- Email: [email protected]
Insights into CCL21's roles in immunosurveillance and immunotherapy for gliomas
Accepted Manuscript Insights into CCL21's role immunotherapy for gliomas
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immunosurveillance
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Thien Nguyen, Carlito Lagman, Lawrance K. Chung, Cheng Hao Jacky Chen, Jessica Poon, Vera Ong, Brittany L. Voth, Isaac Yang PII: DOI: Reference:
S0165-5728(16)30341-1 doi: 10.1016/j.jneuroim.2017.01.010 JNI 476500
To appear in:
Journal of Neuroimmunology
Received date: Accepted date:
8 November 2016 17 January 2017
Please cite this article as: Thien Nguyen, Carlito Lagman, Lawrance K. Chung, Cheng Hao Jacky Chen, Jessica Poon, Vera Ong, Brittany L. Voth, Isaac Yang , Insights into CCL21's role in immunosurveillance and immunotherapy for gliomas. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Jni(2017), doi: 10.1016/j.jneuroim.2017.01.010
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ACCEPTED MANUSCRIPT Review Article
Insights into CCL21’s role in immunosurveillance and immunotherapy for gliomas Thien Nguyena,d, Carlito Lagmana, Lawrance K. Chunga,d, Cheng Hao Jacky Chena, Jessica Poona,d, Vera Onga, Brittany L. Votha, Isaac Yanga,b,c,d
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Corresponding author Isaac Yang, MD Departments of Neurosurgery and Radiation Oncology Jonsson Comprehensive Cancer Center David Geffen School of Medicine University of California, Los Angeles 300 Stein Plaza, Suite 562 5th Floor Wasserman Building Los Angeles, CA 90095-6901 Phone: (310) 267-2621 Fax: (310) 825-9385 Email: [email protected]
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Departments of aNeurosurgery, bRadiation Oncology, and cJonsson Comprehensive Cancer Center, dDavid Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, United States
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ACCEPTED MANUSCRIPT Abstract
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Chemokine (C-C) motif ligand 21 (CCL21) is involved in immunosurveillance and has recently garnered the attention of neuro-oncologists and neuroscientists. CCL21 contains an extended C-terminus, which increases binding to lymphatic glycosaminoglycans (GAGs) and provides a mechanism of cell trafficking by forming a stationary chemokine concentration gradient that allows cell migration via haptotaxis. CCL21 is expressed by endothelial cells of the blood-brain barrier (BBB) in normal physiologic and pathologic conditions and has been implicated in leukocyte extravasation into the CNS. In this review, we summarize the roles of CCL21 in immunosurveillance as a potential immunotherapeutic agent for the treatment of gliomas.
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Keywords: blood-brain barrier; chemokine CCL21; exodus-2 chemokine; glioma
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Abbreviations: CCL21, chemokine (C-C) motif ligand 21; HEV, high endothelial venules; CCR7, C-C chemokine receptor 7; DCs, dendritic cells; CNS, central nervous system; CXCR3, C-XC chemokine receptor 3; BBB, blood-brain barrier; NPCs, neural precursor cells; INF-γ, interferon-γ; ICAM-1, intercellular adhesion molecular-1; MadCAM-1, mucosal addressin cell adhesion molecule 1; Sema7A, semaphorin 7A; TGFBIp, transforming growth factor-β-induced protein; GBM, glioblastoma; WHO, World Health Organization
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ACCEPTED MANUSCRIPT 1. Introduction
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Cytokines are proteins involved in cell signaling and inflammation, and act through long-range endocrine, close-range paracrine, and self-targeting autocrine binding (Zhang and An, 2007). Chemokines are a family of small chemotactic cytokines acting on cell homing, activation, and recruitment, and are segregated into 4 groups (CC, CXC, CX3C and XC) based on the location of the first N-terminal cysteine. Chemokine signal transduction is initiated by binding to G-protein-coupled receptors CCR, CXCR, CX3CR and XCR (Tansey et al., 2011). Historically, cytokines were studied in the context of inflammation and immune response, but within the past decade they have also been implicated in cancer biology (Plotkin et al., 2004, Zlotnik, 2004). Cytokines and chemokines have shown to provide some therapeutic effect against tumor growth (Ashour et al., 2007). One chemokine of interest is chemokine C-C motif ligand 21 (CCL21), also known as 6Ckine, Exodus-2, and thymus derived chemotactic agent-4. In the periphery, CCL21 is expressed by high endothelial venules (HEVs) in secondary lymph organs, lymph nodes, and spleen. Its peripheral receptor, C-C chemokine receptor 7 (CCR7), is expressed on mature dendritic cells (DCs), B-cells, and both naïve and memory T-cells (Columba-Cabezas et al., 2003). Binding of CCL21 to CCR7 initiates a signal transduction cascade ultimately resulting in regulation, migration, and activation of T-cells. During an inflammatory response, CCL21 recruits these various immune cells to the site of inflammation (Gomez-Nicola et al., 2010, Kar et al., 2011, Warnock et al., 2000, Willimann et al., 1998). In the central nervous system (CNS), CCL21 expression has been reported in endangered neurons and aids in the activation of microglial cells through an alternative receptor, C-X-C chemokine receptor 3 (CXCR3) (de Jong et al., 2008). Chemokine-based immunotherapy has proven successful in mammalian tumors, but there remain many challenges in implementing these treatments in patients afflicted with brain cancers. The blood-brain barrier (BBB) hinders effective drug delivery and immune responses, creating a partially immunoprivileged environment (Schwartz and Shechter, 2010). In the healthy brain, almost no immune