Immune-privileged Site Research Articles

Zika virus infects human Sertoli cells and trespass the blood-testes barrier to migrate across the seminiferous epithelium

Abstract The 2015–2016 epidemic of Zika virus (ZIKV) has identified sexual transmission as a new route of disease spread. Presence of ZIKV in the semen when viremia is undetectable in humans suggests the ability of ZIKV to establish infection in the testes. Lack of specific therapeutic drugs to clear testicular infection warrants understanding of the associated mechanisms. Seminiferous tubules, the site of spermatogenesis, is an immune privileged site with an effective blood-testes barrier (BTB) constituted exclusively by specialized tight junctions proteins (TJP) between the adjacent Sertoli cells (SC) that protects germ cells from systemic immune attack. Increased inflammatory mediators such as TNF-α, IL-1β, matrix metalloproteinases and cell-adhesion molecules (CAMs) can disrupt BTB and facilitate testicular infections. We determined the infection kinetics of ZIKV in SC and its association with antiviral defense and BTB integrity. We demonstrate that SC were susceptible to ZIKV and induced cytokines (IFN-α, IFN-γ, TNF-α) and CAMs (VCAM), which correlated with peak virus titers. Interestingly, while ZIKV transmigrated efficiently across the in vitro BTB model, it did not affect barrier integrity, also supported by no difference observed in the levels of TJP such as ZO-1 and claudin between control and infected SC. However, the integrity of the BTB model decreased significantly following exposure to the supernatant from ZIKV-infected macrophages. Collectively, our data suggests infection of SC as one of the routes of virus transmigration into the seminiferous tubules and that inflammatory mediators from infected human macrophages may further compromise integrity of BTB thereby allowing persistent infection of ZIKV in the testes.

Abstract B84: Preclinical analysis of combinatorial glioblastoma therapy with the prodrug-mediated gene therapy vector AdV-TK and immune checkpoint inhibition

Abstract Early clinical trial data show that blockade of PD-1 signaling leads to significant anticancer responses in a subset of patients in certain cancer types. While the brain has traditionally been considered to be an immune-privileged site, evidence supporting the use of immunotherapeutics in brain tumors has been rapidly accumulating. Given that virus-based cancer therapies can be immunostimulatory and immune checkpoint inhibitors block the body's natural checkpoint response, the combination of these two approaches offers a potentially advantageous interaction. One of the molecular underpinnings of T-cell exhaustion is the expression of Programmed Death-1 (PD-1) on T-cells that recognizes its ligand PD-L1. AdV-TK is an immunostimulatory virus-based approach, known as Gene-Mediated Cytotoxic Immunotherapy (GMCI), that involves the intra-tumoral delivery of a non-replicating adenoviral vector carrying the Herpes virus thymidine kinase gene (TK) followed by administration of an anti-herpetic prodrug (ganciclovir GCV) and recently showed encouraging results in a Phase II trial in glioblastoma (Wheeler et al., 2016). The immunological component results from the delivery vehicle being a virus, the mode of cell death, through both necrosis and apoptosis, and the pro-immunogenic properties of the TK protein. We confirm that this approach induces glioblastoma cell death and a consistent anti-tumor immune stimulation. Not surprisingly, however, this immune stimulation also leads to increase in cell surface of immune checkpoint inhibitory ligands on tumor cells, including PD-L1, detected by flow cytometry and immunohistochemistry. We show that GMCI induces a type-I interferon response, and using IFN decoy we demonstrated that the release of IFNβ in vitro is at least partially responsible for autocrine/paracrine PD-L1 up-regulation both in human and mouse glioblastoma cell lines. In vivo studies using an intracranial GL261 model showed high numbers of long term survivors in the GMCI/PD-1 combination (11/14), compared with GMCI (6/16), anti-PD-1 (5/12) and untreated (0/11). In addition, long term survival mice were no longer able to form tumors after rechallenge indicating the establishment of anti-tumor immunity. Finally, tumor infiltrating lymphocytes after GMCI showed an increase in CD8+, CD8+/GranzymeB+, and IFNγ+ cells suggestive of cytotoxic T-cell activation. However, there was also a significant increase in CD4+, CD4+/FoxP3+, and IL-10 indicating a significant infiltration by Tregs, releasing immunosuppressive cytokines. Additionally, there was a significant increase in PD-1+ /TIM3+ T-cells, indicative of an immunosuppressive microenvironment. Overall, our data show that GMCI/anti-PD-1 combinatorial therapy is effective in a syngeneic tumor model, and strongly support clinical trials of GMCI/checkpoint inhibitor combinations in glioblastoma patients. Citation Format: Maria Carmela Speranza, Franz Ricklefs, Carmela Passaro, Sarah R. Klein, Kazue Kasai, Johanna Kaufmann, Hiroshi Nakashima, Bronisz Agnieszka, Estuardo Aguilar-Cordova, Brian W. Guzik, Gordon J. Freeman, David A. Reardon, Patrick Wen, E. Antonio Chiocca, Sean E. Lawler. Preclinical analysis of combinatorial glioblastoma therapy with the prodrug-mediated gene therapy vector AdV-TK and immune checkpoint inhibition. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B84.

