Georgette L. Suidan

Induction of Blood Brain Barrier Tight Junction Protein Alterations by CD8 T Cells

Disruption of the blood brain barrier (BBB) is a hallmark feature of immune-mediated neurological disorders as diverse as viral hemorrhagic fevers, cerebral malaria and acute hemorrhagic leukoencephalitis. Although current models hypothesize that immune cells promote vascular permeability in human disease, the role CD8 T cells play in BBB breakdown remains poorly defined. Our laboratory has developed a novel murine model of CD8 T cell mediated central nervous system (CNS) vascular permeability using a variation of the Theilers virus model of multiple sclerosis. In previous studies, we observed that MHC class II−/− (CD4 T cell deficient), IFN-γR−/−, TNF-α−/−, TNFR1−/−, TNFR2−/−, and TNFR1/TNFR2 double knockout mice as well as those with inhibition of IL-1 and LTβ activity were susceptible to CNS vascular permeability. Therefore, the objective of this study was to determine the extent immune effector proteins utilized by CD8 T cells, perforin and FasL, contributed to CNS vascular permeability. Using techniques such as fluorescent activated cell sorting (FACS), T1 gadolinium-enhanced magnetic resonance imaging (MRI), FITC-albumin leakage assays, microvessel isolation, western blotting and immunofluorescent microscopy, we show that in vivo stimulation of CNS infiltrating antigen-specific CD8 T cells initiates astrocyte activation, alteration of BBB tight junction proteins and increased CNS vascular permeability in a non-apoptotic manner. Using the aforementioned techniques, we found that despite having similar expansion of CD8 T cells in the brain as wildtype and Fas Ligand deficient animals, perforin deficient mice were resistant to tight junction alterations and CNS vascular permeability. To our knowledge, this study is the first to demonstrate that CNS infiltrating antigen-specific CD8 T cells have the capacity to initiate BBB tight junction disruption through a non-apoptotic perforin dependent mechanism and our model is one of few that are useful for studies in this field. These novel findings are highly relevant to the development of therapies designed to control immune mediated CNS vascular permeability.

CD8 T Cell-Initiated Vascular Endothelial Growth Factor Expression Promotes Central Nervous System Vascular Permeability under Neuroinflammatory Conditions

Dysregulation of the blood-brain barrier (BBB) is a hallmark feature of numerous neurologic disorders as diverse as multiple sclerosis, stroke, epilepsy, viral hemorrhagic fevers, cerebral malaria, and acute hemorrhagic leukoencephalitis. CD8 T cells are one immune cell type that have been implicated in promoting vascular permeability in these conditions. Our laboratory has created a murine model of CD8 T cell-mediated CNS vascular permeability using a variation of the Theiler’s murine encephalomyelitis virus system traditionally used to study multiple sclerosis. Previously, we demonstrated that CD8 T cells have the capacity to initiate astrocyte activation, cerebral endothelial cell tight junction protein alterations and CNS vascular permeability through a perforin-dependent process. To address the downstream mechanism by which CD8 T cells promote BBB dysregulation, in this study, we assess the role of vascular endothelial growth factor (VEGF) expression in this model. We demonstrate that neuronal expression of VEGF is significantly upregulated prior to, and coinciding with, CNS vascular permeability. Phosphorylation of fetal liver kinase-1 is significantly increased early in this process indicating activation of this receptor. Specific inhibition of neuropilin-1 significantly reduced CNS vascular permeability and fetal liver kinase-1 activation, and preserved levels of the cerebral endothelial cell tight junction protein occludin. Our data demonstrate that CD8 T cells initiate neuronal expression of VEGF in the CNS under neuroinflammatory conditions, and that VEGF may be a viable therapeutic target in neurologic disease characterized by inflammation-induced BBB disruption.

The CD8 T cell in multiple sclerosis: suppressor cell or mediator of neuropathology?

Multiple sclerosis (MS) is the most common human demyelinating disease of the central nervous system. It is universally accepted that the immune system plays a major role in the pathogenesis of MS. For decades, CD4 T cells have been considered the predominant mediator of neuropathology in MS. This perception was largely due to the similarity between MS and CD4 T-cell-driven experimental allergic encephalomyelitis, the most commonly studied murine model of MS. Over the last decade, several new observations in MS research imply an emerging role for CD8 T cells in neuropathogenesis. In certain experimental autoimmune encephalomyelitis (EAE) models, CD8 T cells are considered suppressors of pathology, whereas in other EAE models, neuropathology can be exacerbated by adoptive transfer of CD8 T cells. Studies using the Theilers murine encephalomyelitis virus (TMEV) model have demonstrated preservation of motor function and axonal integrity in animals deficient in CD8 T cells or their effector molecules. CD8 T cells have also been demonstrated to be important regulators of blood-brain barrier permeability. There is also an emerging role for CD8 T cells in human MS. Human genetic studies reveal an important role for HLA class I molecules in MS susceptibility. In addition, neuropathologic studies demonstrate that CD8 T cells are the most numerous inflammatory infiltrate in MS lesions at all stages of lesion development. CD8 T cells are also capable of damaging neurons and axons in vitro. In this chapter, we discuss the neuropathologic, genetic, and experimental evidence for a critical role of CD8 T cells in the pathogenesis of MS and its most frequently studied animal models. We also highlight important new avenues for future research.

