanoma [142,143]. tively infects cells with hyperactive RAS. It has CTLA4 is expressed on tumor cells and antigen been shown to infect MPNST cells in culture presenting cells and sends an immune suppres- correlating to levels of RAS hyperactivity [148] sive signal to T effector cells. CTLA4 expression and inhibit the growth of MPNST cells in has not yet been evaluated in NF1 tumors, and immunocompromised mice [149] and immune is something to explore in the future. competent mice [150] . A clinical trial of oncolytic herpes simplex virus therapy for MPNST has ●●Nivolumab/pembrolizumab recently been opened (NCT00931931). Viral Both nivolumab and pembrolizumab have been therapy, possibly in combination with therapies developed as anti-PD1 antibodies [144,145]. PD1 that suppress the innate immune response, may is expressed on T cells and mediates immune provide successful therapy for NF1 tumors in suppression. As with CTLA4, the upregulation the future. of PD1 in NF1 tumors has not been thoroughly examined, however, many of the cytokines that ●●Glioma-associated antigens vaccine regulate immune suppression, such as IFN-γ Vaccines for glioma associated antigens have and TNF-α, are upregulated in NF1. A clinical been developed as a way to activate the immune trial of pembrolizumab in MPNST is opening system against gliomas [151] . A study using (NCT02691026). Future studies may point to Nf1;Trp53 mutant astrocytoma and glioblas- an important role for anti-PD1 antibodies in toma cells demonstrated that the glioma antigen treatment of NF1 tumors. EphA2 is expressed in these cells and immu- nized mice generate cytotoxic T cells capable ●●Tocilizumab of killing the astrocytoma cells in vitro [152] . Tocilizumab is a humanized antibody against the These vaccines are currently showing promise IL-6 receptor and interferes with IL-6 signaling. in pediatric low-grade glioma, but have not been It has been shown to downregulate the immu- examined specifically for brain tumors associ- nosuppressive function of MDSCs. Treatment ated with NF1. Characterization of NF1 tumor with tocilizumab enhances T-cell mediated specific antigens could provide another approach antitumor immune responses [100] . As more is to therapy through tumor vaccines or T cells learned about the role of IL-6 and immune sur- engineered to express chimeric antigen receptors. veillance by T cells in NF1, tocilizumab may be a promising therapy. Conclusion & future perspective It is becoming clear that haploinsufficiency for ●●Oncolytic viruses NF1 mutation has a profound effect on the Oncolytic viruses are being developed as therapy immune system, but a clear understanding of due to their ability to replicate in and selectively how these alterations fit together to affect tumor infect cancer cells. They kill cancer cells either progression or tumor suppression is still lacking. by direct lytic activity of the virus life cycle or by Further studies are needed to understand the activating the innate immune response against potential rationale for immunotherapies in NF1 tumor cells. Studies in the Nf1;Trp53 mutant and how response to immunotherapies may or astrocytoma/glioblastoma model show that may not differ in NF1 patients compared with tumor cells are susceptible to oncolytic myx- the general population. Research in mouse mod- oma viral infection and replication in culture, els has demonstrated that mast cells and neuro but are resistant when implanted into immune 0fibroma tumor cells form positive feedback competent mice [146] . Infiltrating macrophages loops, each secreting factors that promote each and monocytes, NK cells, and T cells were all others’ proliferation. Less well understood is how found to play a role in inhibiting viral infec- these mast cells modulate T-cell recruitment and tion in vivo, suggesting that the innate immune whether they are involved in activation or sup- system inhibits anticancer activity in this case. pression of antitumor actions of T cells, dendritic Oncolytic measles virus infects cells expressing cells or NK cells. Cytokine levels are altered in CD46, and has been shown to infect MPNST the serum of NF1 patients, but additional stud- cells selectively compared with normal Schwann ies are needed to assess how these levels change cells. It inhibits tumor growth in an MPNST during different stages of progression of different

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NF1-associated tumors. If cytokine changes are directions for NF1 tumor therapy. Learning how predictive this could become a valuable biomarker to combine different types of immunotherapy for tumor development in NF1 patients. with different molecularly targeted therapies will There is currently great hope for the prom- first require a better understanding of the rela- ise of immunotherapies in cancer, but given the tionships between tumors and different aspects of complexity of immune system alterations in NF1, the immune system in NF1 patients specifically. the role of molecularly targeted therapies should not be overlooked. Immunotherapies harness the Acknowledgements body’s own defenses to attack tumor cells, but The authors thank B Widemann for helpful discussions and therapies targeting signaling pathways may be comments on the manuscript. needed either to combine with immunotherapies in eliminating tumor burden or to normalize the Disclaimer response of the immune system in NF1 patients The content of this publication does not necessarily reflect by adjusting signaling pathways within the the views or policies of the Department of Health and immune cells themselves. For example, a recent Human Services, nor does mention of trade names, com- study has shown that the MEK inhibitor selu- mercial products or organizations imply endorsements by metinib can decrease circulating Treg cells, but the US Government. increase expression of CTLA4 on Treg cells and increase the expression of PD-1 on Treg cells and Financial & competing interests disclosure CD8+ T cells, demonstrating the complex effects The authors are supported by the Intramural Research of altering signaling pathways in the immune Program of the NIH, National Cancer Institute. The authors system [153] . It is still unknown to what extent have no other relevant affiliations or financial involvement NF1-associated tumors mount immune suppres- with any organization or entity with a financial interest in sive defenses in the form of checkpoints such as or financial conflict with the subject matter or materials CTLA4 or PD-L1. Depending on the extent to discussed in the manuscript apart from those disclosed. which the immune response is silenced, immune No writing assistance was utilized in the production of checkpoint therapies may provide exciting new this manuscript.

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