Possible damage to immune-privileged sites in natural killer cell therapy in cancer patients: side effects of natural killer cell therapy.

Natural killer (NK) cells target the cells losing MHC-I in cancer, a phenotype that is similar to certain cells in immune-privileged sites whose milieus are separated from peripheral blood. NK cells are reported to be quantitatively and qualitatively different in immune-privileged sites from those cytotoxic ones in the blood. We hypothesize that cytotoxic and expanded NK cells induced in cancer patients may be turned into pathogenic factors if they enter immune-privileged microenvironments in susceptible individuals, such as, patients with brain cancer or a blood-brain barrier dysfunction. Therefore, in susceptible individuals, different levels of caution should be taken based on the seriousness of the side effect as discussed in this perspective.

SUBCONJUNCTIVAL INJECTION

Introduction: Normally the cornea in human eye is crystalline clear membranepresent in the anterior most portion of the eyeball. Regarding the various functions of thecornea in human eye it provides protection, clear vision, refractive media the visual systemand maintains itself as an immune privileged site. Neovascularization is mostly associated withan inflammation and always indicate a state of disease. Similarly Corneal Neovascularizationcan cause Graft rejection reaction after keratoplasty the different types of anti-VEGF agentsnow are used to prevent neovascular eye diseases. These different Anti-VEGF inhibitors areLucentis, Macugen and bevacizumab/Avastin and used in case of ocular neovascularization.Objectives: To determine the efficacy of subconjuctival injection of Avastin on patients havingcorneal neovascularization following keratoplasty. Settings: Department of OphthalmologyAllied Hospital, Faisalabad and Independent University Hospital, Faisalabad. Study Duration:The duration of study was 11-02-2015 to 11-07-2015. Results: A total of 86 cases fulfilling theinclusion/exclusion criteria were enrolled to determine the efficacy of subconjuctival injectionof Avastin on patients having corneal neovascularization following keratoplasty. Discussion:Regarding the success of Keratoplasty is determined by many factors especially avascularity ofcornea after surgery. Corneal neovasculrization is disease process secondary to various ocularinsults in which growth of vessels towards central cornea occur from the limbal vascular plexus.But now a days Bevacizumab/Avastin is commonly used in Ophthalmology as “off label” drug inthe treatment of Exudative age related macular degeneration as well as in diabetic retinopathy.Conclusion: We concluded that the frequency of efficacy of subconjuctival bevacizumab ishigher in patients having corneal neovascularization after corneal transplantation.

Expression of lectin-like transcript-1 in human tissues

Background: Receptor-ligand pairs of C-type lectin-like proteins have been shown to play an important role in cross talk between lymphocytes, as well as in immune responses within concrete tissues and structures, such as the skin or the germinal centres. The CD161-Lectin-like Transcript 1 (LLT1) pair has gained particular attention in recent years, yet a detailed analysis of LLT1 distribution in human tissue is lacking. One reason for this is the limited availability and poor characterisation of anti-LLT1 antibodies. Methods: We assessed the staining capabilities of a novel anti-LLT1 antibody clone (2H7), both by immunohistochemistry and flow cytometry, showing its efficiency at LLT1 recognition in both settings. We then analysed LLT1 expression in a wide variety of human tissues. Results: We found LLT1 expression in circulating B cells and monocytes, but not in lung and liver-resident macrophages. We found strikingly high LLT1 expression in immune-privileged sites, such as the brain, placenta and testes, and confirmed the ability of LLT1 to inhibit NK cell function. Conclusions: Overall, this study contributes to the development of efficient tools for the study of LLT1. Moreover, its expression in different healthy human tissues and, particularly, in immune-privileged sites, establishes LLT1 as a good candidate as a regulator of immune responses.

