Citations

Abstract Immune-mediated blood brain barrier (BBB) disruption is a prominent feature of various neurological conditions for which an emerging role of CD8 T cells is being realized. Our laboratory has developed a model of CD8 T cell-mediated BBB disruption which employs a variation of the Theiler’s murine encephalomyelitis virus (TMEV) infection. At seven days post TMEV infection, the majority of antiviral CD8 T cells recognize an immunodominant virus peptide, VP2121–130, in the context of the H-2Db class I molecule. Upon intravenous administration of this VP2 peptide at seven days post infection, Db:VP2121–130 epitope specific CD8 T cells induce BBB in a perforin dependent process. In this model, we addressed the role of microglia in tandem with CD8+ T cells. Using real time two-photon in vivo imaging, we demonstrate that microglia adopt distinct morphological features including enlarged cell body and fewer ramified processes as early as 6 hours post administration of VP2 peptide. Notably, perforin-deficient mice failed to display BBB disruption and had significantly diminished microglial expression of MHC-II and CD68 in this model. This study demonstrates CD8 T cell promote microglia activation through a perforin-dependent process during BBB disruption. Importantly, microglia are activated concurrently with the onset of vascular permeability and could serve as a critical cell type in this process.

Abstract Cerebral malaria is a serious health complication that can arise during Plasmodium infections. Symptoms of human cerebral malaria (HCM) include coma, permanent neurological deficits, and potentially death. While the mechanism is not well understood, brain swelling and edema have been shown to correlate with a poor disease outcome for afflicted individuals. This work attempts to identify the cellular changes occurring during breakdown of the blood-brain barrier (BBB) by utilizing the murine model of experimental cerebral malaria (ECM). Previous work determined that the tight junctions claudin-5 and occludin, proteins essential for regulating BBB integrity, were disorganized on cerebral endothelial cells during ECM. Here, we focus on the constituents of the BBB to characterize the changes occuring within endothelial cells during disease progression. Importantly, endothelial cell changes were not strictly induced by parasitic infection itself, but also required the presence of functional CD8 T cells. Using perforin deficient mice to impair CD8 T cell effector function, we demonstrate that brain endothelial cells retain the characteristics of uninfected animals in contrast to wild-type infected mice. Interestingly, the CD8 T cells appear to be acting independently of direct killing as there is not an increase in activation of the apoptotic pathway nor is there an increase in dead or dying endothelial cells. From these data we propose that non-cytotoxic CD8 T cell functions are required for ECM induction.

Abstract Microglia are central nervous system (CNS) resident immune cells for which the impact of MHC class I antigen presentation is still being defined. To directly address if microglia contribute to CD8 T cell-mediated immunity of the CNS, we generated a novel tamoxifen inducible CX3CR1-creER H-2Kb MHC class I conditional knockout mouse. In this transgenic mouse, the H-2Kb MHC class I molecule is deleted on all CX3CR1-expressing cells following tamoxifen treatment. By two weeks post tamoxifen treatment, peripheral CX3CR1-expressing cells repopulate, restoring normal expressing of H-2Kb. This leaves only microglia as the sole MHC class I-deficient cell type. We hypothesized that deletion of H-2Kb on microglia would reduce CD8 T cell responses in the CNS. We challenged these animals and cre-negative littermates with intracranial picornavirus infection with Theiler’s murine encephalomyelitis virus expressing ovalbumin (TMEV-OVA). We also challenged mice with experimental GL261 gliomas. We found that microglia-specific H-2Kb deletion reduces the CD8 T cell response in the CNS and attenuates the activation status of brain-infiltrating CD8 T cells in both models of neuroinflammation. We conclude that microglia are functional antigen presenting cells, and are required for a complete antigen-specific CD8 T cell response in the brain. These findings are the first to definitively address the role of microglia-expressed MHC class I in CNS immunological challenges.

Abstract One of the most severe complications of Plasmodium falciparum infection in humans is cerebral malaria. Cerebral malaria causes coma, neurological deficits, and potentially death. The disease is characterized by blood-brain barrier (BBB) disruption leading to vascular permeability. In this study, we investigated cerebral endothelial cell tight junction protein alterations and BBB breakdown in the Plasmodium berghei ANKA(PbA) model of experimental cerebral malaria. PbAinfected C57BL/6 mice displayed significant areas of vascular leakage within the central nervous system at six days post infection. Regions of permeability co-localized with disruption of the BBB tight junction proteins claudin-5 and occludin on microvasculature as shown using confocal microscopy. Additionally, 3D MRI analysis revealed that PbA infected mice exhibited vascular leakage specific to the hypothalamus, thalamus, and brain stem. In contrast, PbA infected perforin deficient mice retained tight junction integrity and displayed significantly reduced vascular leakage. We propose that CD8 T cells initiate vascular permeability through disruption of cerebral endothelial cell tight junctions. In turn, the resulting imbalance of homeostasis in vital brain regions likely contributes to the pathology and morbidity associated with PbA infection.

