Maynes, Mark Andrew

Abstract The impact of microglia antigen presentation on CNS infiltrating CD8 T cells responses during neurotropic virus infection has been difficult to define. Using Theiler’s murine encephalomyelitis virus (TMEV) infection of neurons as a model system, our laboratory has previously determined that H-2D b restricted, but not H-2K b restricted CD8 T cells are required for viral clearance, demonstrating the role of discrete MHC class I alleles. To determine the extent microglia antigen presentation impacts brain-infiltrating CD8 T cells, our laboratory generated novel single MHC class I conditional knockout mice in which H-2K b or H-2D b can be specifically deactivated in TMEM119+ microglia with tamoxifen administration. During TMEV infection, conditional knockout of H-2K b in microglia reduced antigen-specific CD8 T cell proliferation in the brain. Meanwhile, mice with deletion of D b in microglia had reduced levels of perforin in antigen-specific CD8 T cells. Furthermore, microglia specific deletion of H-2D b reduced CD8 T cell numbers in the brain and preserved blood-brain barrier (BBB) integrity. Microglial D b restricted antigen presentation was also essential for the reactivation of CD8 tissue resident memory (TRM) cells and BBB integrity during memory recall responses. These findings further our understanding of how brain infiltrating antiviral CD8 T cell responses are impacted by microglia, as well as define how this cellular interaction contributes to BBB disruption during neuroinflammation. These findings also have high significance to our understanding of how microglia impact CD8 TRM cell populations that reside in the brain long after virus infection is cleared. Graphical abstract

Abstract Description Tauopathies are a subset of neurological diseases associated with pathologic tau protein accumulation and the formation of neurofibrillary tangles in the brain. There is growing evidence of immune cell infiltration including T cells and an increase of inflammatory microglia in human brains from patients that have Tauopathies. However, the effect of T cells and how they interact with CNS-resident immune cells in these pathologies is not well understood. To answer this question, we developed a neurotropic AAV1 encoding human mutant P301L-Tau. AAV-Tau transduction of neurons in neonatal mice recapitulates features of Tauopathies found in human patients. As microglia are the predominant resident antigen presenting cells within the CNS, we hypothesized that CD8 T cells may be dependent on MHC class I presentation by microglia in mice with Tauopathy to achieve full infiltration and expansion in the brain. To address this hypothesis, we introduced AAV-Tau into our novel transgenic mice for which MHC class I antigen presentation by H-2Kb on microglia can be conditional ablated. We observed lower frequencies of CD8 T cell populations and less CD8 T cell CD69+CD103+ expression in mice with Tauopathy when MHC class I was ablated in microglia. We conclude that microglia class I restricted antigen presentation contributes to the expansion of resident memory CD8 T cells in experimental Tauopathy and therefore are an important driver of the immune response observed in this dementia model. Funding Sources NIH/NINDS: R01NS122174 Mayo Graduate School of Biomedical Sciences Topic Categories Neuroimmunology (NEUR)

Abstract Description Brain Barrier (BBB) disruption plays a role in various neurodegenerative diseases such as ALS, stroke, Huntington’s disease and Cerebral Malaria. In our laboratory, we aim to interrogate the immunological underpinnings associated with neuroinflammation and barrier leakage. In clinical studies, aging is associated with a decline in T cell production and a gradual shift to resident memory T cells in the brain characterized by reduced T cell repertoire diversity and weak activation. To model these clinical observations, we sought to define the effect of MHC class I restricted antigen presentation on endothelial cells of brain vasculature on CD8 T cell infiltration of the CNS. We conditionally ablated H-2Kb and H-2Db molecules on endothelial cells using Cdh5-Cre mice crossed with our novel Kb LoxP and Db LoxP mice generated by our research program. In these animals we studied CD8 T cell accumulation in the brain using Theiler’s Murine Endothelial Virus (TMEV) infection of young mice (<12 months) compared to old mice (>12 months). Our results indicate that the loss of endothelial cell Kb induces a defect in establishment of antigen experienced CD8 T cell populations in acute infection. These finding will allow us to further investigate the role of endothelial cell MHC Class I molecules in the immune response to chronic TMEV infection, as well as other neurologic disease models. Funding Sources NIH Grants R01 NS103212 and R01 NS122174 Topic Categories Neuroimmunology (NEUR)

