Citations

Abstract Immunosuppression following damage to the CNS is a common, yet poorly understood, feature of neurological diseases as diverse as stroke, traumatic brain injury, and glioblastoma. This immunosuppression is a barrier to successful patient outcomes. We sought to define the effect of various brain insults on the thymus and T-cell responses. We tested thymic function following various neurological insults, including viral infection, brain tumor, sterile inflammation, physical injury, and seizures. All brain insults resulted in significant thymic involution that was reversible if the insult was cleared. Thymic involution did not occur following similar peripheral insults. Using parabiosis, we demonstrated that thymic involution was transferable via circulatory routes from glioma-bearing to non-tumor-bearing parabionts. Similarly, serum obtained from mice with ongoing neurological insults potently inhibited T-cell proliferation in vitro. We next fractionated the serum based on molecular weight and tested the resulting fractions’ immunosuppressive potential. Interestingly, we found that serum fractions with large molecular weights of greater than 100 kiloDaltons were responsible for the immunosuppressive properties of serum obtained from glioma-bearing mice. In short, CNS-specific insults, regardless of nature, induce immunosuppression by prompting thymic involution and systemic immunosuppression mediated through circulating factors with large molecular weight. These studies provide evidence of the mechanisms leading to immune deficiencies observed in patients following neurological injuries.

Abstract Cerebral malaria (CM) is a severe complication arising from Plasmodium falciparum infection which results in high mortality. Lack of mechanistic insight into this pathology has resulted in ineffective therapeutic strategies. Using the Plasmodium berghei ANKA model of experimental cerebral malaria (ECM) our lab recapitulated hallmarks of human CM using small animal imaging, including blood-brain barrier (BBB) permeability, severe edema and microhemorrhage visible via T1 and T2-weighted MRI. Previously it had been demonstrated that infiltration of CD8 T cells with cytotoxic capacity is required to induce BBB permeability and ECM. The observation that not only infiltration, but cytotoxic killing is required provoked us to analyze differential MHC class I-restricted priming among discrete antigen presenting cell (APC) types. Using new MHC class I conditional knockout mice generated by our team, we addressed CD8 T cell priming and capacity to disrupt the BBB in ECM. Our findings revealed that mice lacking the H-2Kb MHC class I molecule expression in CD11c+ cells, but not LysM+ cells, were protected from the development of lethal ECM. Subsequently, conditional ablation of the H-2Db on these same subsets yielded similar results. Therefore, we demonstrate that both Db and Kb class I molecule expression by CD11c+ cells enable CD8 T cell priming that results in BBB permeability during ECM. In contrast, CD8 T cells primed by H-2Kb or H-2Db expressing LysM+ APCs are not capable of inducing BBB disruption. These results imply discrete APC subsets prime functionally distinct CD8 T cell responses in the CNS and modulate the outcome of cerebral malaria. This work begins to bridge the mechanistic gap and will impact future therapeutic strategies.

Abstract Brain resident memory T cells (TRM) have recently been phenotypically characterized. However, the function of these cells in the brain, and their changes during neurological insults, is not well understood. Using the described phenotype of brain TRMs (TCRB+, CD69+, CD4 or CD8+, CD103−, CD44+), we evaluated cellular responses of this population in the naïve brain and upon various neurological insults. We found that numbers of brain TRMs increased as a function of age. Additionally, TRM cells in the naïve brain produce TNFα constitutively, setting them apart from TRMs in other organs. Parabiosis studies revealed that circulating pools of memory T cells contributes to maintenance of TRM cells in the naïve brain. During neurological injury, we observed an increase in numbers of TRMs as early as 24 hours following physical insult induced by intracranial injection of PBS. During brain infection with Theiler’s Murine Encephalomyelitis virus, brain TRMs increased in number prior to detection of any virus-specific CD8 T cells. This implies TRMs respond to CNS viral infections prior to generation of virus antigen specific responses. This response is due to proliferation of TRM cells in the brain, as treatment with FTY720 did not inhibit TRM proliferation 24 hours post infection. In short, naïve brains harbor populations of TNFα producing TRM cells. And TRMs rapidly respond to neurological injuries through proliferation in an antigen independent manner. Understanding brain TRMs is crucial in investigating their role in neurodegenerative disorders or targeting this potent population of brain resident T cells in cancers of the CNS.

