Citations

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 Brain atrophy is a common feature of many neurological diseases as diverse as Alzheimer’s disease, cerebral palsy, Huntington’s disease, multiple sclerosis, 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 using mice with a C57BL/6 background. The H-2DbClass I molecule not only contributed to significantly more ventricular enlargement in a T2-weighted MRI at 45 d.p.i., but this ventricular enlargement correlated to CD8 T cell infiltration. To further define the cellular and molecular mechanisms of MHC class I molecules in TMEV induced brain atrophy we investigated the cell specific contribution of brain resident antigen presenting cells (APCs) in ventricular atrophy. We developed novel bi-transgenic mouse lines with tamoxifen induced conditional ablation of the H-2Db class I molecule in Cx3CR1+ brain resident myeloid cells. Cx3CR1cre+/Db Lox P mice presented with significantly less ventricular atrophy at 45 d.p.i., compared to cre- littermates along with a decrease in CD8 T cell infiltration. These results strongly imply that antigen presentation by H-2Db on myeloid cells, at least in part is responsible for the development of brain atrophy and provided insight into the molecular mechanism of this poorly understood neuropathology. NS103212, NS122174

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)

AbstractBackgroundThe nervous and immune systems interact in a reciprocal manner, both under physiologic and pathologic conditions. Literature spanning various CNS pathologies including brain tumors, stroke, traumatic brain injury and de-myelinating diseases describes a number of associated systemic immunologic changes, particularly in the T-cell compartment. These immunologic changes include severe T-cell lymphopenia, lymphoid organ contraction, and T-cell sequestration within the bone marrow.MethodsWe performed an in-depth systematic review of the literature and discussed pathologies that involve brain insults and systemic immune derangements.ConclusionsIn this review, we propose that the same immunologic changes hereafter termed ‘systemic immune derangements’, are present across CNS pathologies and may represent a novel, systemic mechanism of immune privilege for the CNS. We further demonstrate that systemic immune derangements are transient when associated with isolated insults such as stroke and TBI but persist in the setting of chronic CNS insults such as brain tumors. Systemic immune derangements have vast implications for informed treatment modalities and outcomes of various neurologic pathologies.

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 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.