cells enter the brain parenchyma. T-cells routinely survey the brain for antigens via the choroid plexus and cerebrospinal fluid (Engelhardt, 2006, Galea et al., Ransohoff et al., 2003, Schwartz and Shechter, 2010). CCL21 has been shown to traffic T-lymphocytes into the CNS in response to infections (Williams et al., 2014). During injury and stress, CCL21 demonstrates cytoprotective effects (Byram, 2004, Derecki et al., 2010, Ploix et al., 2011, Schwartz and Cohen, 2000, Schwartz and Kipnis, 2007, Stalder et al., 1998). Given appropriate expression of chemokines, the CNS can become permeable to lymphocytes. Once Tlymphocytes have infiltrated the BBB, they have the potential to activate microglia, immune-like cells within the CNS, to initiate an immune response (Schwartz and Shechter, 2010). In this review, we summarize the current understanding of CCL21 activity in normal and brain cancer cells, detail its mechanism of action in lymphocyte recruitment through the BBB, and address CCL21’s potential application as a chemokinebased immunotherapeutic agent for gliomas. 2. Physiologic conditions
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2.1. CCL21 chemotaxis
2.2. T-cell recruitment
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CCL21 acts as a chemoattractant in response to injury by recruiting lymphocytes to the afflicted area (Fig. 1) (Tansey, Turbic, 2011). During injury, CCL21 is released from astrocytes and microglia, which in turn recruit leukocytes to repair damage (Ambrosini and Aloisi, 2004, Babcock et al., 2003, Belmadani, 2006). Microglia are key players in innate and adaptive CNS immune responses, release of proteases and cytokines, and phagocytosis of pathogens (Kreutzberg, 1996, Zhai et al., 2011). Microglia are innate immune cells of the CNS that also interact with CCL21. Under inflammatory conditions, murine microglia were shown to be activated by neuronal CCL21 via its alternate receptor CXCR3 (de Jong, Vinet, 2008, Dijkstra et al., 2004). Similarly, in mice that underwent intracerebral lipopolysaccharide injections (triggering an inflammatory response), CCR7 was upregulated in astrocytes, which in turn activate microglia (Gomez-Nicola, Pallas-Bazarra, 2010). This suggests that CCL21 produced after CNS inflammation may be a possible mediator in astrocyte activation, potentially repairing damage and inflammation induced by tumor cells (Gomez-Nicola, PallasBazarra, 2010). CCL21 has also been shown to induce neuronal differentiation of adult neural precursor cells (NPCs) to neurons, as well as aid neuronal migration of NPCs in response to injury (Tansey, Turbic, 2011). NPCs provide important repair for CNS damage by replacing destroyed cells through either local differentiation or migration to the injured site (Tansey, Turbic, 2011). CCL21 is also highly expressed in the lymph nodes and HEVs, acting as a chemotactic factor for CCR7+ T-cells (Zlotnik, 2004).
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CCL21 facilitates binding of circulating lymphocytes to the vascular endothelium by stimulating the adhesion of lymphocytes to intercellular adhesion molecule-1 (ICAM1) via 2 integrin-mediated adhesion (Campbell et al., 1998, Columba-Cabezas, Serafini, 2003, Constantin et al., 2000). Lymphocytes also adhere to mucosal addressin cell adhesion molecule 1 (MadCAM-1), expressed in mucosal HEV via CCL21-induced α4,β7 integrin-mediated adhesion (Columba-Cabezas, Serafini, 2003, Zlotnik and Yoshie, 2000). The combination of ICAM-1, MadCAM-1, and CCL21 expression on the BBB provides a potential mechanism by which circulating lymphocytes can enter the CNS during inflammation and disease (Columba-Cabezas, Serafini, 2003). Semaphorin 7A (Sema7A), a membrane protein expressed in various activated lymphocytes, regulates CCL21-induced DC migration (van Rijn et al., 2015). In mice that lack Sema7A, CCL21induced skin DC migration was found to be impaired (van Rijn, Paulis, 2015). CCL21 expression is regulated by transforming growth factor-β-induced protein (TGFBIp) through the extracellular signal-regulated kinases (ERK) -dependent signaling pathway (Maeng et al., 2015). Maeng and co-workers found that lymphatic endothelial cells treated with TGFBIp in vitro had increased expression of CCL21, and promoted CCR7+ cells entry into lymphatic vessels (Maeng, Aguilar, 2015). CCL21 can also alternate between cell-bound and soluble phases. Cell-bound CCL21 induces cell adhesion and haptotaxis, while soluble CCL21 initiates chemotaxis (Lorenz et al., 2016).
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This transition is driven by plasmin, which exposes the C-terminal peptide in CCL21 by cleaving its cell-bound form, substantiating the role of plasmin in the regulation of CCL21 function (Lorenz, Loef, 2016). CCL21 gene mutations in mouse models are associated with decreased lymphocyte trafficking and migration (Chen et al., 2002). Similarly, Forster and coworkers found that mice which lack the CCL21 receptor, CCR7, have reduced migration of lymphocytes into secondary lymph organs, further demonstrating the importance of CCL21 and its receptor in the recruitment of lymphocytes (Chen, Leach, 2002, Forster et al., 1999). CCL21 is involved in the regulation of both innate and adaptive immunity. DCs present antigens and migrate to lymph nodes as an essential step in the immune response (Tyrinova et al., 2015). The development of immature DCs, occurring in the setting of inflammation or pathogen associated molecular patterns, results in the expression of chemokines (e.g., CCL21) and their receptors (e.g., CCR7) (Lin et al., 2014, Tyrinova, Leplina, 2015). Specifically, DCs upregulate CCR7 upon maturation, initiating their migration toward lymphoid organ T-cell zones containing CCL21 (Randolph et al., 2008).
