Hansen, Michael

Abstract Introduction The H-2Db class I restricted CD8 T cell response is critical for clearance of neurotropic Theiler’s virus encephalomyelitis virus (TMEV) infection. However, the key central nervous system cell types required to present H-2Db for CD8 T cell trafficking and expansion in the brain are not known. Mural cells are a critical cellular component of the neurovascular unit for maintaining vascular integrity which also express MHC class I molecules. However, the importance of mural cell antigen-restricted engagement by CD8 T cells has not been investigated. Methods To determine the function of mural cell 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 mural cells during neuroinflammation. Results We observed robust knockdown of H-2Db on mural cells during acute TMEV infection. Conditional H-2Db knockdown in mural cells resulted in increased brain infiltration of CD8 T cells specific to the immunodominant Db:VP2 121-130 epitope of TMEV, demonstrating an important role of mural cells for immune cell recruitment. Conclusion To our knowledge, this is the first study demonstrating MHC class I antigen presentation by mural cells impacts CD8 T cell recruitment during neuroinflammation. Funding Source R01-NS103212, T32-AI170478 Topic Categories Neuroimmunology (NEUR)

Abstract Introduction T cell activation is a dynamic process governed by the strength and duration of antigen recognition via the T cell receptor (TCR). The timing of in vivo TCR signaling— when and how strongly memory CD8+ T cells become reactivated in the inflamed brain following antigen exposure— is a fundamental mechanism that remains unclear. The Nr4a3-Tocky reporter (Tocky) mouse employs a fluorescent timer that shifts from blue to red after TCR-driven Nr4a3 activation, which allows researchers to track activation kinetics through two parameters: the Timer Angle (recency of T cell activation), and the Timer Intensity (strength of signaling). Methods To understand CD8+ T cell activation during this neuroinflammation, we generated memory CD8+ T cells by intracranial Theiler’s murine encephalomyelitis virus infection of these Tocky mice. After viral clearance, we reactivated memory CD8+ T cells by intravenously injecting their cognate antigen. Tissues were collected 0-72 hours post-injection and profiled by flow cytometry. An R pipeline was constructed to transform event-level flow data to quantify the TCR activation. Data were analyzed with a Metropolis—Hastings MCMC algorithm to model activation dynamics and estimate probabilistic transitions between biologically defined states (newly activated, persistent, arrested states). Results These empirical transition probabilities enabled Bayesian comparison of antigen-specific versus nonspecific CD8+ T cells. It was revealed that antigen-specific CD8+ T cells rapidly transition to a high-intensity activation state within 24 hours and maintain persistent signaling compared to nonspecific populations. Conclusion This establishes a biologically grounded, in vivo framework for modeling and comparing TCR signaling during neuroinflammation. Funding Source R01 - NS103212 Topic Categories Neuroimmunology (NEUR)

Abstract Introduction Glioblastoma (GBM) is the most aggressive primary brain tumor in adults with an incident rate of 12,000 cases in the United States every year. Current treatment options for these patients include surgical resection, radiation, and chemotherapy. Despite these conventional treatments, patients have a dismal prognosis mostly due to the inability to deliver therapeutic agents across the blood-brain barrier. In recent years, the use of oncolytic picornavirus has been employed as a novel virotherapy for GBM. We hypothesized that we could enhance antibody delivery to the brain through harnessing the immune response associated with this oncolytic virus. Prior work from our lab has shown that CD8 T cell engagement with MHC class I on brain endothelium induces CNS vascular permeability which is dependent on perforin. We therefore asked if picornavirus antigen specific CD8 T cells could enhance antibody delivery to the brain. Methods To test this, we evaluated the potential of activating MHC class I restricted CD8 T cells in vivo through intravenous administration of their cognate picornavirus antigens. Results We determined that this approach enhanced delivery of antibody to brain parenchyma. Importantly, antibody delivery to the brain was still enhanced when CD8 T cells lacked perforin expression which significantly reduced the toxicity of this combination approach. Conclusion These findings demonstrate that theCD8 T cell response towards virotherapy can be harnessed to promote delivery of therapeutic antibody to the brain. Funding Source NCI — R01 CA 94953 NINDS — R01 NS122174 NINDS —R01 NS103212 Mayo Clinic Graduate School of Biomedical sciences Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

