Johnson, Aaron J.

Abstract Aging has been shown to be one of the major risk factors for host morbidity and mortality following infections with respiratory viruses including influenza virus and SARS-CoV-2. The unique immune properties of the aged hosts not only influence pathogen clearance and tissue damage responses, but also modulate the pathogenesis of the long-term sequalae post acute viral clearance. Currently, the dynamics and features of mucosal immune responses following acute respiratory viral infections in the aged hosts remain to be fully elucidated. Here, we characterized the lung cellular and molecular profiles following influenza virus infection in young (~3-month-old) or aged (~24-month-old) mice with a chronological manner from acute to chronic phase. Using DNA-oligo-conjugated CD45 antibody and i.v. labeling, we were able to distinguish the unique responses of circulating vs lung-resident immune cells to influenza virus infection during aging. These responses were further validated by spectral flow cytometry analysis of the lung innate and adaptive immune cells. Further data analysis and integration are warranted for this unique large data set detailing the single cell molecular profiles of mucosal responses to influenza virus infection comparing young and aged hosts.

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 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 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.