Gut microbiota as a source of a surrogate antigen that triggers autoimmunity in an immune privileged site

ABSTRACTRecent discoveries on the role of commensal microbiota have significantly changed our understanding of human physiology. The host-microbiota interplay is now an important aspect to take into account to understand immune responses and immunological diseases. Autoimmune uveitis is a sight-threatening disease that arises without a known infectious etiology. It is unknown where and how autoreactive T cells become primed to trigger disease in the eye, which is an immune privileged site. We recently reported data supporting the notion that retina-specific T cells receive a signal in the gut from commensal microbiota-derived cross-reactive antigen(s) and trigger autoimmune uveitis in the R161H mouse model. Here we discuss our published findings, as well as our recent attempts to identify the responsible microbe(s) by using different antibiotic treatments, 16S rDNA sequencing and homology searches for candidate antigenic mimic(s) of the retinal antigen.

Is the Cannabinoid CB2 Receptor a Major Regulator of the Neuroinflammatory Axis of the Neurovascular Unit in Humans?

The central nervous system (CNS) is an immune privileged site where the neurovascular unit (NVU) and the blood-brain barrier (BBB) act as a selectively permeable interface to control the passage of nutrients and inflammatory cells into the brain parenchyma. However, in response to injury, infection, or disease, CNS cells become activated, and release inflammatory mediators to recruit immune cells to the site of inflammation. Increasing evidence suggests that cannabinoids may have a neuroprotective role in CNS inflammatory conditions. For many years, it was widely accepted that cannabinoid receptor type 1 (CB1) modulates neurological function centrally, while peripheral cannabinoid receptor type 2 (CB2) modulates immune function. As knowledge about the physiology and pharmacology of the endocannabinoid system advances, there is increasing interest in targeting CB2 as a potential treatment for inflammation-dependent CNS diseases (Ashton & Glass, 2007), where recent rodent and human studies have implicated intervention at the level of the NVU and BBB. These are incredibly important in brain health and disease. Therefore, this review begins by explaining the cellular and molecular components of these systems, highlighting important molecules potentially regulated by cannabinoid ligands and then takes an unbiased look at the evidence in support (or otherwise) of cannabinoid receptor expression and control of the NVU and BBB function in humans.

The Pathway From Genes to Gene Therapy in Glaucoma: A Review of Possibilities for Using Genes as Glaucoma Drugs.

Treatment of diseases with gene therapy is advancing rapidly. The use of gene therapy has expanded from the original concept of re-placing the mutated gene causing the disease to the use of genes to con-trol nonphysiological levels of expression or to modify pathways known to affect the disease. Genes offer numerous advantages over conventional drugs. They have longer duration of action and are more specific. Genes can be delivered to the target site by naked DNA, cells, nonviral, and viral vectors. The enormous progress of the past decade in molecular bi-ology and delivery systems has provided ways for targeting genes to the intended cell/tissue and safe, long-term vectors. The eye is an ideal organ for gene therapy. It is easily accessible and it is an immune-privileged site. Currently, there are clinical trials for diseases affecting practically every tissue of the eye, including those to restore vision in patients with Leber congenital amaurosis. However, the number of eye trials compared with those for systemic diseases is quite low (1.8%). Nevertheless, judg-ing by the vast amount of ongoing preclinical studies, it is expected that such number will increase considerably in the near future. One area of great need for eye gene therapy is glaucoma, where a long-term gene drug would eliminate daily applications and compliance issues. Here, we review the current state of gene therapy for glaucoma and the possibilities for treating the trabecular meshwork to lower intraocular pressure and the retinal ganglion cells to protect them from neurodegeneration.

Expression of lectin-like transcript-1 in human tissues.