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2.3. Traversing the BBB
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The BBB consists of microvascular endothelium, basement membrane, neurons, astrocytes, pericytes, and microglia (Nimjee, 2011, Williams, Holman, 2014). These structures together serve as immune checkpoints that allow important molecules like oxygen and glucose into the CNS, while preventing the entry of other molecules in circulation (Nimjee, 2011, Williams, Holman, 2014). CCL21 is expressed at the BBB, and hypothesized to regulate the migration of circulating leukocytes into the CNS during neuroinflammation (Fig. 2). During inflammation, CCL21 is expressed in venules surrounded by inflammatory cells, while its receptors CCR7 and CXCR3 were expressed on inflammatory cells in the CNS (Alt et al., 2002). Lymphocytes traverse the BBB in a similar fashion to peripheral leukocyte extravasation. This process has been described prior studies (Alt, Laschinger, 2002, Engelhardt, 2006b):
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1) Circulating lymphocytes roll on the endothelial cell surfaces after coming into contact with the vascular endothelium. This process is regulated by adhesion molecules (selectin-family) and their carbohydrate ligands. 2) Lymphocytes scan the endothelial cell surfaces for chemotactic gradients. 3) Chemokines on the vascular endothelium bind to their receptors on lymphocytes, resulting in the activation of integrins that are expressed on the surface of lymphocytes. The activation of integrins allows lymphocytes to arrest onto the vascular wall. Because naive T-cells lack affinity to integrins on the vascular endothelium, only activated T-cells will activate integrins and interact with chemokines on the vascular endothelium. 4) Transendothelial migration of lymphocytes across the BBB and into the CNS. 3. Immunosurveillance and the Tumor Microenvironment
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3.1. Immunosurveillance
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CCL21 plays a role in immunosurveillance of the CNS (Alt, Laschinger, 2002). Immunosurveillance occurs when thymus-dependent cells regularly scan host tissues for transformed cells, and upon recognition of transformed cells or cancer precursors, the immune system destroys the cells before they manifest into malignant states (Drakes and Stiff, 2014, Dunn et al., 2004, Hamai et al., 2010). To evade detection from immunosurveillance, tumor cells frequently remain dormant until they can form primary tumors (Koebel et al., 2007, Slaney et al., 2013). Immunosurveillance serves as the elimination phase of immuno-editing, when immune cells can recognize and destroy premalignant tumors cells. Following the elimination phase, tumor cells enter the equilibrium phase, develop resistance against antitumor responses, and finally enter the escape phase to form clinically significant tumors (Dunn, Old, 2004, Zitvogel et al., 2013, Zitvogel et al., 2006). Various immune components are responsible for immunosurveillance, including CD4+, CD8+ and γδ T-cells as well as natural killer cells, macrophages, interferon-γ (INF-γ), perforin, and tumor necrosis factor-related apoptosis-inducing ligands (Drakes and Stiff, 2014, Waldron et al., 2010, Yang et al., 2010). Interestingly, the density of tumor-infiltrating T-lymphocytes is an accurate prognostic tool, often more useful than tumor histological features, where higher tumor infiltration of CD3+ and CD8+ T-cells correlate with better patient outcomes (Drakes and Stiff, 2014, Fong et al., 2012, Ooi et al., 2014, Yang, Tihan, 2010). In mice with autoimmune encephalomyelitis (a model for multiple sclerosis), encephalitogenic T-cells expressed both CCL21 receptors, CCR7, and CXCR3 on its surface (Alt, Laschinger, 2002). Thus, with the functional expression of CCL21 at the BBB, activated T-cells will chemotax through the BBB and into the CNS during both immunosurveillance and inflammation (Alt, Laschinger, 2002). Ploix and coworkers showed that CCL21 induction by glial or neuronal cells within the CNS promotes CD4+ T-cell homeostatic proliferation, resulting in a decreased CNS autoimmunity threshold (Ploix et al., 2001). 3.2. Tumor biology
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CCL21 expression can promote glioma growth by recruiting microglia (Zhai, Heppner, 2011). CCL21 has also been shown to suppress antitumor responses and promote metastatic invasion into the lymphatic system in melanoma (Shields et al., 2007, Shields et al., 2010). Immune cells, particularly macrophages, paradoxically promote tumor growth and destroy tumor cells. Macrophages can inhibit tumor growth by secreting cytokines that activate T-cells to destroy tumor cells, and may be recruited by tumor-secreted cytokines to facilitate its proliferation in the microenvironment (Han et al., 2010). Likewise, although microglia serve as protective agents during possible infections, these functions are altered in the presence of gliomas (Han, Kaur, 2010, Jian et al., 2010, Kaur et al., 2010, Yang et al., 2011, Yang et al., 1995). Gliomas secrete numerous growth factors which stimulate growth and recruitment of tumor-infiltrating macrophages and microglia, including granulocyte-
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macrophage stimulating factor, hepatocyte growth factor, monocyte chemoattractant protein-1, and colony stimulating factor-1 (Alterman and Stanley, 1994, Badie et al., 1999, Kielian et al., 2002, Leung et al., 1997, Nitta et al., 1992). Gliomas create an immunosuppressive microenvironment, which is, in part, mediated through CCL21 expression. Microglia have been shown to increase glioma cell growth in culture (Zhai, Heppner, 2011). The immunoprotective effect of CCL21 on gliomas occurs only in the presence of microglia; this effect is not observed when microglia are absent from the microenvironment (Kaur, Han, 2010, Zhai, Heppner, 2011). Thus, glioma-infiltrating microglia and macrophages enhance glioma proliferation and invasion (Kaur, Han, 2010, Zhai, Heppner, 2011). Upon silencing of CCL21, through a neutralizing antibody, microglial cells are unable to promote glioma growth, severing this stromal effect (Zhai, Heppner, 2011). 4. Immunotherapeutic Potential
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Despite its dual pro- and anti-tumoral activity, CCL21 has proven to be a potential therapeutic agent that immunologically inhibits tumor growth in various types of murine cancers, including melanoma, pancreatic, lung, and breast cancers (Ashour, Lin, 2007, Kirk et al., 2001, Sharma et al., 2000, Turnquist et al., 2007). In pancreatic cancer, increased DC infiltration co-localized with CD3+ T-cells and natural killer cells, after intratumoral injection of CCL21 (Turnquist, Lin, 2007). CD8+ T-cells were also increased in melanoma, lung, and breast cancer, while CD4+ T-cells were increased only in melanoma and lung cancer (Ashour, Lin, 2007, Kirk, Hartigan-O'Connor, 2001, Sharma, Stolina, 2000). The immune response initiated by injected CCL21 was observed not only at the tumor, but was also present in the draining lymph nodes (Sharma, Stolina, 2000). While the evidence suggests a nuanced role of CCL21 in tumor growth/immunosurveillance, there is a rationale for using it as an immunotherapeutic agent. 4.1. CCL21 and glioma treatment
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To the best of our knowledge, treatment of gliomas specifically with CCL21 has not been described in the literature. Gliomas are the most frequently occurring primary brain tumors in the USA and Europe with the most common glioma being glioblastoma (GBM) (World Health Organization [WHO] grade IV)—a highly malignant tumor with high recurrence and low survival rates (Han et al., 2010, Nagasawa et al., 2012, Tanaka et al., 2013, Yang and Aghi, 2009). Even after standard therapy of maximal safe resection, radiation, and chemotherapy, the median survival of patients with GBM is just over a year (Yang et al., 2004). The potential for chemokine-related immunotherapy has been previously reported. Gliomas are known to produce CCL22 and CCL2, which bind to the CCR4 receptor on glioma-infiltrating regulatory T-cells and induces chemotaxis (Perng and Lim, 2015). Studies that administered blocking antibodies of CCR4 in vitro showed that regulatory T-cell migration towards the glioma was arrested, thus confirming the capability of chemokine regulation of glioma-infiltrating immune cells (Jordan et al., 2007, Perng and Lim, 2015).