Abstract Introduction CD8 T cells are key immune cells in viral clearance, targeting infected cells following recognition of MHC class I restricted antigen presentation. Previous work has determined that microglia and perivascular macrophages are essential for normal CD8 T cell responses against neurotropic viral infection, indicating that cells at the neurovascular unit can act as antigen-presenting cells. However, the specific importance of microglia MHC class I restricted antigen presentation to brain-infiltrating CD8 T cells has been difficult to define. Methods To address this question, our laboratory generated novel single MHC Class I conditional knockout mice in which H-2Kb or H-2Db can be deactivated specifically in Tmem119+ microglia with tamoxifen administration. Recombinant Theiler’s murine encephalomyelitis virus (TMEV) encoding the model OVA antigen enabled analysis of virus antigen-specific CD8 T cells restricted to H-2Kb class I molecules while Daniel’s strain TMEV H-2Db class I molecules enabled analysis of Db:VP2 specific CD8 T cells. Results Our results revealed profound differences in the response of CD8 T cells upon deletion of MHC class I molecules on microglia. Conditional knockout of H-2Kb in Tmem119+ microglia reduced Kb:OVA epitope specific CD8 T cells in the brain compared to Cre negative littermate controls, and BrdU staining revealed differences in T cell proliferation. Meanwhile, mice with deletion of Db in Tmem119+ microglia had reduced levels of perforin in Db:VP2 CD8 T cells. Furthermore, in a model of CNS vascular disease, the deletion of H-2Db of Tmem119+ microglia reduced CD8 T cell numbers in the brain, as well as the blood-brain barrier (BBB) breakdown observed by a FITC albumin assay. Conclusion These findings demonstrate discrete roles for specific MHC Class I molecules on microglia during CNS viral infection. This data will further our understanding of brain infiltrating CD8 T cell responses in neurologic diseases as well as the regulation of BBB by microglia. Funding Source R01 NS 103212 R01 NS122174 Topic Categories Neuroimmunology (NEUR)

Abstract The impact of microglia antigen presentation on CNS infiltrating CD8 T cells responses during neurotropic virus infection remains undefined. Using Theiler’s murine encephalomyelitis virus (TMEV) infection of neurons as a model system, we generated H-2K b and H-2D b Tmem119 conditional knockout mice. During TMEV infection, we determined using Tmem119 K b cKO mice that microglia H-2K b contributes to antiviral CD8 T cell proliferation in the brain. Meanwhile, using Tmem119 D b cKO mice we determined that microglia H-2D b increased antiviral CD8 T cell perforin levels and the capacity to induce blood-brain barrier (BBB) disruption. Furthermore, microglial H-2D b was essential for antigen-specific reactivation of CD8 tissue resident memory (TRM) cell recall responses and BBB disruption long after virus had been cleared. These findings demonstrate discrete roles of individual MHC class I genes on microglia in CD8 T cell proliferation, effector functions, memory recall and neuropathology.

Abstract Skull bone marrow (BM) is a recently identified site for immune activation responsive to CNS insults. The extent to which adaptive immunity is generated through skull BM is poorly understood. In this study, we evaluated how skull BM supports the early expansion of antiviral CD8 T cells during acute neurotropic virus infection. Virus antigen-specific CD8 T cells were rapidly identified in skull BM at a rate comparable to lymphoid compartments during acute Theiler’s murine encephalomyelitis viral (TMEV) infection. We therefore employed MHC class I conditional knockout mice and demonstrated the necessity of CD11c+ antigen-presenting cells (APCs) for the generation of various subsets of TMEV-specific CD8 T cells in skull BM. Importantly, the early expansion of skull BM-derived antiviral T cells was impeded by FTY720 treatment and restored upon shared circulation with a mouse exhibiting intact antigen presentation by CD11c+ APCs. Additionally, TMEV-specific CD8 T cells were retained in skull BM during both chronic CNS infection and long after infection has been resolved. Overall, we determined that skull BM responds to acute neurotropic viral infection through the rapid recruitment of virus-specific CD8 T cells primed in the periphery before serving as a reservoir for immunologic memory.