Background: Receptor-ligand pairs of C-type lectin-like proteins have been shown to play an important role in cross talk between lymphocytes, as well as in immune responses within concrete tissues and structures, such as the skin or the germinal centres. The CD161-Lectin-like Transcript 1 (LLT1) pair has gained particular attention in recent years, yet a detailed analysis of LLT1 distribution in human tissue is lacking. One reason for this is the limited availability and poor characterisation of anti-LLT1 antibodies. Methods: We assessed the staining capabilities of a novel anti-LLT1 antibody clone (2H7), both by immunohistochemistry and flow cytometry, showing its efficiency at LLT1 recognition in both settings. We then analysed LLT1 expression in a wide variety of human tissues. Results: We found LLT1 expression in circulating B cells and monocytes, but not in lung and liver-resident macrophages. We found strikingly high LLT1 expression in immune-privileged sites, such as the brain, placenta and testes, and confirmed the ability of LLT1 to inhibit NK cell function. Conclusions: Overall, this study contributes to the development of efficient tools for the study of LLT1. Moreover, its expression in different healthy human tissues and, particularly, in immune-privileged sites, establishes LLT1 as a good candidate as a regulator of immune responses.

The Impact of the Bone Marrow Microenvironment on T Cell Redirection Therapeutics

Redirecting T cells to specifically target and kill malignant cells has been validated as an effective anti-cancer strategy in the clinic with the approval of CD19xCD3 BiTE (Blincyto) for acute lymphoblastic leukemia. However, the immunosuppressive nature of the tumor microenvironment potentially poses a significant hurdle to T cell therapies. For instance, the bone marrow (BM) niche is appreciated to be a site of immune privilege at steady state to allow for normal hematopoiesis and immune cell generation. Additionally, in hematological malignancies, the BM niche is protective to leukemic stem cells, a phenomenon that has minimized the efficacy of several anti-cancer drugs including chemotherapy, targeted small molecule inhibitors and antibody based therapies.In this study, we investigated the impact of the BM microenvironment on T cell redirection. Using antibodies made with the Genmab DuoBody® technology targeting specific tumor antigens (CD123 and BCMA) and CD3, we observed that co-culture of acute myeloid leukemia (AML; KG-1, MOLM-13 and OCI-AML5) or multiple myeloma (MM; H929 and RPMI8226) cell lines with bone marrow stromal cell lines (HS-5 and HS-27a) significantly protected leukemic cells from DuoBody®-T cell mediated lysis in vitro. Specifically, co-culture of bone marrow stromal cells in killing assays led to a 20-50% decrease in the maximum observed cytotoxicity and a 3-10 fold weaker EC50, reflecting an impact on both the efficacy and potency of bispecific DuoBody® antibodies. Similar results were also observed with primary stromal cells obtained from healthy donors. Furthermore, presence of stromal cells in a humanized xenograft AML model attenuated tumor growth inhibition (TGI) observed with DuoBody® treatment (78% TGI with MOLM-13 vs 15% TGI with MOLM-13+HS-5). Impaired TGI correlated with reduced T cell activation (7 fold decrease in CD25 upregulation) and production of granzyme B (8 fold reduction), providing one potential mechanism to explain loss of activity of the DuoBody® antibody. In vitro trans-well redirection assays revealed that cell-cell contact with stromal cells was crucial for reduced T cell activation and target cell survival relative to controls. Additionally, leukemic cells not killed by T cells were observed to preferentially cluster around stromal cells. We propose that target cells can evade T cell death by a stromal cell dependent mechanism involving activation of multiple pro-survival and anti-apoptotic pathways in leukemic cells in addition to suppressed activation of T cells. We are currently studying pathways mediating the cross-talk between cancer, immune and stromal cells. A better understanding of the mechanisms underlying the protective and immunosuppressive nature of the BM microenvironment will be instrumental to the design of more effective CD3 redirected therapeutics or novel combinatorial regimens for robust anti-cancer responses. DisclosuresNair-Gupta:Janssen: Employment. Rudnick:Janssen Pharmaceuticals R&D: Employment. Luistro:Janssen: Employment. Chin:Janssen: Employment. Smith:Janssen Research & Development, LLC: Employment, Equity Ownership. McDaid:Janssen Pharmaceuticals Research and Development: Employment. Li:Janssen: Employment. Pillarisetti:Janssen Research and Development, LLC: Employment. Baldwin:Janssen: Employment. Packman:Janssen: Employment. Elsayed:Janssen: Employment. Attar:Janssen: Employment. Gaudet:Janssen Pharmaceuticals R&D: Employment, Other: Stock options, Patents & Royalties: pending, not yet issued.