Abstract MHC multimers including tetramers and pentamers provide a powerful way to visualize antigen specific T cell responses in both experimental and clinical immune assays. The multimers can be acquired commercially, from the NIH, or assembled in the laboratory for measuring immune responses to commonly-studied or novel-hypothetical antigens. The multimer reagents can be expensive and time consuming to acquire and in many cases are uncontrolled reagents as robust positive control T cells used to assess the reagents functionally may not be available. This is particularly problematic when using MHC multimers to investigate less well-defined T cell responses, such as those characterized by low T cell frequencies or by specificity to hypothetical antigens relevant to cancer, infection, or autoimmune disease. These problems are magnified when studying irreplaceable clinical trial samples where having confidence that reagents are biochemically intact is critical. To address this problem, we have devised a versatile, flow cytometry-based, capture assay to probe the structural integrity of MHC multimer reagents. In principle, the idea is to immobilize on a bead antibody specific for conformational determinants expressed on a properly folded MHC molecule. The particle becomes labeled and can be visualized and quantified by flow cytometry when a fluorescently tagged tetramer is adsorbed to the particulate upon binding to the antibody.

Abstract The most severe clinical complication of Plasmodium falciparum infection is cerebral malaria (CM). Plasmodium berghei ANKA (PbA) infection of C57BL/6 mice is an established model of CM. Previous work has implied a role for CD8 T cells and perforin expression in fatal stages of PbA infection. We therefore infected C57BL/6 and C57BL/6 Perforin-/- mice with PbA and assessed the CNS for cerebral endothelial cell blood-brain barrier (BBB) disruption using FITC albumin leakage into tissue, confocal microscopy and 3D volumetric T2 and T1 gadolinium enhanced MRI. PbA infected C57BL/6 mice: 1.) had extensive disruption of BBB tight junction proteins Occludin and Claudin 5, 2.) developed severe CNS vascular permeability of FITC albumin and gadolinium leakage, and 3.) became moribund. We also observed increased VEGF cytokine expression coinciding with CNS vascular permeability in these animals. Despite having similar CD8 T cell infiltration into the brain, PbA infected C57BL/6 Perforin-/- mice had intact BBB tight junction, were devoid of CNS vascular permeability, presented with normal brain MRI, and had normal behavioral scores on the rotarod assay. This study is therefore the first to demonstrate the critical importance of perforin expression in promoting BBB tight junction alteration during PbA infection. Therefore, development of therapies to block factors related perforin mediated delivery could have significant benefit to the treatment of CM.

Abstract MHC class Ia molecules are regulators of antiviral immunity and are thought to function similarly in the presentation of self and antigenic peptides to T cells. However, differences in natural polymorphisms found among class Ia loci in humans and mice indicate these genes may not function equivalently. We used transgenic mice to evaluate the equivalence of MHC Ia gene function in directing cellular immunity against the picornavirus, Theiler’s encephalomyelitis virus, and found that effective antiviral immunity was promoted by H-2D transgenes but not by H-2K in this system. Because transgenes evaluated in our study encoded the peptide binding domain of the Db antigen presenting molecule expressed in the context of the Db or Kb genetic framework, our findings indicate that structural features distinguishing these genes mapping outside of these coding sequences determine ability to direct effective viral immunity. Irrespective of which transgene was expressed, the mice developed robust anti viral T cell responses against the immunodominant TMEV epitope and the T cells were recruited to infected tissue. However, only when the Db antigen binding domain was expressed in the context of the H-2D genetic framework was virus cleared. Our findings suggest that differences in tissue specific class I gene expression at the site of infection is a key factor in this differential function of class Ia genes. Such difference could underlie natural variations in class Ia polymorphism.

