Jin, Fang

Abstract CD8 T cell responses are essential for clearance of infections in the CNS but contribute to neuropathology. T cell interaction with antigen presenting cells (APCs) at CNS barriers is thought to allow activated antigen-specific T cells to infiltrate the CNS during neuroinflammation. However, the role of CNS-resident APCs during CD8 T cell infiltration of the CNS is unclear. To address this, we utilized Theiler’s murine encephalomyelitis virus (TMEV) and Plasmodium berghei ANKA (PbA) to assess the role of APCs during CD8 T cell responses to CNS infection in mice. During infection, we observed robust upregulation of MHC class I on microglia and brain associated macrophages (BAM). Microglia and BAMs were located in close proximity to inflamed vasculature where antigen presentation to T cells could occur. Thus, we hypothesized that microglia and BAMs are critical APCs for CNS infiltration of antigen specific CD8 T cells. To test this, we generated mice with inducible deletion of MHC class I on microglia and BAMs by crossing CX3CR1creERT2 mice with H-2Db or H-2Kb floxed mice (Db cKO or Kb cKO). We found that brain infiltrating virus-specific CD8 T cells were reduced in Db cKO mice compared to controls during TMEV infection, while peripheral priming of T cells was unaffected. Conversely, antigen-specific CD8 T cell infiltration was not impacted in Kb cKO mice infected with TMEV-OVA257–264. In Db cKO mice, brain-infiltrating CD8 T cells were increased during PbA induced experimental cerebral malaria (ECM), indicating a potential inhibitory role for CNS APCs. These results demonstrate that local antigen presentation by microglia/BAMs can both positively and negatively regulate CD8 T cell infiltration of the brain in discrete neurologic disease models.

Abstract Glioblastoma multiforme (GBM) is a deadly CNS malignancy with an average survival of ~12–18 months post diagnosis. The unique immune environment of the brain and the immunosuppressive features of GBM complicate the use of immunotherapies against these tumors. We aimed to promote the efficacy of αPD-1 against the GL261 murine model of GBM by supplementing 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 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. Neither αPD-1 nor MSA-IL-2 are effective monotherapies for GL261 tumors, but the combination provides durable tumor clearance and prevents subsequent rechallenge. Most strikingly, this combination therapy cleared established GL261 tumors even in mice incapable of MHC class I restricted antigen presentation. Instead, therapeutic efficacy was abrogated by the depletion of CD4 T cells. Our combination therapy also reversed multiple key features of GBM-associated peripheral immune suppression, including depressed CD4 T cell counts and dampened MHC class II expression. Our combination therapy’s ability to overcome these immune derangements associated with glioma growth and to function independent of MHC class I restricted antigen presentation enhance the appeal of this therapy for future translational adaptation. This work was supported by the National Institutes of Health (R01 NS103212) and the Mayo Clinic-Koch Institute Cancer Solutions Team Grant

Abstract Glioblastoma (GBM) is an incurable brain cancer which is associated with severe peripheral immune suppression. Using experimental GBM, we recently determined that peripheral immunosuppression involves thymic atrophy, stunted T cell proliferation, and release of circulating factors that inhibit immune responses. RNA sequencing and proteomic studies have recently determined that transcripts and protein levels of Serpina3n are upregulated in the brain and serum of GBM patients and correlates with poor prognosis. We determined Serpina3n is a highly upregulated gene in the brain and the thymus of glioma-bearing mice using RNA sequencing technologies. We therefore sought to determine the role of Serpina3n in peripheral immunosuppression in GL261 glioma harboring wild type and Serpina3n knockout mice. Glioma implantation in Serpina3n knockout mice resulted in similar survival and peripheral immunosuppression compared to controls. Western blotting experiments confirmed that Serpina3n is expressed and secreted by GL261 gliomas isolated from WT and Serpina3n knockout mouse brains. Together this data suggests that tumor-derived, but not host derived, Serpina3n needs to be evaluated as a putative factor associated with peripheral immunosuppression. Supported by Brains Together for a Cure grant

Abstract The contribution of antiviral resident memory T cells (TRM) to blood-brain barrier disruption has not been defined despite the implication of this lymphocyte in clinical encephalitis. We therefore evaluated the capacity of TRM cells to induce CNS vascular permeability in mice which resolved Theiler’s murine encephalomyelitis (TMEV) infection. Following TMEV infection of the brain, persistent populations of virus antigen specific resident memory T cells are induced within the brain. This population of resident memory T cells can be further reactivated at 60 dpi through administration of the cognate immunodominant virus peptide antigen, VP2, inducing blood brain barrier disruption in the process. We determined that brain resident memory T cells expand following reactivation with administered VP2 peptide. This reactivation is associated with infiltration of T cells, and inflammatory myeloid cells into the brain. Importantly, using depletion and T cell sequestrating strategies with low dose anti CD8 antibodies and FTY720, we determined that TRM induced blood-brain barrier disruption is independent of peripheral T cells. We conclude that reactivation of brain TRM cells during peptide administration occurs without dependency on peripheral responses, highlighting the importance of this cell type in inducing neuroinflammation and underlying neuropathology.