Abstract Brain tissue resident memory T cells (TRM) are an emerging lymphocyte of interest. While the phenotype and gene expression pattern of brain TRMs have recently been discussed, their function and response to neurologic insults is not yet understood. Using the described phenotype of TRMs (TCRB+, CD69+, CD4 or CD8+, CD103−, CD44+), we analyzed the reaction of this lymphocyte in the brain to various neurological insults. We found that physical insult induced by an intracranial(ic) injection of PBS resulted in increased TRM numbers within 24 hours post insult. This TRM population later decreased yet remained above baseline by 50 days post insult. Interestingly, TRM populations also increased in the brains of animals as a function of age, suggesting natural insults experienced by mice leads to an ever increasing TRM population in the brain. We then assessed the reaction of TRMs to virus in the brain. Mice infected ic with Theiler’s Murine Encephalomyelitis virus (TMEV) had a marked increase in brain TRMs one day later, which is 4 days prior to detection of traditional TMEV antigenspecific CD8 T cells. This implies TRMs respond to CNS viral infections prior to generation of virus antigen specific responses. Finally, we investigated the role of dendritic cell (DC) antigen presentation in generation of TRMs using our novel MHC class I conditional knockout mice. Conditional ablation of H-2Kb and H-2Db did not disrupt TRM populations in the brain. Our data is the first study to show: 1. Activation of TRMs in the brain preceded antigen specific cell infiltration and 2. MHC class I molecules on DCs are not required to generate brain resident TRMS. Understanding brain TRMs is crucial in elucidating their role in neurodegeneration and/or targeting them in CNS cancers.

Abstract Cerebral malaria (CM) is a deadly neurological complication of Plasmodium falciparum infection resulting in high mortality or severe cognitive deficit. Current therapeutics are highly ineffective, namely due to drug resistance and lack of mechanistic insight into pathophysiology. CM is characterized by VEGF upregulation, disruption of blood-brain barrier (BBB), vascular permeability, and severe edema detectable by MRI. Using small animal MRI and the established animal model of CM Plasmodium berghei ANKA, we recapitulate these features of human disease. Here we show that regions of brain permeability during CM as identified by T1-weighted gadolinium-enhanced and T2-weighted MRI coincide with VEGF mRNA upregulation. Regions of analysis include granular and glomerular cell layers of main olfactory bulb and superior medullary brainstem. We determined through RNA in situ hybridization that neurons are the major cell type responsible for the increased VEGF production during CM. Interestingly, we identified another cell type found to minimally upregulate VEGF in close proximity to these neurons. These VEGF expressing cells may play a role in support or induction of the neuronal response. Intriguingly, astrocytes show no VEGF upregulation during this pathology. Overall, these findings suggest a novel role for neuronal-VEGF in promoting CNS pathology, and a putative mechanism of BBB disruption and vascular permeability during CM.

Abstract Systemic immunosuppression following neurological insults including stroke, traumatic brain injury, and glioblastoma mutiforme (GBM) causes mortality and leads to failure of therapies. Exact immunological nature and the underlying mechanisms of this immunosuppression are unknown. Our goal was to define effects of neurological insults given exclusively to the brain on the thymus; a primary immune organ. Specifically, we evaluated the effects on the thymus following physical injury with intracranial PBS injection, intracranial (ic) Theiler’s Murine Encephalomyelitis Virus infection, intracranial injection with lipopolysaccharide, CNS tumors (B16 melanoma and GL261 glioma ic), and seizure induction with intraperitoneal Kainic acid injection. These insults resulted in significant thymic involution that is reversible upon clearance of the neurological insult. The extent of thymic involution correlated with the extent of brain injury. Further analysis of the brain-thymus axis in mice with GL261 gliomas was conducted using RNA-Seq. Principle component analysis of RNA-Seq data revealed unbiased separation of thymi of naïve and glioma harboring mice, suggesting that thymus is directly affected by brain injury. To determine the extent to which thymic involution was caused by a soluble factor, we employed parabiosis. This approach demonstrated a soluble factor is responsible for thymic involution. These findings are the first demonstration that neurological insults ubiquitously contribute to immune suppression. We also provide evidence that thymic involution is mediated by a soluble factor, the identification of which could be critical for ameliorating the effect of neurological injuries on immune suppression.

Abstract Glioblastoma multiforme (GBM) is incurable and among the most lethal of cancers. Immunotherapy for GBM is hampered by the fact that GBM patients are severely immunosuppressed with dangerously low T cell counts. Importantly, immunotherapy for GBM will be ineffective without defining the mechanism of immunosuppression. Given that GBM does not metastasize, we investigated the possibility of neuro-immune interactions using the GL261 glioma model in C57BL/6 mice. C57BL/6 mice harboring GL261 gliomas have abnormal thymuses which are acutely and significantly involuted compared to healthy control mice. Glioma bearing mice also have significant thymic atrophy which increases with tumor burden. Further structural analysis of the thymus in glioma bearing mice revealed disrupted thymic epithelial cells and reduced EPCAM+ cell expression of MHC II. Thymic subsets in tumor bearing mice were also developmentally skewed with increased generation of single positive T cells and a block at double negative 2 (DN2) to DN3 transition. Peripherally, MHCII expression was also significantly decreased in blood derived cells in glioma bearing mice. This correlated with an overall reduction in total CD4 T cell numbers. The results of this study demonstrate the GL261 model is recapitulating the immune suppression observed in GBM patients. These results also support the presence of a brain-thymus interaction that could negatively impact the development of immunotherapy strategies to treat GBM.