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ACCEPTED MANUSCRIPT 4.2. CCL21-vault nanoparticles
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In our lab, we are currently conducting preliminary studies utilizing bioengineered vault nanoparticles coupled with CCL21 injected in mouse glioma 261 models to assess CCL21’s potential as an immunotherapeutic in glioma. Vault nanoparticles are endogenous barrel-shaped ribonucleoproteins used as a novel drug delivery system (Nagasawa et al., 2012, Ung and Yang, 2015, Yang et al., 2012). C57BL/6 mice inoculated with mouse glioma 261 (GL261) cells were used as the immunocompetent, syngeneic malignant glioma models. GL261 cells were subcutaneously injected into the left flank of eight-week-old female mice. Mice with established tumors were separated into 3 treatment arms (unpublished results): 1) CCL21-vault nanoparticles, 2) free recombinant CCL21, 3) empty vault nanoparticles. Average initial tumor volumes were 45.20 mm3, 46.55 mm3, and 78.95 mm3 for the three treatment groups, respectively. Average final tumor volumes after treatment were 31.11 mm3, 94.46 mm3, and 272.64 mm3 for the three treatment groups. Average changes in tumor volume were -14.06 mm3, 41.97 mm3, and 193.69 mm3 for the three treatment groups (p = .035). Future experiments will evaluate the potential for CCL21-vaults to traverse the blood-brain barrier and effectively decrease intracranial tumor volume.
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5. Conclusion
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Chemokines have shown great potential in regulating immune responses against tumors. CCL21 has the ability to activate both innate and adaptive immune responses, making it an important immunotherapeutic agent against pathogens in both the periphery and the CNS. CCL21’s functional expression at the BBB aids in immunosurveillance of the healthy brain, lymphocyte recruitment, and transendothelial migration across the BBB during CNS inflammation. Therefore, CCL21 has strong implications in the treatment of gliomas and further studies validating this interaction are necessary.
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ACCEPTED MANUSCRIPT Acknowledgements: We would like to thank Andrea Bottaro, Ph.D. (Department of Biomedical Sciences, Cooper Medical School of Rowan University, Camden, NJ) for his thorough review and critical evaluation of this article.
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Funding: Thien Nguyen is a recipient of the David Geffen Medical Scholarship. Carlito Lagman was partially supported by a Gurtin Skull Base Research Fellowship. Lawrance K. Chung was partially supported by an AMA Foundation Seed Grant and an AΩA Carolyn L. Kuckein Student Research Fellowship. Isaac Yang was partially supported by a Visionary Fund Grant, an Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research UCLA Scholars in Translational Medicine Program Award, the Jason Dessel Memorial Seed Grant, the UCLA Honberger Endowment Brain Tumor Research Seed Grant, and the STOP CANCER Research Career Development Award.
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ACCEPTED MANUSCRIPT Figure Legends
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Figure 1. Chemokine concentration gradient mediates chemotaxis Figure 2. CCL21 at the blood-brain barrier (BBB)
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Figure 1. Chemokine concentration gradient mediates chemotaxis
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With a noxious stimulus such as inflammation, antigen presenting cells (APCs) release chemokines to attract T-lymphocytes to the afflicted area. This chemotaxis is mediated by the chemokine concentration, allowing these immune cells to arrive at the exact area. CCL21 is one such chemokine.
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Figure 2. CCL21 at the blood-brain barrier (BBB)
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Neuroinflammation triggers the expression of CCL21. At the blood brain barrier, neuroinflammatory cells express CCL21, which then bind to the CCR7 and CXCR3 receptors on dendritic cells. This enhances chemotaxis of CD4+ lymphocytes and upon binding allows these CD4+ lymphocytes to extravasate through the blood brain barrier. In the choroid plexus binding of CCL21 to these CD4+ lymphocytes result in their proliferation and decreases the threshold for immunosurveillance. As a result, these CD4+ lymphocytes are better able to recognize gliomas. In turn, gliomas combat this immunosurveillance by secreting CCL21, resulting in immunosuppression of these Tlymphocytes.