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 Antigen receptor (CAR) T cells can be generated in vivo within the natural lymph node (LN) environment as the immune response to a virus infection unfolds. This in situ approach entails priming LNs with intradermal inoculations of replication defective adenoviruses encoding MHC class I and class II allo/xeno-antigens. Universal transplant antigens were devised for LN priming by introducing tryptophan into amino acid position 162 of the MHC HLA-A2:01 class I heavy chain and by using the mouse MHC class II beta chain to form chimeric class II antigens with endogenous class II alleles. The virus encoded mismatched MHC antigen presenting molecules are expressed functionally in the LN following intradermal inoculation provoking polyclonal T cells responses. Three days later CAR T cells are generated by directly injecting retroviruses encoding CARs of interest into the responding LN. Mouse CD19-specifc CAR T cells created in this manner in B6-DQ8 and DQ8/A2 mice effectively target endogenous B cells and are therapeutic in male and female Emu-myc lymphomas demonstrating the potential for the generating functional CAR T cells directly in vivo. While in situ CAR T cells are readily produced in B6, B6-IA0/DQ8, B6-IA0/DQ8/A2, and B6-IA0/DQ6 LNs, functional differences among the strains emerged in the periphery suggesting that properties of the autoimmune-associated HLA DQ8 class II allele is particularly reactive during the production of CAR T cells in vivo. Funding Sources MustangBio, Inc Mayo Clinic Center of Biomedical Research Mayo Clinic Department of Immunology Mayo Clinic Graduate School of Biomedical Science Fraternal Order of Eagles Mayo Clinic Ventures NIH R21 AI 097337 NIH R01 CA 178804 NIH T32 AI 07425 Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

Abstract Glioblastoma (GBM) is an aggressive form of brain cancer with poor survival despite standard of care. Checkpoint inhibitors aimed to revive dysfunctional T cells have significantly improved outcomes in other cancers, but have failed GBM. GBM patients have severe peripheral immunosuppression, including severe lymphopenia, immune organ atrophy, and defective T cell responses. This immunosuppression is a critical barrier to patient survival and the success of immunotherapies. Using parabiosis, we demonstrated that serum-derived factors were sufficient to drive hallmark features of immunosuppression, including inhibition of ex vivo T cell proliferation. This factor was nonsteroidal in nature and had molecular weights greater than 100kDa. Mass-spectrometry and pathway analysis of the suppressive serum implicated proteins involved in DNA response elements, cell death, and DNA-histone complexes. We hypothesized that the factor is cell-free DNA (cfDNA). Confirming this, serum levels of cfDNA are increased in glioma-bearing mice. Moreover, exposure to cfDNA from serum of glioma-bearing mice was sufficient to directly and potently inhibit T cell proliferation ex vivo. Interestingly, exposure to genomic DNA was not sufficient to induce proliferation defects in T cells, highlighting the unique features of cfDNA. Importantly, reducing cfDNA content using DNaseI partially restored proliferation capacity. We determined that the T cell proliferation defects were independent of DNA sensing mechanisms through AIM2, implicating a novel pathway of DNA sensing in T cells. Finally, to determine the origin of the cfDNA, we employed methyl-seq. The chromatin signature most closely represented neutrophils. This highlights a potential role for neutrophils-derived cfDNA as a targetable strategy in GBM. Together, we contend that cfDNA induces immunosuppressive effects in GBM and devising strategies to reduce circulating cfDNA could be a therapeutic approach to improve T cell functions and outcomes in GBM patients.

Abstract Glioblastoma (GBM) is aggressive form of brain cancer that is associated with severe peripheral immunosuppression. This immunosuppression is a critical barrier to patient survival and the success of immunotherapies. Immunocompetent mice harboring intracranial gliomas present with the hallmark features of peripheral immunosuppression observed in GBM patients, including T cell lymphopenia. Using parabiosis, we determined that T cell lymphopenia can be transferred from glioma-bearing to healthy mice via circulation This led to the identification of a high molecular weight immunosuppressive factor in sera of glioma-bearing mice that inhibits T cell proliferation ex vivo. Proteomics analysis identified 119 proteins uniquely upregulated in the sera of glioma-bearing mice compared to controls. These proteins are associated with pathways involved in DNA response elements, cell death, and DNA-histone complexes which imply DNA release. Additional analysis revealed serum isolated from glioma-bearing mice harbor significantly higher concentrations of double stranded cell free DNA (cfDNA) compared to controls. Reducing DNA content using DNase partially reverses the proliferation defects observed in T cells when exposed to serum of glioma-bearing mice. This is while, addition of cfDNA purified from serum of glioma-bearing mice, but not genomic DNA, potently inhibited T cell function. These results were AIM2 independent implicating a novel pathway of cfDNA sensing in T cells. We contend that cfDNA induces immunosuppressive effects in experimental GBM. Devising strategies to reduce circulating cfDNA is therefore a therapeutic approach that could improve outcomes in GBM patients.