Abstract Glioblastoma (GBM) is an incurable and aggressive form of brain cancer that is associated with severe peripheral immunosuppression. This immunosuppression is a critical barrier to patient survival and the success of immune modulatory therapies. Using the murine GL261 model of GBM, we recapitulated major features of peripheral immunosuppression observed in patients including spleen atrophy, T cell lymphopenia, and T cell sequestration within the bone marrow. We determined that peripheral immunosuppression in GBM is multifaceted and involves release of novel circulating soluble factors that are found in the serum of glioma-bearing mice. This high molecular weight species potently inhibits T cell proliferation in vitro. Cell-free extracellular DNA has been recently identified as an immune suppressive factor in circulation. We, therefore, assessed cell-free DNA levels in the GL261 model. We determined that serum isolated from glioma-bearing mice harbor significantly higher concentrations of double stranded and single stranded cell-free DNA compared to healthy controls. Serial assessment of DNA concentrations in serum of glioma-bearing mice indicated that the most prominent increase was concurrent with end-stage disease. These findings provide a tractable approach to investigate the cellular source and immune suppressive effects of cell-free DNA in the leading murine model of GBM. Supported by K99NS117799

Abstract Brain atrophy is a common feature of many neurological diseases as diverse as Alzheimer’s disease, cerebral palsy, Huntington’s disease, Krabbe disease, multiple sclerosis, Pick’s disease, epilepsy, encephalitis, neurosyphilis, neuroAIDS, and Covid-19 infection. We have developed a murine model of brain atrophy using the Theiler’s murine encephalomyelitis virus (TMEV) infection mouse model of multiple sclerosis. In this study, we investigated the contribution of the MHC class I molecule, H-2Db, in generating an immune response associated with the onset of brain atrophy. To define the roll of the MHC class I molecule in the cellular and molecular mechanisms of brain atrophy, we developed a novel bi-transgenic mouse lines with tamoxifen induced conditional ablation of the H-2Db class I molecule in Cx3CR1+ brain resident myeloid cells on the C57BL/6 background. Interestingly, both CX3CR1cre/Db LoxP mice and their cre- littermate controls experienced short-term memory loss using novel object recognition tests. However, Cx3CR1cre+/Db LoxP mice presented with significantly less brain atrophy as assessed by analysis of lateral ventricular volume at 45 d.p.i. using T2-weighted MRI, compared to cre- littermates. This change in ventricular volume was sustained at 6 months post infection. These results strongly imply a requirement for antigen presentation by H-2Db on myeloid cells for the development of brain atrophy and provided insight into the molecular mechanism of this poorly understood neuropathology. Supported by NIH (RFI NS122174)

Abstract Immune mechanisms of neuropathology in human cerebral malaria are not fully understood. Using the Plasmodium berghei ANKA model of experimental cerebral malaria (ECM), we sought to define the contribution of cerebral endothelial cell (CEC) MHC class I antigen presentation, to the pathogenesis and lethality of ECM. CD8 T cell interactions with class I molecules on CECs has been put forward as a putative mechanism for ECM. However, the importance of this interaction has not been confirmed molecularly in vivo. We therefore hypothesized that conditional ablation of antigen presentation by H-2Kb and H-2Db class I molecules specifically on brain endothelium would result in controlled modulation of activation and entry of parasite-specific CD8 T cells, attenuating pathology. To test this, we crossed endothelium-specific CDH5-Cre mice with our novel H-2Kb and H-2Db LoxP mice to create endothelium-specific cKO of either or both class I molecules. 2-photon imaging revealed that each discreet class I molecule influenced differential T cell dynamics at the neurovasculature during lethal stage of disease. Furthermore, CD8 T cell activation and infiltration of the brain was distinctly altered, per class I interaction, and vascular permeability at the blood-brain barrier was attenuated, resulting in improved survival of H-2Kb and H-2Db endothelial cKO mice. Together these data demonstrate that parasite-specific CD8 T cells undergo Ag-dependent engagement with H-2Kb or H-2Db class I molecules on CECs in order to coordinate activation, entry, and induction of neuropathology. These studies indicate that modulation of CEC class I restricted antigen presentation may hold therapeutic benefit in cases of severe malaria. Supported by Ruth L. Kirschstein National Research Service Award Individual Predoctoral Fellowship F31 1F31NS116924-01. NIH R01 5R01NS103212-05.

Abstract CD8 T cell priming after viral infection of the central nervous system (CNS) is incompletely understood. In peripheral infections, T cell priming generally relies upon stimulation by antigen-bearing dendritic cells (DCs) migrating into draining lymph nodes. The parenchyma of the brain, however, is largely devoid of dendritic cells and evidence demonstrating that activated DCs leave the brain parenchyma is lacking. We therefore sought to understand the role of DCs and specific MHC class I molecules in priming CD8 T cells following CNS infection. We employed H-2Db−/− H-2Kb−/− (MHC-I deficient) mice that express a floxed H-2Db or floxed Kb molecule, a unique transgenic mouse system developed by our research program. Crossed with an MHC class I deficient CD11c-Cre expressing animal results in DC-specific ablation of Db or Kb Class I restricted antigen presentation ability. We found that CD11c+ APCs are critical for the early priming of CD8 T cells against the Db restricted VP2121–130 epitope of the model pathogen, TMEV. However, DCs are ultimately dispensable for long-term development of these antigen specific T cells. Discordantly, the loss of MHC class I restricted antigen presentation ability on CD11c+ APCs does not impact the priming of T cells against the H-2Kb restricted OVA257–264 epitope following intracranial TMEV-OVA infection, but does impact the ability of these T cells to successfully occupy the CNS. This work suggests that differing patterns of MHC class I restriction for CNS pathogen-derived epitopes might have functional consequences on the APC requirements for a successful CD8 T cell response against neurotropic viruses.

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 unique model of CD8 T cell-mediated BBB disruption which employs a variation of the Theiler’s murine encephalomyelitis virus infection (TMEV) infection. Upon intravenous administration of VP2121–130 viral peptide during peak anti-viral adaptive response, Db:VP2121–130 epitope specific CD8 T cells induce BBB disruption in a perforin dependent manner. 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 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.