Abstract Glioblastoma multiforme (GBM) is a deadly CNS malignancy with an average survival of ~12 months post diagnosis despite aggressive treatment. Immunotherapy approaches to treat GBM are being developed, but the unique immune environment of the brain and the immunosuppressive features of GBM have complicated early efforts. Even αPD-1 checkpoint blockade, highly useful in the context of melanoma, has proved ineffective in the treatment of GBM. In order to promote the efficacy of αPD-1 against the GL261 murine model of GBM, we supplemented this therapy with an engineered IL-2 cytokine. IL-2 therapy has been used clinically for metastatic melanoma and renal cancer, but its short half-life in circulation necessitated dangerously high doses to keep the drug concentration sufficiently high. Fusing IL-2 to the mouse serum albumin molecule (MSA-IL-2), extends the cytokine’s half-life. While neither αPD-1 nor MSA-IL-2 are effective monotherapies against the GL261 model tumor, we have found this combination immunotherapy to provide durable tumor clearance and to create immunologic memory capable of resisting GL261 rechallenge. Most strikingly, this combination therapy is independent of the CD8 T cell function, clearing established GL261 tumors in mice incapable of MHC class I restricted antigen presentation. Independence of MHC class I restricted antigen presentation makes this combination immunotherapy using αPD-1 checkpoint blockade both highly novel and translatable to clinical use.

Abstract Peripheral immune suppression is a hallmark of highly malignant Glioblastoma multiforme (GBM). Given that effective immunotherapy for GBM relies on a functional immune system, it is crucial to define the underlying mechanism of peripheral immunosuppression. We therefore evaluated immunosuppression using the GL261 model of GBM. We first examined cytokine production by microglia and brain-infiltrating lymphocytes for immune suppressing factors. We observed relatively unchanged levels of IL-10 and TGF-β in glioma bearing mice compared to controls. Therefore, we next investigated cellular components of the blood for evidence of suppression. However, we did not observe enhanced frequencies of Tregs (Foxp3+ CD4+) in tumor bearing mice. Finally, we analyzed cytokine levels in the serum of mice harboring gliomas compared to glioma negative animals. This assay revealed fluctuations in chemokine and cytokine levels between these two groups, prompting the analysis of a potential serum derived factor in promoting immunosuppression. We therefore evaluated T cell activation and proliferation in the presence of serum isolated from glioma harboring mice and healthy controls. CFSE-labeled T cells stimulated with CD3/CD28 beads in the presence of glioma negative mouse serum had normal proliferation. In contrast, CD4+ and CD8+ T cell proliferation was completely inhibited when cultured with serum from glioma bearing mice. These studies demonstrate the importance of a serum derived factor in mediating immunosuppression. Defining and inhibiting this circulating factor could be the first step towards improving immunotherapy strategies for GBM.

Abstract Blood brain barrier (BBB) disruption is a feature of multiple neurological disorders for which there is an increasingly recognized role for CD8+ T cells. Theiler’s murine encephalomyelitis virus (TMEV) infection serves as an excellent model to study CD8+ T cell mediated BBB disruption because of the well-characterized immune response to the immunodominant VP2121–130 peptide presented in the context of the H-2Db class I molecule. Intravenous injection of VP2121–130 peptide seven days post TMEV infection results in severe BBB disruption. BBB disruption is dependent on CD8+ T cell interaction with the Db: VP2121–130 epitope, but the antigen presenting cell required for BBB disruption is undefined. We hypothesized that dendritic cells are responsible for presentation of the immunodominant VP2121–130 peptide. We tested this hypothesis using a novel H-2Db Cre-LoxP conditional knockout mouse bred to MHC class I deficient CD11c-Cre expressing mice. We then evaluated these animals at 7 days post infection for induction of BBB disruption, following VP2121–130 peptide administration, using gadolinium enhanced MRI, fluorescent albumin leakage into the brain, and confocal microscopy for the tight junction protein occludin. Control CD11c-Cre(−) mice generated robust Db: VP2121–130 epitope specific CD8+ T cell responses and displayed extensive BBB disruption. In contrast, CD11c-Cre(+) littermates generated no brain infiltrating antiviral CD8+ T cell responses and were protected from BBB disruption. We conclude that CD11c+ dendritic cells are required for priming of CD8+ T cell responses and are necessary for BBB disruption during neuroinflammation in this model.