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CCL21 may have reparative capacity in the setting of tumor-induced damage CCL21 may regulate migration of circulating leukocytes into CNS during inflammation CCL21 inhibition of tumor growth has been observed in several non-CNS cancers Treatment of glioma with CCL21 has yet to be described
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Thien Nguyen, Carlito Lagman, Lawrance K. Chung, Cheng Hao Jacky Chen, Jessica Poon, Vera Ong, Brittany L. Voth, Isaac Yang PII: DOI: Reference:
S0165-5728(16)30341-1 doi: 10.1016/j.jneuroim.2017.01.010 JNI 476500
To appear in:
Journal of Neuroimmunology
Received date: Accepted date:
8 November 2016 17 January 2017
Please cite this article as: Thien Nguyen, Carlito Lagman, Lawrance K. Chung, Cheng Hao Jacky Chen, Jessica Poon, Vera Ong, Brittany L. Voth, Isaac Yang , Insights into CCL21's role in immunosurveillance and immunotherapy for gliomas. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Jni(2017), doi: 10.1016/j.jneuroim.2017.01.010
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ACCEPTED MANUSCRIPT Review Article
Insights into CCL21’s role in immunosurveillance and immunotherapy for gliomas Thien Nguyena,d, Carlito Lagmana, Lawrance K. Chunga,d, Cheng Hao Jacky Chena, Jessica Poona,d, Vera Onga, Brittany L. Votha, Isaac Yanga,b,c,d
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Corresponding author Isaac Yang, MD Departments of Neurosurgery and Radiation Oncology Jonsson Comprehensive Cancer Center David Geffen School of Medicine University of California, Los Angeles 300 Stein Plaza, Suite 562 5th Floor Wasserman Building Los Angeles, CA 90095-6901 Phone: (310) 267-2621 Fax: (310) 825-9385 Email: [email protected]
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Departments of aNeurosurgery, bRadiation Oncology, and cJonsson Comprehensive Cancer Center, dDavid Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, United States
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Chemokine (C-C) motif ligand 21 (CCL21) is involved in immunosurveillance and has recently garnered the attention of neuro-oncologists and neuroscientists. CCL21 contains an extended C-terminus, which increases binding to lymphatic glycosaminoglycans (GAGs) and provides a mechanism of cell trafficking by forming a stationary chemokine concentration gradient that allows cell migration via haptotaxis. CCL21 is expressed by endothelial cells of the blood-brain barrier (BBB) in normal physiologic and pathologic conditions and has been implicated in leukocyte extravasation into the CNS. In this review, we summarize the roles of CCL21 in immunosurveillance as a potential immunotherapeutic agent for the treatment of gliomas.
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Keywords: blood-brain barrier; chemokine CCL21; exodus-2 chemokine; glioma
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Abbreviations: CCL21, chemokine (C-C) motif ligand 21; HEV, high endothelial venules; CCR7, C-C chemokine receptor 7; DCs, dendritic cells; CNS, central nervous system; CXCR3, C-XC chemokine receptor 3; BBB, blood-brain barrier; NPCs, neural precursor cells; INF-γ, interferon-γ; ICAM-1, intercellular adhesion molecular-1; MadCAM-1, mucosal addressin cell adhesion molecule 1; Sema7A, semaphorin 7A; TGFBIp, transforming growth factor-β-induced protein; GBM, glioblastoma; WHO, World Health Organization
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ACCEPTED MANUSCRIPT 1. Introduction
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Cytokines are proteins involved in cell signaling and inflammation, and act through long-range endocrine, close-range paracrine, and self-targeting autocrine binding (Zhang and An, 2007). Chemokines are a family of small chemotactic cytokines acting on cell homing, activation, and recruitment, and are segregated into 4 groups (CC, CXC, CX3C and XC) based on the location of the first N-terminal cysteine. Chemokine signal transduction is initiated by binding to G-protein-coupled receptors CCR, CXCR, CX3CR and XCR (Tansey et al., 2011). Historically, cytokines were studied in the context of inflammation and immune response, but within the past decade they have also been implicated in cancer biology (Plotkin et al., 2004, Zlotnik, 2004). Cytokines and chemokines have shown to provide some therapeutic effect against tumor growth (Ashour et al., 2007). One chemokine of interest is chemokine C-C motif ligand 21 (CCL21), also known as 6Ckine, Exodus-2, and thymus derived chemotactic agent-4. In the periphery, CCL21 is expressed by high endothelial venules (HEVs) in secondary lymph organs, lymph nodes, and spleen. Its peripheral receptor, C-C chemokine receptor 7 (CCR7), is expressed on mature dendritic cells (DCs), B-cells, and both naïve and memory T-cells (Columba-Cabezas et al., 2003). Binding of CCL21 to CCR7 initiates a signal transduction cascade ultimately resulting in regulation, migration, and activation of T-cells. During an inflammatory response, CCL21 recruits these various immune cells to the site of inflammation (Gomez-Nicola et al., 2010, Kar et al., 2011, Warnock et al., 2000, Willimann et al., 1998). In the central nervous system (CNS), CCL21 expression has been reported in endangered neurons and aids in the activation of microglial cells through an alternative receptor, C-X-C chemokine receptor 3 (CXCR3) (de Jong et al., 2008). Chemokine-based immunotherapy has proven successful in mammalian tumors, but there remain many challenges in implementing these treatments in patients afflicted with brain cancers. The blood-brain barrier (BBB) hinders effective drug delivery and immune responses, creating a partially immunoprivileged environment (Schwartz and Shechter, 2010). In the healthy brain, almost no