EXTH-23. PRECLINICAL ANALYSIS OF COMBINATORIAL GLIOBLASTOMA THERAPY WITH THE PRODRUG-MEDIATED GENE THERAPY VECTOR AdV-tk AND IMMUNE CHECKPOINT INHIBITION

While the brain has traditionally been considered to be an immune-privileged site, evidence supporting the use of immunotherapeutics has been rapidly accumulating. Given that virus-based cancer therapies can be immunostimulatory and immune-checkpoint inhibitors block tumor-induced T-cell exhaustion, the combination of these two approaches offers a potentially synergistic interaction. One of the molecular underpinnings of T-cell exhaustion is the expression of Programmed Death-1 (PD1) on T-cells that recognizes its ligand PD-L1. AdV-TK is an immunostimulatory virus-based approach, known as Gene-Mediated Cytotoxic Immunotherapy (GMCI), that involves the intra-tumoral delivery of a non-replicating adenoviral vector carrying the Herpes virus thymidine kinase gene(TK) followed by administration of an anti-herpetic prodrug(ganciclovir-GCV) and recently showed encouraging results in a Phase II trial in glioblastoma(Wheeler et al.,2016). To provide a rationale for this therapeutic combination we investigated PD-L1 expression during GMCI therapy in human and mouse glioma cells in vitro and found that there was a consistent increase in cell surface PD-L1 levels. Interestingly, this was not associated with an increase of mRNA or protein. We also show that GMCI induces a type-I interferon response, and that the release of IFNβ is at least partially responsible for autocrine/paracrine PD-L1 up-regulation. In vivo studies using an intracranial GL261 model showed high levels of long term survivors in the GMCI/PD1 combination(11/14), compared with GMCI(6/16), anti-PD1(5/12) and controls(0/11). In addition, tumor infiltrating lymphocytes after GMCI showed an increase in CD8+, CD8+/GranzymeB+, and CD8+/IFNγ+/TNFα+cells suggestive of cytotoxic T-cell activation. However, there was also a significant increase in CD4+, CD4+/FoxP3+, and IL-10 indicating a significant infiltration by Tregs, releasing immunosuppressive cytokines. Additionally, there was a significant increase in PD1+/TIM3+ T-cells, indicative of an immunosuppressive microenvironment. Overall, our data show that GMCI/anti-PD1 combinatorial therapy is effective in a syngeneic tumor model, and strongly support clinical trials of GMCI/checkpoint inhibitor combinations in glioblastoma patients.

Abstract A075: Preclinical analysis of combinatorial glioblastoma therapy with the prodrug-mediated gene therapy vector AdV-TK and immune checkpoint inhibition in GBM therapy

Abstract While the brain has traditionally been considered to be an immune-privileged site, evidence supporting the use of immunotherapeutics has been rapidly accumulating. Given that virus-based cancer therapies can be immunostimulatory and immune-checkpoint inhibitors block tumor-induced T-cell exhaustion, the combination of these two approaches offers a potentially synergistic interaction. One of the molecular underpinnings of T-cell exhaustion is the expression of Programmed Death-1 (PD1) on T-cells that recognizes its ligand PD-L1. AdV-tk is an immunostimulatory virus-based approach, known as Gene-Mediated Cytotoxic Immunotherapy (GMCI), that involves the intra-tumoral delivery of a non-replicating adenoviral vector carrying the Herpes virus thymidine kinase gene(TK) followed by administration of an anti-herpetic prodrug(ganciclovir-GCV) and recently showed encouraging results in a Phase II trial in glioblastoma(Wheeler et al.,2016). To provide a rationale for this therapeutic combination we investigated PD-L1 expression during GMCI therapy in human and mouse glioma cells in vitro and found that there was a consistent increase in cell surface PD-L1 levels. Interestingly, this was not associated with an increase of mRNA or protein. We also show that GMCI induces a type-I interferon response, and that the release of IFNβ is at least partially responsible for autocrine/paracrine PD-L1 up-regulation. In vivo studies using an intracranial GL261 model showed high levels of long term survivors in the GMCI/PD1 combination (11/14), compared with GMCI (6/16), anti-PD1 (5/12) and controls (0/11). In addition, tumor infiltrating lymphocytes after GMCI showed an increase in CD8+, CD8+/GranzymeB+, and CD8+/IFNγ+/TNFα+cells suggestive of cytotoxic T-cell activation. However, there was also a significant increase in CD4+, CD4+/FoxP3+, and IL-10 indicating a significant infiltration by Tregs, releasing immunosuppressive cytokines. Additionally, there was a significant increase in PD1+/TIM3+ T-cells, indicative of an immunosuppressive microenvironment. Overall, our data show that GMCI/anti-PD1 combinatorial therapy is effective in a syngeneic tumor model, and strongly support clinical trials of GMCI/checkpoint inhibitor combinations in glioblastoma patients. Citation Format: Maria Carmela Speranza, Kazue Kasai, Franz Ricklefs, Sarah R. Klein, Carmela Passaro, Hiroshi Nakashima, Johanna Kaufmann, Agnieszka Bronisz, Estuardo Aguilar-Cordova, Brian W. Guzik, Gordon J. Freeman, David A. Reardon, Patrick Wen, E. Antonio Chiocca, Sean E. Lawler. Preclinical analysis of combinatorial glioblastoma therapy with the prodrug-mediated gene therapy vector AdV-TK and immune checkpoint inhibition in GBM therapy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A075.

