Johnson, Aaron J.

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 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. Using in vivo two-photon imaging, we found that myeloid cells infiltrate into tumor mass since early time points and have intimate interaction with tumor cells. Notably, by full-spectrum flow cytometry, we found that Hv1−/− mice have reduced monocyte/macrophage infiltration, 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 controls myeloid infiltration and could be a novel therapeutic target for the treatment of GBM. Supported by R01NS110825, R01NS088627

Abstract Cytotoxic T cell infiltration into the central nervous system (CNS) is required for clearance of pathogen or tumor. Whether microglia, the resident innate immune cells of the brain, are involved in orchestrating CD8 T cell infiltration of the brain in an antigen dependent manner remains to be elucidated. We hypothesized that antigen presentation by major histocompatibility complex (MHC) class I molecules expressed by microglia is required to orchestrate antigen specific CD8 T cell infiltration into the brain parenchyma. To test this hypothesis, we generated a novel tamoxifen-inducible microglial MHC class I conditional knockout mouse (CX3CR1-creERT2H-2Kb cKO). In these mice, administration of tamoxifen mediates deletion of the H-2Kbmolecule in cells expressing CX3CR1. Short-lived peripheral CX3CR1+ cells repopulate within 4–6 weeks after tamoxifen treatment and restore expression of H-2Kb, leaving only long-lived microglia deficient for MHC class I. We challenged these mice with intracranial infection of Theiler’s murine encephalomyelitis virus expressing the model antigen ovalbumin (TMEV-OVA) to assess antigen specific CD8 T cell responses within the brain parenchyma. In microglia MHC I cKO mice, the frequency of brain infiltrating OVA-specific CD8 T cells was attenuated while the frequency of intravascular OVA-specific CD8 T cells was increased when compared to cre− littermate controls. These results suggest that microglial antigen presentation is important for antigen specific CD8 T cell infiltration into the brain parenchyma. These studies directly address the critical role of microglial MHC class I on antigen specific CD8 T cell responses to an in vivo neuroinflammatory insult.

Abstract CD8 T cell responses are essential for clearance of infections and tumors in the central nervous system (CNS). Under homeostatic conditions, T cell access to the CNS parenchyma is restricted. An intricate process of T cell reactivation by antigen presenting cells (APCs) at CNS barriers is thought to allow activated antigen-specific T cells to infiltrate the parenchyma during neurological disease. The role of CNS-resident APCs during CD8 T cell infiltration of the CNS parenchyma is unclear. We hypothesized that microglia and brain-associated macrophages (BAMs) are APCs critical for antigen specific CD8 T cell reactivation and CNS infiltration. To test this hypothesis, we generated mice with tamoxifen-inducible deletion of MHC class I on microglia and BAMs by crossing CX3CR1creER mice with H-2Db fl/fl mice (Db cKO). We challenged these mice with intracranial infection of Theiler’s murine encephalomyelitis virus (TMEV), which generates a robust CD8 T cell response against a Db restricted antigen (VP2121–130) in the brain. At 7 days post infection (dpi), brain infiltrating Db:VP2-specific CD8 T cells were reduced in Db cKO mice compared to control, while peripheral priming of CD8 T cells was unaffected. Unexpectedly, inflammatory monocytes infiltrating the brain at 7 dpi were also reduced in Db cKO mice, indicating a role for microglia/BAMs and CD8 T cells in recruitment of inflammatory monocytes. Db cKO mice were able to clear virus, indicating functional antiviral responses against TMEV occur despite reduced immune infiltration. These results demonstrate that local antigen presentation by microglia and BAMs facilitates inflammatory monocyte recruitment and antigen-specific CD8 T cell responses in the brain after TMEV infection.

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 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 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 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.