immune cells enter the brain parenchyma. T-cells routinely survey the brain for antigens via the choroid plexus and cerebrospinal fluid (Engelhardt, 2006, Galea et al., Ransohoff et al., 2003, Schwartz and Shechter, 2010). CCL21 has been shown to traffic T-lymphocytes into the CNS in response to infections (Williams et al., 2014). During injury and stress, CCL21 demonstrates cytoprotective effects (Byram, 2004, Derecki et al., 2010, Ploix et al., 2011, Schwartz and Cohen, 2000, Schwartz and Kipnis, 2007, Stalder et al., 1998). Given appropriate expression of chemokines, the CNS can become permeable to lymphocytes. Once Tlymphocytes have infiltrated the BBB, they have the potential to activate microglia, immune-like cells within the CNS, to initiate an immune response (Schwartz and Shechter, 2010). In this review, we summarize the current understanding of CCL21 activity in normal and brain cancer cells, detail its mechanism of action in lymphocyte recruitment through the BBB, and address CCL21’s potential application as a chemokinebased immunotherapeutic agent for gliomas. 2. Physiologic conditions
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2.1. CCL21 chemotaxis
2.2. T-cell recruitment
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CCL21 acts as a chemoattractant in response to injury by recruiting lymphocytes to the afflicted area (Fig. 1) (Tansey, Turbic, 2011). During injury, CCL21 is released from astrocytes and microglia, which in turn recruit leukocytes to repair damage (Ambrosini and Aloisi, 2004, Babcock et al., 2003, Belmadani, 2006). Microglia are key players in innate and adaptive CNS immune responses, release of proteases and cytokines, and phagocytosis of pathogens (Kreutzberg, 1996, Zhai et al., 2011). Microglia are innate immune cells of the CNS that also interact with CCL21. Under inflammatory conditions, murine microglia were shown to be activated by neuronal CCL21 via its alternate receptor CXCR3 (de Jong, Vinet, 2008, Dijkstra et al., 2004). Similarly, in mice that underwent intracerebral lipopolysaccharide injections (triggering an inflammatory response), CCR7 was upregulated in astrocytes, which in turn activate microglia (Gomez-Nicola, Pallas-Bazarra, 2010). This suggests that CCL21 produced after CNS inflammation may be a possible mediator in astrocyte activation, potentially repairing damage and inflammation induced by tumor cells (Gomez-Nicola, PallasBazarra, 2010). CCL21 has also been shown to induce neuronal differentiation of adult neural precursor cells (NPCs) to neurons, as well as aid neuronal migration of NPCs in response to injury (Tansey, Turbic, 2011). NPCs provide important repair for CNS damage by replacing destroyed cells through either local differentiation or migration to the injured site (Tansey, Turbic, 2011). CCL21 is also highly expressed in the lymph nodes and HEVs, acting as a chemotactic factor for CCR7+ T-cells (Zlotnik, 2004).
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CCL21 facilitates binding of circulating lymphocytes to the vascular endothelium by stimulating the adhesion of lymphocytes to intercellular adhesion molecule-1 (ICAM1) via 2 integrin-mediated adhesion (Campbell et al., 1998, Columba-Cabezas, Serafini, 2003, Constantin et al., 2000). Lymphocytes also adhere to mucosal addressin cell adhesion molecule 1 (MadCAM-1), expressed in mucosal HEV via CCL21-induced α4,β7 integrin-mediated adhesion (Columba-Cabezas, Serafini, 2003, Zlotnik and Yoshie, 2000). The combination of ICAM-1, MadCAM-1, and CCL21 expression on the BBB provides a potential mechanism by which circulating lymphocytes can enter the CNS during inflammation and disease (Columba-Cabezas, Serafini, 2003). Semaphorin 7A (Sema7A), a membrane protein expressed in various activated lymphocytes, regulates CCL21-induced DC migration (van Rijn et al., 2015). In mice that lack Sema7A, CCL21induced skin DC migration was found to be impaired (van Rijn, Paulis, 2015). CCL21 expression is regulated by transforming growth factor-β-induced protein (TGFBIp) through the extracellular signal-regulated kinases (ERK) -dependent signaling pathway (Maeng et al., 2015). Maeng and co-workers found that lymphatic endothelial cells treated with TGFBIp in vitro had increased expression of CCL21, and promoted CCR7+ cells entry into lymphatic vessels (Maeng, Aguilar, 2015). CCL21 can also alternate between cell-bound and soluble phases. Cell-bound CCL21 induces cell adhesion and haptotaxis, while soluble CCL21 initiates chemotaxis (Lorenz et al., 2016).
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This transition is driven by plasmin, which exposes the C-terminal peptide in CCL21 by cleaving its cell-bound form, substantiating the role of plasmin in the regulation of CCL21 function (Lorenz, Loef, 2016). CCL21 gene mutations in mouse models are associated with decreased lymphocyte trafficking and migration (Chen et al., 2002). Similarly, Forster and coworkers found that mice which lack the CCL21 receptor, CCR7, have reduced migration of lymphocytes into secondary lymph organs, further demonstrating the importance of CCL21 and its receptor in the recruitment of lymphocytes (Chen, Leach, 2002, Forster et al., 1999). CCL21 is involved in the regulation of both innate and adaptive immunity. DCs present antigens and migrate to lymph nodes as an essential step in the immune response (Tyrinova et al., 2015). The development of immature DCs, occurring in the setting of inflammation or pathogen associated molecular patterns, results in the expression of chemokines (e.g., CCL21) and their receptors (e.g., CCR7) (Lin et al., 2014, Tyrinova, Leplina, 2015). Specifically, DCs upregulate CCR7 upon maturation, initiating their migration toward lymphoid organ T-cell zones containing CCL21 (Randolph et al., 2008).