Acanthamoeba castellanii Genotype T4 Stimulates the Production of Interleukin-10 as Well as Proinflammatory Cytokines in THP-1 Cells, Human Peripheral Blood Mononuclear Cells, and Human Monocyte-Derived Macrophages.

Free-living amoebae of the genus Acanthamoeba can cause severe and chronic infections in humans, mainly localized in immune privileged sites, such as the brain and the eye. Monocytes/macrophages are thought to be involved in Acanthamoeba infections, but little is known about how these facultative parasites influence their functions. The aim of this work was to investigate the effects of Acanthamoeba on human monocytes/macrophages during the early phase of infection. Here, THP-1 cells, primary human monocytes isolated from peripheral blood, and human monocyte-derived macrophages were either coincubated with trophozoites of a clinical isolate of Acanthamoeba (genotype T4) or stimulated with amoeba-derived cell-free conditioned medium. Production of proinflammatory cytokines (tumor necrosis factor alpha [TNF-α], interleukin-6 [IL-6], and IL-12), anti-inflammatory cytokine (IL-10), and chemokine (IL-8) was evaluated at specific hours poststimulation (ranging from 1.5 h to 23 h). We showed that both Acanthamoeba trophozoites and soluble amoebic products induce an early anti-inflammatory monocyte-macrophage phenotype, characterized by significant production of IL-10; furthermore, challenge with either trophozoites or their soluble metabolites stimulate both proinflammatory cytokines and chemokine production, suggesting that this protozoan infection results from the early induction of coexisting, opposed immune responses. Results reported in this paper confirm that the production of proinflammatory cytokines and chemokines by monocytes and macrophages can play a role in the development of the inflammatory response during Acanthamoeba infections. Furthermore, we demonstrate for the first time that Acanthamoeba stimulates IL-10 production in human innate immune cells, which might both promote the immune evasion of Acanthamoeba and limit the induced inflammatory response.

Abstract 2349: Evaluation of the combination of the prodrug-mediated gene therapy vector AdV-tk and immune checkpoint inhibitor for glioblastoma treatment in a syngeneic mouse model

Abstract There has been a recent surge of interest in cancer immunotherapies due to newly FDA approved immunologic treatments based on targeting immune checkpoint inhibition pathways. Despite the dramatic responses in animal models and early clinical human trials, particularly in melanoma, durable clinical responses are observed only in approximately 30% of patients, therefore combinatorial immune therapeutic approaches may be needed to improve outcomes further. While the brain has traditionally been considered to be an immune-privileged site, evidence supporting the use of immunotherapeutics in brain tumors has been rapidly accumulating. Given that virus-based cancer therapies can be immunostimulatory and immune checkpoint inhibitors block the body's natural checkpoint (ICI) response, the combination of these two approaches offers a potentially advantageous interaction. AdV-tk is an immunostimulatory virus-based approach that involves the intra-tumoral delivery of a non-replicating adenoviral vector carrying the Herpes virus thymidine kinase gene (AdV-tk) followed by administration of an anti-herpetic prodrug (ganciclovir - GCV). This approach has shown benefit in clinical trials of glioblastoma among other tumor types. The immunological component results from the delivery vehicle being a virus, the mode of cell death, through both necrosis and apoptosis, and the pro-immunogenic properties of the thymidine kinase protein (TK). This approach has consistently demonstrated anti-tumor immune stimulation and an increased intra-tumoral CD8+ T-cell infiltrate. Not surprisingly, however, this immune stimulation also leads to increased expression of immune checkpoint inhibitory ligands on tumor cells, including PD-L1. Our initial in vitro studies showed that the immune checkpoint ligand PD-L1 is expressed across a panel of human and mouse conventional and stem-like glioblastoma-derived cells (GSCs). After AdV-tk infection and GCV treatment PD-L1 is consistently up-regulated both at protein and mRNA levels, while no changes were detected after AdV-tk alone. In vivo experiments in immunocompetent mouse models with GL261 glioma cell lines demonstrate a significant improvement in survival when we combine AdV-tk+GCV and anti-PD-1 antibodies. This data suggests that combination of immune checkpoint blockade with AdV-tk treatment should be explored in glioblastoma clinical trials. Citation Format: Maria Carmela Speranza, Kazue Kasai, Johanna Kaufmann, Estuardo Aguilar-Cordova, Brian W. Guzik, E. Antonio Chiocca, Sean Lawler. Evaluation of the combination of the prodrug-mediated gene therapy vector AdV-tk and immune checkpoint inhibitor for glioblastoma treatment in a syngeneic mouse model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2349.

