Fain, Cori E.

Abstract Bone marrow is a primary immune organ responsible for stem cell maintenance and hematopoiesis. While the bone marrow niche is mainly studied in the context of hematopoiesis, aging, and mechanisms underlying stem cell and plasma cell survival, changes of the niche during brain viral infections and primary T cell responses is unknown. Intracranial infection with the neurotropic Theiler’s Murine Encephalomyelitis Virus (TMEV) results in an acute infection in the C57BL/6 mouse which is cleared 30 days post infection due to generation of an antigen specific CD8 T cell response raised against the immunodominant epitope VP2121–130. We determined that 7–10% of CD8 T cells present in the sternal and femoral bone marrows and 15–20% in the cranial bone marrow were antigen specific 7 days post intracranial TMEV infection. Over 70% of these CD8 T cells were located outside of the vasculature as determined by IV labeling. Importantly, continuous treatment with FTY720, which sequesters T cells outside of the blood, did not eliminate antigen specific CD8 T cells in any bone marrow compartment. CD8 T cells in the bone marrow established durable memory following viral clearance and were reactivated upon cognate antigen reencounter. Both acute TMEV infection and antigen specific reactivation caused an increase in lineage−, Sca-1+, ckit+ (LSK) cells in the bone marrow. This LSK dysregulation was abrogated upon CD8 T cell depletion. We conclude that brain viral infections induce in situ effector and memory T cell responses within the bone marrow compartment. Both acute and memory recall CD8 T cell responses cause stem cell dysregulation. Our data hence paves the way for crucial studies of bone marrow resident antigen specific CD8 T cells in health and disease. Supported by grants from NIH (K99NS117799-01A1 (KA), R01NS103212 (AJJ), and RF1NS122174(AJJ)

Abstract Cerebral malaria (CM) is a severe complication of P. falciparum infection and a leading cause of death, globally. Plasmodium berghei ANKA (PbA) infection of C57BL/6 mice serves as a murine model of CM called experimental cerebral malaria (ECM) due to the neurological and neuropathological features that simulate the infection of human CM. A putative mechanism of disease in human CM requires CD8 T cell to infiltration into the brain and blood-brain barrier disruption. The specific antigen-presenting cell type(s) that prime this CNS infiltrating CD8 T cell response capable of inducing lethal blood-brain barrier disruption has not been defined. To answer this, single class I expressing Kb loxP transgenic mice were generated. Crossing these mice to class I deficient CD11c-cre or LysM-cre mice created the LysM-cre Kb cKO and CD11c-cre Kb cKO mice, respectively. PbA infected Kb loxP and LysM-cre Kb cKO were susceptible to ECM. In contrast, CD11c-cre Kb cKO mice survived acute PbA infection and avoided neurological manifestations. We therefore employed spectral flow cytometry to analyze splenocytes and brain infiltrating immune cells during acute PbA infection in these genotypes. We observed a reduction in activated CD8 T cells in the spleens of PbA infected CD11c-cre Kb cKO mice versus the Kb loxP mice and the LysM-cre Kb cKO. This reduced peripheral CD8 T cell activation in PbA infected CD11c-cre Kb cKO mice also corresponded with reduced brain infiltration of CD8 T cells. We conclude a CD11c+ APC is therefore required for priming and potentially recruitment of CD8 T cells to the brain during lethal ECM pathology. 5R01NS103212-06