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2.3. Traversing the BBB
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The BBB consists of microvascular endothelium, basement membrane, neurons, astrocytes, pericytes, and microglia (Nimjee, 2011, Williams, Holman, 2014). These structures together serve as immune checkpoints that allow important molecules like oxygen and glucose into the CNS, while preventing the entry of other molecules in circulation (Nimjee, 2011, Williams, Holman, 2014). CCL21 is expressed at the BBB, and hypothesized to regulate the migration of circulating leukocytes into the CNS during neuroinflammation (Fig. 2). During inflammation, CCL21 is expressed in venules surrounded by inflammatory cells, while its receptors CCR7 and CXCR3 were expressed on inflammatory cells in the CNS (Alt et al., 2002). Lymphocytes traverse the BBB in a similar fashion to peripheral leukocyte extravasation. This process has been described prior studies (Alt, Laschinger, 2002, Engelhardt, 2006b):
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1) Circulating lymphocytes roll on the endothelial cell surfaces after coming into contact with the vascular endothelium. This process is regulated by adhesion molecules (selectin-family) and their carbohydrate ligands. 2) Lymphocytes scan the endothelial cell surfaces for chemotactic gradients. 3) Chemokines on the vascular endothelium bind to their receptors on lymphocytes, resulting in the activation of integrins that are expressed on the surface of lymphocytes. The activation of integrins allows lymphocytes to arrest onto the vascular wall. Because naive T-cells lack affinity to integrins on the vascular endothelium, only activated T-cells will activate integrins and interact with chemokines on the vascular endothelium. 4) Transendothelial migration of lymphocytes across the BBB and into the CNS. 3. Immunosurveillance and the Tumor Microenvironment
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3.1. Immunosurveillance
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CCL21 plays a role in immunosurveillance of the CNS (Alt, Laschinger, 2002). Immunosurveillance occurs when thymus-dependent cells regularly scan host tissues for transformed cells, and upon recognition of transformed cells or cancer precursors, the immune system destroys the cells before they manifest into malignant states (Drakes and Stiff, 2014, Dunn et al., 2004, Hamai et al., 2010). To evade detection from immunosurveillance, tumor cells frequently remain dormant until they can form primary tumors (Koebel et al., 2007, Slaney et al., 2013). Immunosurveillance serves as the elimination phase of immuno-editing, when immune cells can recognize and destroy premalignant tumors cells. Following the elimination phase, tumor cells enter the equilibrium phase, develop resistance against antitumor responses, and finally enter the escape phase to form clinically significant tumors (Dunn, Old, 2004, Zitvogel et al., 2013, Zitvogel et al., 2006). Various immune components are responsible for immunosurveillance, including CD4+, CD8+ and γδ T-cells as well as natural killer cells, macrophages, interferon-γ (INF-γ), perforin, and tumor necrosis factor-related apoptosis-inducing ligands (Drakes and Stiff, 2014, Waldron et al., 2010, Yang et al., 2010). Interestingly, the density of tumor-infiltrating T-lymphocytes is an accurate prognostic tool, often more useful than tumor histological features, where higher tumor infiltration of CD3+ and CD8+ T-cells correlate with better patient outcomes (Drakes and Stiff, 2014, Fong et al., 2012, Ooi et al., 2014, Yang, Tihan, 2010). In mice with autoimmune encephalomyelitis (a model for multiple sclerosis), encephalitogenic T-cells expressed both CCL21 receptors, CCR7, and CXCR3 on its surface (Alt, Laschinger, 2002). Thus, with the functional expression of CCL21 at the BBB, activated T-cells will chemotax through the BBB and into the CNS during both immunosurveillance and inflammation (Alt, Laschinger, 2002). Ploix and coworkers showed that CCL21 induction by glial or neuronal cells within the CNS promotes CD4+ T-cell homeostatic proliferation, resulting in a decreased CNS autoimmunity threshold (Ploix et al., 2001). 3.2. Tumor biology
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CCL21 expression can promote glioma growth by recruiting microglia (Zhai, Heppner, 2011). CCL21 has also been shown to suppress antitumor responses and promote metastatic invasion into the lymphatic system in melanoma (Shields et al., 2007, Shields et al., 2010). Immune cells, particularly macrophages, paradoxically promote tumor growth and destroy tumor cells. Macrophages can inhibit tumor growth by secreting cytokines that activate T-cells to destroy tumor cells, and may be recruited by tumor-secreted cytokines to facilitate its proliferation in the microenvironment (Han et al., 2010). Likewise, although microglia serve as protective agents during possible infections, these functions are altered in the presence of gliomas (Han, Kaur, 2010, Jian et al., 2010, Kaur et al., 2010, Yang et al., 2011, Yang et al., 1995). Gliomas secrete numerous growth factors which stimulate growth and recruitment of tumor-infiltrating macrophages and microglia, including granulocyte-
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macrophage stimulating factor, hepatocyte growth factor, monocyte chemoattractant protein-1, and colony stimulating factor-1 (Alterman and Stanley, 1994, Badie et al., 1999, Kielian et al., 2002, Leung et al., 1997, Nitta et al., 1992). Gliomas create an immunosuppressive microenvironment, which is, in part, mediated through CCL21 expression. Microglia have been shown to increase glioma cell growth in culture (Zhai, Heppner, 2011). The immunoprotective effect of CCL21 on gliomas occurs only in the presence of microglia; this effect is not observed when microglia are absent from the microenvironment (Kaur, Han, 2010, Zhai, Heppner, 2011). Thus, glioma-infiltrating microglia and macrophages enhance glioma proliferation and invasion (Kaur, Han, 2010, Zhai, Heppner, 2011). Upon silencing of CCL21, through a neutralizing antibody, microglial cells are unable to promote glioma growth, severing this stromal effect (Zhai, Heppner, 2011). 4. Immunotherapeutic Potential
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Despite its dual pro- and anti-tumoral activity, CCL21 has proven to be a potential therapeutic agent that immunologically inhibits tumor growth in various types of murine cancers, including melanoma, pancreatic, lung, and breast cancers (Ashour, Lin, 2007, Kirk et al., 2001, Sharma et al., 2000, Turnquist et al., 2007). In pancreatic cancer, increased DC infiltration co-localized with CD3+ T-cells and natural killer cells, after intratumoral injection of CCL21 (Turnquist, Lin, 2007). CD8+ T-cells were also increased in melanoma, lung, and breast cancer, while CD4+ T-cells were increased only in melanoma and lung cancer (Ashour, Lin, 2007, Kirk, Hartigan-O'Connor, 2001, Sharma, Stolina, 2000). The immune response initiated by injected CCL21 was observed not only at the tumor, but was also present in the draining lymph nodes (Sharma, Stolina, 2000). While the evidence suggests a nuanced role of CCL21 in tumor growth/immunosurveillance, there is a rationale for using it as an immunotherapeutic agent. 4.1. CCL21 and glioma treatment
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To the best of our knowledge, treatment of gliomas specifically with CCL21 has not been described in the literature. Gliomas are the most frequently occurring primary brain tumors in the USA and Europe with the most common glioma being glioblastoma (GBM) (World Health Organization [WHO] grade IV)—a highly malignant tumor with high recurrence and low survival rates (Han et al., 2010, Nagasawa et al., 2012, Tanaka et al., 2013, Yang and Aghi, 2009). Even after standard therapy of maximal safe resection, radiation, and chemotherapy, the median survival of patients with GBM is just over a year (Yang et al., 2004). The potential for chemokine-related immunotherapy has been previously reported. Gliomas are known to produce CCL22 and CCL2, which bind to the CCR4 receptor on glioma-infiltrating regulatory T-cells and induces chemotaxis (Perng and Lim, 2015). Studies that administered blocking antibodies of CCR4 in vitro showed that regulatory T-cell migration towards the glioma was arrested, thus confirming the capability of chemokine regulation of glioma-infiltrating immune cells (Jordan et al., 2007, Perng and Lim, 2015).