Central Nervous System-Peripheral Immune System Dialogue in Neurological Disorders: Possible Application of Neuroimmunology in Urology.

Previous concepts of immune-privileged sites obscured the role of peripheral immune cells in neurological disorders and excluded the consideration of the potential benefits of immunotherapy. Recently, however, numerous studies have demonstrated that the blood–brain barrier in the central nervous system is an educational barrier rather than an absolute barrier to peripheral immune cells. Emerging knowledge of immune-privileged sites suggests that peripheral immune cells can infiltrate these sites via educative gates and that crosstalk can occur between infiltrating immune cells and the central nervous system parenchyma. This concept can be expanded to the testis, which has long been considered an immune-privileged site, and to neurogenic bladder dysfunction. Thus, we propose that the relationship between peripheral immune cells, the brain, and the urologic system should be considered as an additional possible mechanism in urologic diseases, and that immunotherapy might be an alternative therapeutic strategy in treating neurogenic bladder dysfunction.

Ebola Lingers: A Patient Relapses with Meningoencephalitis

Persistence of Ebola virus in immune-privileged sites, such as semen and the aqueous humor, has been well documented. Investigators now report relapse

Late Ebola virus relapse causing meningoencephalitis: a case report

SummaryBackgroundThere are thousands of survivors of the 2014 Ebola outbreak in west Africa. Ebola virus can persist in survivors for months in immune-privileged sites; however, viral relapse causing life-threatening and potentially transmissible disease has not been described. We report a case of late relapse in a patient who had been treated for severe Ebola virus disease with high viral load (peak cycle threshold value 13·2).MethodsA 39-year-old female nurse from Scotland, who had assisted the humanitarian effort in Sierra Leone, had received intensive supportive treatment and experimental antiviral therapies, and had been discharged with undetectable Ebola virus RNA in peripheral blood. The patient was readmitted to hospital 9 months after discharge with symptoms of acute meningitis, and was found to have Ebola virus in cerebrospinal fluid (CSF). She was treated with supportive therapy and experimental antiviral drug GS-5734 (Gilead Sciences, San Francisco, Foster City, CA, USA). We monitored Ebola virus RNA in CSF and plasma, and sequenced the viral genome using an unbiased metagenomic approach.FindingsOn admission, reverse transcriptase PCR identified Ebola virus RNA at a higher level in CSF (cycle threshold value 23·7) than plasma (31·3); infectious virus was only recovered from CSF. The patient developed progressive meningoencephalitis with cranial neuropathies and radiculopathy. Clinical recovery was associated with addition of high-dose corticosteroids during GS-5734 treatment. CSF Ebola virus RNA slowly declined and was undetectable following 14 days of treatment with GS-5734. Sequencing of plasma and CSF viral genome revealed only two non-coding changes compared with the original infecting virus.InterpretationOur report shows that previously unanticipated, late, severe relapses of Ebola virus can occur, in this case in the CNS. This finding fundamentally redefines what is known about the natural history of Ebola virus infection. Vigilance should be maintained in the thousands of Ebola survivors for cases of relapsed infection. The potential for these cases to initiate new transmission chains is a serious public health concern.FundingRoyal Free London NHS Foundation Trust.