Acute hemorrhagic demyelination in a murine model of multiple sclerosis

Acute hemorrhagic leukoencephalomyelitis (AHLE) is a rare neurological condition characterized by the development of acute hemorrhagic demyelination and high mortality. The pathomechanism of AHLE, as well as potential therapeutic approaches, have remained elusive due to the lack of suitable animal models. We report the first murine model of AHLE using a variation of the Theilers Murine Encephalitis Virus (TMEV) MS model. During acute TMEV infection, C57BL/6 mice do not normally undergo demyelination. However, when 7 day TMEV infected C57BL/6 mice are intravenously administered the immunodominant CD8 T cell peptide, VP2121–130, animals develop characteristics of human AHLE based on pathologic, MRI and clinical features including microhemorrhages, increased blood-brain barrier permeability, and demyelination. The animals also develop severe disability as assessed using the rotarod assay. This study demonstrates the development of hemorrhagic demyelination in TMEV infected C57BL/6 mice within 24 hours of inducing this condition through intravenous administration of CD8 T cell restricted peptide. This study is also the first demonstration of rapid demyelination in a TMEV resistant non-demyelinating strain without transgenic alterations or pharmacologically induced immunosuppression.

A potential role for CD8+ T-cells as regulators of CNS vascular permeability

Abstract The role of immune cells in promoting central nervous system (CNS) vascular permeability is poorly understood. In recent years, there is a growing body of literature that suggests CD8+ T-cells are potent mediators of vascular permeability in peripheral viral infections as well as in immune mediated neurological diseases. This review outlines the recent advances in tissue culture and animal models used to study vascular permeability. In addition, we put forth our hypothesis that CD8+ T-cells promote the opening of tight junctions between cerebral endothelial cells, enabling the infiltration of white blood cells and in certain models even leading to microhemorrhages in the CNS. Determining the mechanism by which CD8+ T-cells and other immune cells promote CNS vascular permeability in animal models could define new targets for immune mediated neurological conditions characterized by vascular permeability.

Preserved vascular integrity and enhanced survival following neuropilin-1 inhibition in a mouse model of CD8 T cell-initiated CNS vascular permeability.

BackgroundAltered permeability of the blood–brain barrier (BBB) is a feature of numerous neurological conditions including multiple sclerosis, cerebral malaria, viral hemorrhagic fevers and acute hemorrhagic leukoencephalitis. Our laboratory has developed a murine model of CD8 T cell-initiated central nervous system (CNS) vascular permeability in which vascular endothelial growth factor (VEGF) signaling plays a prominent role in BBB disruption.FindingsIn this study, we addressed the hypothesis that in vivo blockade of VEGF signal transduction through administration of peptide (ATWLPPR) to inhibit neuropilin-1 (NRP-1) would have a therapeutic effect following induction of CD8 T cell-initiated BBB disruption. We report that inhibition of NRP-1, a co-receptor that enhances VEGFR2 (flk-1) receptor activation, decreases vascular permeability, brain hemorrhage, and mortality in this model of CD8 T cell-initiated BBB disruption. We also examine the expression pattern of VEGFR2 (flk-1) and VEGFR1 (flt-1) mRNA expression during a time course of this condition. We find that viral infection of the brain leads to increased expression of flk-1 mRNA. In addition, flk-1 and flt-1 expression levels decrease in the striatum and hippocampus in later time points following induction of CD8 T cell-mediated BBB disruption.ConclusionThis study demonstrates that NRP-1 is a potential therapeutic target in neuro-inflammatory diseases involving BBB disruption and brain hemorrhage. Additionally, the reduction in VEGF receptors subsequent to BBB disruption could be involved in compensatory negative feedback as an attempt to reduce vascular permeability.

A translatable molecular approach to determining CD8 T-cell epitopes in TMEV infection

Defining the epitope specificity of CD8+ T cells is an important goal in autoimmune and immune-mediated disease research. We have developed a translational molecular approach to determine the epitope specificity of CD8+ T cells using the Theilers murine encephalomyelitis virus (TMEV) model of multiple sclerosis (MS). TMEV-specific CD8+ T cells were isolated from brains and spleens of 7-day TMEV-infected C57BL/6J mice and stimulated by Cos-7 cells that were co-transfected with expression vectors encoding the D(b) class I molecule along with overlapping segments of the TMEV genome. Both brain-infiltrating and spleen-derived CD8+ T cells expressed IFN-gamma when Cos-7 cells were co-transfected with D(b) class I molecule and the TMEV genomic segment that encoded the immunodominant TMEV epitope. This demonstrated that peripheral and brain-infiltrating CD8+ T-cell responses were focused on peptide epitope(s) encoded by the same region of the TMEV genome. We propose that a similar molecular approach could also be used to determine the antigen specificity of suppressor CD8 T cells by the measurement of transforming growth factor-beta (TGF-beta) production. In addition, with a randomly generated library and peripheral blood or isolated CSF CD8+ T cells, this would be an effective method of predicting the epitope specificity of CD8+ T cells in human inflammatory CNS diseases, in animal models of MS or other organ-specific inflammatory diseases with a protective or pathogenic role of CD8 T cells.