Abstract Glioblastoma (GBM) is an incurable brain cancer that is associated with severe peripheral immunosuppression. This immunosuppression is a critical barrier to patient survival and the success of immunotherapies. C57BL/6 mice harboring GL261 gliomas present with the hallmark features of peripheral immunosuppression observed in GBM patients, including T cell lymphopenia. We determined that peripheral immunosuppression involves release of non-steroidal soluble factors with large molecular weight that are found in sera of glioma-bearing mice. Sera of glioma-bearing mice inhibits T cell proliferation ex vivo. To determine the identity of the immunosuppressive molecules in serum, we used two parallel approaches: 1.) evaluation of abundant macromolecules and 2.) proteomics analysis using tandem mass tag mass-spectrometry (TMT-MS). We determined that serum of glioma-bearing mice contains significantly higher levels of cell free DNA (cfDNA) compared to controls. TMT-MS identified histones as a highly abundant protein in serum, further implicating DNA-histone complexes as top candidates. DNAse treatment of sera isolated from GL261 glioma-bearing mice reduced the suppressive effect on T cell proliferation. We next performed T cell proliferation assays in the presence of pure cfDNA isolated from sera of glioma-bearing mice while genomic DNA was used as control. cfDNA from serum potently inhibited T cell proliferation while control genomic DNA did not. These results were AIM2 independent implicating a novel pathway of DNA sensing in T cells. We contend that cfDNA induces immunosuppressive effects in experimental GBM. Devising strategies to reverse immunosuppression through targeting cfDNA could improve outcomes in GBM patients. Supported by grants from NIH (K99NS117799-01A1 (KA), R01NS103212 (AJJ), and RF1NS122174(AJJ) and Brains Together for a Cure Foundation (KA).

Abstract Aging is considered to be a major risk factor for host morbidity and mortality upon acute respiratory virus infections. To better elucidate the immune determinants of viral pathogenesis during aging, we chronological characterized lung cellular and molecular profiles following influenza virus (PR8) infection in young or aged mice till 60 days post infection. Time-course scRNAseq coupled with high dimensional flow cytometry enabled the description of changes in both immune and stromal compartment between young and aged hosts. We further zoomed into the myeloid compartment and focused on the dynamic responses of lung macrophages given their complex roles in viral pathogenesis, tissue repair and lung fibrosis. Enlarged Monocyte-derived Macrophage (MoM) population as well as decreased resident-Alveolar Macrophage (AM) population were observed in the aged mice when compared to those of the young mice. Despite the self-sustain nature of AMs during homeostasis, it has been reported that Ly6Chi monocytes can gain AM identity following severe lung damage like influenza virus infection. We found that the gain of AM identity from monocytes was abrogated in the aged hosts, which likely contributed to the age-associated impairment in lung recovery after influenzas virus infection. Additionally, trajectory analysis with scRNAseq allowed us to identify IFNα/γ signaling to be potential pathways contributing to such defect. This notion is also supported by elevated IFNγ level in BAL from the aged host during lung recovery. Our results thus have improved our understanding on the cellular and molecular mechanisms by which aged hosts exhibit severe diseases and delayed recovery after respiratory viral infection.

Abstract Intracranial infection with neurotropic Theiler’s Murine Encephalomyelitis Virus induces robust generation of brain TRMs against the immunodominant antigen, VP2121–130, following viral clearance in C57BL/6 mice. While the presence of TRMs in the brain is documented, neurological consequences of TRM reactivation and their causal role in neuropathology remain understudied. We disabled peripheral CD8 T cells via anti CD8 depleting strategies and FTY720 treatment to determine the causal link between brain TRM reactivation and neuropathology. We depleted peripheral T cells and then administrated VP2 peptide IV to reactivate TRMs 60–200 days post infection as this elegant technique isolates responses of brain TRMs away from other branches of memory. We used state-of-the-art flow cytometry and MRI analysis to evaluate neurological consequences of TRM reactivation. TRM reactivation induced severe neuroinflammation in the brain as evident by infiltration of myeloid cells and blasting of T cells. MRI detected gadolinium leakage marking severe blood-brain-barrier (BBB) disruption when brain TRMs were reactivated. Depletion of peripheral CD8 T cells with low-dose anti CD8 antibodies or their sequestration via FTY720 did not alter neuroinflammation indicating brain TRM reactivation causally initiates neuropathology. Confirming this, tissue depletion of all TRMs in the brain using high-dose anti CD8 antibodies prevented both neuroinflammation and neuropathology. Hence, TRM reactivation causes severe neuroinflammation and BBB disruption from within the brain independently of peripheral T cells. Targeting brain TRM reactivation is a novel potential therapeutic for patients with many acute and chronic neurological diseases. Supported by grants from NIH (K99NS117799-01A1 (KA), R01NS103212 (AJJ), and RF1NS122174(AJJ)