Splenic B cells contribute to intraocular tumor progression

Abstract Primary intraocular melanomas heavily infiltrated by CD8+ T cells and macrophages are generally larger, more heavily vascularized tumors with a genetic profile indicating greater risk of metastasis to the liver. To better understand how the tumoricidal activity of these immune cell populations is inhibited within the eye, a site of immune privilege, we developed a mouse model of intraocular tumor development in which immunogenic E.G7-OVA tumor cells transplanted in the anterior chamber form progressively growing intraocular tumors in C57Bl/6 mice despite infiltration by tumor-specific CD8+ T Cells and macrophages. Recently, we demonstrated that splenectomy prior to tumor challenge restored tumoricidal activity of CD8+ T cells and macrophages and promoted complete elimination of established intraocular tumors. Herein, we show that E.G7-OVA tumors transplanted in the eye of B cell deficient μMT mice are rejected by CD8+ T cells. The rejection of intraocular tumors in μMT mice was not due to abnormal splenic architecture but rather stemmed from the absence of tumor-specific B cells as mice transgenic for an immunoglobulin molecule to an irrelevant antigen (hen egg lysozyme) also rejected intraocular E.G7-OVA tumors. These data suggest that intraocular tumor development induces the generation of tumor-specific regulatory B cells (Breg) in the spleen. How these Breg contribute to intraocular tumor growth is not completely understood. However, intraocular E.G7-OVA tumors grew progressively in mice deficient in activation-induced cytidine deaminase that cannot undergo isotype class switching suggesting that secreted tumor specific IgG does not contribute to inhibiting tumoricidal activity within the eye.

Delay in CD8 T-cell priming impairs viral control after vaginal LCMV transmission

Abstract Parameters required for eliciting CD8-T-cell-mediated immunity in the tolerogenic environment of female reproductive tract (FRT) are not well understood. We established a mouse model of intravaginal infection with acute lymphocytic choriomeningitis virus (LCMV) and showed that, compared to systemic infection, CD8-T-cell-mediated protective immune response was delayed in the FRT. After i.vag. LCMV infection, live virus is transmitted to the draining iliac lymph node (iLN) from the lower female reproductive tract (LFRT), which helps to prime virus-specific CD8 T cells in the iLN and restricts further systemic spread of the virus. However, T-cell priming in the iLN of i.vag. mice is delayed compared to that in i.p. infected mice, causing a delay in the appearance of effector CD8 T cells in the LFRT. This delayed CD8 T-cell response in LFRT provides LCMV a window of opportunity to replicate robustly in this immune privileged site. Mechanistically, the delay in CD8 T-cell priming is due to suboptimal activation of LFRT migratory DCs, which require further maturation upon migration into the iLN. As a potential cause of the suboptimal DC activation, we found that LFRT expresses limited steady-state levels of viral RNA-sensing pattern-recognition-receptors and elicits minimal induction of interferons and other inflammatory mediators required for anti-viral immunity. These findings provide a mechanistic explanation for the cause of the delay in CD8 T-cell activation after intravaginal viral infection and emphasize the notion that successful vaccination against RNA viruses may require adjuvants that elicit a stronger innate immune response than that provided by live viruses.

Dissecting the mechanisms underling acute lymphoblastic leukemia brain metastasis

Abstract Despite improved treatments, patient with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) experience relapse with devastating tumor cell dissemination into the central nervous system (CNS). As an “immune privilege site”, CNS is protected from infiltration of immune cells by specialized structure of blood-brain barrier with astrocyte endfeet that surround the blood vessels. To assess whether leukemia cells hijack mechanisms employed by inflamed immune cells to invade into the brain parenchyma, we set up leukemia mouse xenograft models with well-characterized human BCP-ALL cell lines and primary cells derived from patients. We found massive infiltration/colonization of GFP+ leukemia cells inside brain vessels and parenchyma addition to meningeal space. Immunohistochemistry staining showed that activation of astrocytes following the induction of VEGF-A in brains of leukemia burden mice. We address phenotype changes in leukemia cells, that are associated with brain metastasis by comparing the expression of chemokine receptors and cytokines in leukemia cells recovered from bone marrow and other extramedullary sites (spleen, liver) for comparison to the cells recovered from brain tissue. By investigation of extravasation steps in intact brain, we also try to figure how leukemia cells bleach the BBB and find new niche to colonize at brain parenchyma. Ongoing studies will provide new insights into the mechanisms that regulate leukemia CNS infiltration.