Abstract Description CD8 T cell induced blood-brain barrier (BBB) disruption is an established mechanism of neuropathology. The pericyte is a MHC class I expressing neurovascular unit cell type which is critical for maintaining BBB integrity. However, the importance of pericyte antigen-restricted engagement by CD8 T cells is not fully understood. To determine the function of pericyte MHC Class I antigen presentation, we crossed the Pdgfrβ-creERT2 mouse to our novel H-2Db LoxP mouse developed by our laboratory. This transgenic mouse approach enabled us to target pericytes during neuroinflammation. We observed robust knockdown of H-2Db on Pdgfrβ+ pericytes during TMEV infection in these animals. The effect of conditional H-2Db knockdown on this cell type resulted in increased CD4 T cell, CD8 T cell, and B cells infiltration of the brain during TMEV infection, demonstrating an important role of Pdgfrβ+ pericytes for immune cell recruitment into the CNS. To our knowledge, this is the first study demonstrating an important role for MHC class I antigen presentation by pericytes in a murine model of neuroinflammation. Funding Sources Supported by R01NS122174 and R01NS103212 Topic Categories Neuroimmunology (NEUR)

Abstract Description The dura mater of the meninges recruits immune cells for CNS surveillance and response to infections, serving as a critical interface between the brain and the peripheral immune system. Theiler’s murine encephalomyelitis virus (TMEV) infection is a model neurotropic virus for which MHC class I molecules are critical for sterilizing immunity. Using spectral flow cytometry, we profiled the immune response in the dura mater in healthy and TMEV-infected wildtype mice. Acute TMEV infection resulted in the infiltration of diverse immune cell types into the dura mater, including increased cDC1, pDCs, monocytes, NK cells, CD4 T cells, and CD8 T cells. We next examined the importance of MHC class I molecules on diverse immune cell populations in the dura mater during acute TMEV infection. MHC class I deficiency resulted in increased numbers of CD4 T cells with high expression of PD1 in MHC class I-/- (H-2Kb-/-, H-2Db-/-) mice compared to WT mice 7 days post TMEV infection. This demonstrated the importance of MHC class I and CD8 T cells in modulating dura immune cell populations and implies a compensatory shift toward a CD4 T cell response during acute infection. Subsequent studies employing in vivo cell-specific ablation of MHC class I molecules in discrete cell types will further define the impact of antigen presentation on immune regulation and inflammatory outcomes within the dura mater. Topic Categories Neuroimmunology (NEUR)

Abstract Aberrant vasculature is a hallmark of glioblastoma (GBM), contributing to both blood–brain barrier (BBB) dysfunction and frequent intracranial hemorrhage. Myeloid cells, including microglia and macrophages, are known to co-opt tumor vasculature during tumor progression, but the nature and consequences of their physical interactions with blood vessels require clearer definition. To address this, we characterize vessel-associated myeloid cells in murine gliomas with high spatial and temporal resolution, leveraging in vivo two-photon imaging, MRI, and transgenic mouse lines. To investigate the role of microglia in glioma vasculature, we pretreated glioma-bearing mice with CSF1R inhibitors (PLX3397 & PLX5622) to ablate microglia prior to tumor inoculation. Strikingly, over half of the microglia-depleted mice developed acute, lethal paralysis between days 11–15, whereas control tumor-bearing mice remained asymptomatic. MRI revealed extensive intracranial hemorrhage and edema, and two-photon imaging showed nodular, abnormal vasculature. This vascular dysfunction led to a dramatic reduction in median survival—from 30 days in control glioma-bearing mice to 15 days in PLX-treated mice with gliomas. To identify the protective microglial population involved, we examined vascular–myeloid interactions. We found that the peritumoral region harbored dense capillary networks supporting tumor expansion, along with a marked increase in myeloid cells with somas directly contacting blood vessels. Using TMEM119CreER/+:R26tdTomato/+ mice, we confirmed these vessel-associated cells as brain-resident microglia, not infiltrating macrophages. Quantitatively, ~60% of peritumoral microglia were vessel-associated, compared to ~30% in naïve or contralateral hemispheres. These microglia displayed elongated somata and processes aligned with vessels or wrapping around vessels, which is a phenotype also observed following single-vessel photothrombosis, suggesting a response to impaired blood flow and BBB leakage. Our findings uncover a critical neurovascular role for vessel-associated microglia in maintaining vascular integrity during glioma progression and highlight the importance of preserving, reprogramming, and further investigating this population in GBM therapies.

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