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In our lab, we are currently conducting preliminary studies utilizing bioengineered vault nanoparticles coupled with CCL21 injected in mouse glioma 261 models to assess CCL21’s potential as an immunotherapeutic in glioma. Vault nanoparticles are endogenous barrel-shaped ribonucleoproteins used as a novel drug delivery system (Nagasawa et al., 2012, Ung and Yang, 2015, Yang et al., 2012). C57BL/6 mice inoculated with mouse glioma 261 (GL261) cells were used as the immunocompetent, syngeneic malignant glioma models. GL261 cells were subcutaneously injected into the left flank of eight-week-old female mice. Mice with established tumors were separated into 3 treatment arms (unpublished results): 1) CCL21-vault nanoparticles, 2) free recombinant CCL21, 3) empty vault nanoparticles. Average initial tumor volumes were 45.20 mm3, 46.55 mm3, and 78.95 mm3 for the three treatment groups, respectively. Average final tumor volumes after treatment were 31.11 mm3, 94.46 mm3, and 272.64 mm3 for the three treatment groups. Average changes in tumor volume were -14.06 mm3, 41.97 mm3, and 193.69 mm3 for the three treatment groups (p = .035). Future experiments will evaluate the potential for CCL21-vaults to traverse the blood-brain barrier and effectively decrease intracranial tumor volume.
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5. Conclusion
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Chemokines have shown great potential in regulating immune responses against tumors. CCL21 has the ability to activate both innate and adaptive immune responses, making it an important immunotherapeutic agent against pathogens in both the periphery and the CNS. CCL21’s functional expression at the BBB aids in immunosurveillance of the healthy brain, lymphocyte recruitment, and transendothelial migration across the BBB during CNS inflammation. Therefore, CCL21 has strong implications in the treatment of gliomas and further studies validating this interaction are necessary.
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ACCEPTED MANUSCRIPT Acknowledgements: We would like to thank Andrea Bottaro, Ph.D. (Department of Biomedical Sciences, Cooper Medical School of Rowan University, Camden, NJ) for his thorough review and critical evaluation of this article.
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Funding: Thien Nguyen is a recipient of the David Geffen Medical Scholarship. Carlito Lagman was partially supported by a Gurtin Skull Base Research Fellowship. Lawrance K. Chung was partially supported by an AMA Foundation Seed Grant and an AΩA Carolyn L. Kuckein Student Research Fellowship. Isaac Yang was partially supported by a Visionary Fund Grant, an Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research UCLA Scholars in Translational Medicine Program Award, the Jason Dessel Memorial Seed Grant, the UCLA Honberger Endowment Brain Tumor Research Seed Grant, and the STOP CANCER Research Career Development Award.
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ACCEPTED MANUSCRIPT Figure Legends
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Figure 1. Chemokine concentration gradient mediates chemotaxis Figure 2. CCL21 at the blood-brain barrier (BBB)
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Figure 1. Chemokine concentration gradient mediates chemotaxis
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With a noxious stimulus such as inflammation, antigen presenting cells (APCs) release chemokines to attract T-lymphocytes to the afflicted area. This chemotaxis is mediated by the chemokine concentration, allowing these immune cells to arrive at the exact area. CCL21 is one such chemokine.
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Figure 2. CCL21 at the blood-brain barrier (BBB)
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Neuroinflammation triggers the expression of CCL21. At the blood brain barrier, neuroinflammatory cells express CCL21, which then bind to the CCR7 and CXCR3 receptors on dendritic cells. This enhances chemotaxis of CD4+ lymphocytes and upon binding allows these CD4+ lymphocytes to extravasate through the blood brain barrier. In the choroid plexus binding of CCL21 to these CD4+ lymphocytes result in their proliferation and decreases the threshold for immunosurveillance. As a result, these CD4+ lymphocytes are better able to recognize gliomas. In turn, gliomas combat this immunosurveillance by secreting CCL21, resulting in immunosuppression of these Tlymphocytes.
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ACCEPTED MANUSCRIPT Highlights
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CCL21 may have reparative capacity in the setting of tumor-induced damage CCL21 may regulate migration of circulating leukocytes into CNS during inflammation CCL21 inhibition of tumor growth has been observed in several non-CNS cancers Treatment of glioma with CCL21 has yet to be described
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