Abstract Background Glioblastoma (GBM) is the most common malignant brain tumor and has a poor prognosis. Imaging findings at diagnosis and in response to treatment are nonspecific. Developing noninvasive assays to augment imaging would be helpful. Plasma extracellular vesicles (EVs) are a promising biomarker source for this. Here, we develop spectral flow cytometry techniques that demonstrate differences in bulk plasma EV phenotype between GBM patients and normal donors that could serve as the basis of a liquid biopsy. Methods Plasma EVs were stained for EV-associated tetraspanins (CD9/CD63/CD81), markers indicating cell of origin (CD11b/CD31/CD41a/CD45), and actin/phalloidin (to exclude cell debris). EVs were analyzed using spectral flow cytometry. Multiparametric analysis using t-distributed stochastic neighbor embedding (t-SNE) and self-organizing maps on flow cytometry data (FlowSOM) was performed comparing GBM and normal donor (ND) plasma EVs. Results Size exclusion chromatography plus spectral-based flow cytometer threshold settings enriched plasma EVs while minimizing background noise. GBM patients had increased CD9+, CD63+, CD81+, and myeloid-derived (CD11b+) EVs. Multiparametric analysis demonstrated distinct surface marker expression profiles in GBM plasma EVs compared to ND EVs. Fifteen plasma EV sub-populations differing in size and surface marker expression were identified, six enriched in GBM patients and two in normal donors. Conclusions Multiparametric analysis demonstrates that GBM patients have a distinct nonneoplastic plasma EV phenotype compared to ND. This simple rapid analysis can be performed without purifying tumor EVs and may serve as the basis of a liquid biopsy.

Abstract Glioblastoma (GBM) is the most common malignant brain tumor in adults, responsible for approximately 225,000 deaths per year. Despite preclinical successes, most interventions have failed to extend patient survival by more than a few months. Treatment with anti—programmed cell death protein 1 (anti–PD-1) immune checkpoint blockade (ICB) monotherapy has been beneficial for malignant tumors such as melanoma and lung cancers but has yet to be effectively employed in GBM. This study aimed to determine whether supplementing anti–PD-1 ICB with engineered extended half-life IL2, a potent lymphoproliferative cytokine, could improve outcomes. This combination therapy, subsequently referred to as enhanced checkpoint blockade (ECB), delivered intraperitoneally, reliably cures approximately 50% of C57BL/6 mice bearing orthotopic GL261 gliomas and extends median survival of the treated cohort. In the CT2A model, characterized as being resistant to CBI, ECB caused a decrease in CT2A tumor volume in half of measured animals similar to what was observed in GL261-bearing mice, promoting a trending survival increase. ECB generates robust immunologic responses, features of which include secondary lymphoid organ enlargement and increased activation status of both CD4 and CD8 T cells. This immunity is durable, with long-term ECB survivors able to resist GL261 rechallenge. Through employment of depletion strategies, ECB's efficacy was shown to be independent of host MHC class I–restricted antigen presentation but reliant on CD4 T cells. These results demonstrate ECB is efficacious against the GL261 glioma model through an MHC class I–independent mechanism and supporting further investigation into IL2-supplemented ICB therapies for tumors of the central nervous system.

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 group has developed a unique model of CD8 T cell-mediated BBB disruption which employs Theiler’s murine encephalomyelitis virus (TMEV) infection. Upon intravenous administration of VP2121–130 viral peptide during the peak antiviral 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 relation to antigen specific CD8+ T cells and permeable cerebral vessels. 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, microglial population exhibit robust down-regulation of homeostatic markers including CX3CR1 during BBB disruption. This study demonstrates CD8 T cells can promote microglia activation in a perforin-dependent manner during BBB disruption. Importantly, microglial activation occurs concurrently with the onset of vascular permeability and could serve as a critical cell type in this process.

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 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.