Abstract Blood brain barrier (BBB) disruption is a common feature of neurological disease. We developed a novel model of CD8 T cell mediated BBB disruption using the Theilers murine encephalomyelitis virus (TMEV) model of multiple sclerosis. At 7 days post TMEV infection, 50-70% of CNS infiltrating CD8 T cells are specific for an immunodominant virus peptide VP2 121-130 presented in the context of the Db class I molecule. Intravenous injection of VP2 121-130 peptide at 7 days post TMEV infection results in severe CNS vascular permeability and death in the C57BL/6 mouse strain. Despite having an identical CD8 T cell response, 129 SvIm mice survive administration of the VP2 121-130 peptide. We performed bone marrow transplants to determine if a hematopoetic factor was responsible for this fatal syndrome. 129 SvIm mice were reconstituted with C57BL/6 or autologous 129 SvIm bone marrow. Similar brain infiltration and purity of transplanted CD8 T cells was observed during acute TMEV infection in reconstituted mice. However, 129 SvIm mice reconstituted with C57BL/6 bone marrow had increased microhemorrhage formation as measured by T2* weighted MRI and became moribund. Meanwhile, 129 SvIm mice receiving an autologous bone marrow transplant had significantly less microhemorrhage formation and remained resistant to becoming moribund. These studies demonstrate that a hematopoetic factor is responsible for the onset of brain microhemorrhage in immune mediated fatal BBB disruption.

Abstract Myeloid cells comprising up to 50% of total tumor mass in glioblastoma (GBM) contribute to tumor progression and immunosuppression. Restraining glioma-induced myeloid cell infiltration increases functional T cells within the tumors, improves the efficacy of checkpoint blockade, and significantly extends mouse survival. Here, we focus on an ion channel, voltage-gated proton channel Hv1, which is mainly expressed in myeloid cells and shapes the physiological functions of myeloid cells. Our bioinformatics analysis demonstrated that elevated Hv1 expression in the tumor mass correlates with poor disease prognosis in patients. Using the leading immunocompetent model of GBM in mice, we showed that Hv1 knockout mice (Hv1-/-) exhibit slower glioma progression and prolonged survival. Notably, in vivo two-photon imaging, immunofluorescence, and full-spectrum flow cytometry revealed that Hv1-/- mice have reduced monocyte/macrophage infiltration, higher frequency of MHCII+ among all infiltrating myeloid cells, and increased PD-1+CD4+ T cells in the tumor-burdened hemisphere. As a result, we combined anti-PD-1 treatment with Hv1 knockout in the middle stage of glioma, and found that approximately 30% of Hv1-/- mice were cured. Together, our results demonstrated that Hv1 regulates myeloid infiltration and could be a novel therapeutic target for the treatment of GBM.

Abstract INTRODUCTION: Plasma extracellular vesicles (EVs) have been shown as a promising source for biomarker identification in glioblastoma (GBM) and could help differential diagnosis, treatment evaluation and tumor progression monitoring. These EVs are enriched in molecular signatures indicative of their cell origins, giving an indication of the key players in this pathology. In this project, we aimed to identify diagnostic biomarkers for GBM plasma EVs and their cells of origin. METHODS: Plasma EV samples were prepared following the MIFlowCyt-EV guideline of the International Society for Extracellular Vesicles, then stained for EV markers (CD9/CD63/CD81) and markers indicative of cell origins (CD31/CD45/CD41a/CD11b). Actin phalloidin was used as a negative marker. Stained plasma samples were analyzed using a Cytek Aurora flow cytometer. Percentages of different EV subpopulations were analyzed and compared between GBM and normal donor (ND) plasma EVs (reference group). Further clustering analysis was performed on EV events by t-distributed stochastic neighbor embedding (t-SNE) and self-organizing maps on flow cytometry data (FlowSOM) analysis. The predictive value of multiparametric qualities derived from the reference group was tested in blinded test group samples. RESULTS: Percentages of CD9, CD81, and CD11b positive EVs were higher in GBM patient plasma, while ND plasma had more CD41a positive EVs. GBM plasma EVs had unique multiparametric signatures compared to ND plasma EVs based on t-SNE and FlowSOM analysis. Our analysis also identified 15 distinct EV subpopulations which differed in size and various surface marker expression levels. Eight of these subpopulations were enriched for GBM EVs, while three were enriched for ND EVs. Our method of multiparametric analysis demonstrates high sensitivity and specificity in predicting disease status in human samples. CONCLUSIONS: GBM plasma EVs have a unique surface marker expression profile and distinct EV subpopulations compared to ND plasma EVs. Multiparametric signatures show promise as potential diagnostic markers of GBM.