Jin, Fang

Abstract Introduction Peripheral nervous system innervation of various organs has been associated with the development and maintenance of the immune system. Yet, the intricate details of thymic innervation remain less well-studied. Interestingly, various neurological insults including brain tumors, and neurotropic infections have been shown to have profound impacts on thymic size and cellularity and T cell development. Methods We used flow cytometry, confocal microscopy, neuro tracers, and genetic knockout mice to evaluate developing T cell-neuron interactions within the thymus. Results We have identified neuronal cell bodies within the thymus. These cells express neuronal markers such as NeuN and tyrosine hydroxylase and lack TEC identifiers, such as EpCAM, UEA-1, and Ly51. Thymic neurons can be infected with a neurotropic rabies vector, further cementing their neuronal identity. Considering the thymus consists mostly of T cells, we next investigated a potential interaction between T cells and thymic neurons. Interestingly, loss of T cell development results in a lack of thymic neuron development indicating T cell-neuron coregulation during development. However, thymic neurons can be recovered following a bone marrow transplant, suggesting the provision of a differentiation and/or survival signal for neurons provided by the developing T cells. Using knockout mice lacking the T cell lineage at various stages of development, we determined that TCR beta selection is required for the development of thymic neurons. Finally, we determined that a lack of transition from DN4 to DP severely diminishes the development of thymic neurons. Analysis of the publicly available bulk RNA-sequencing data comparing thymic DP to DN cell populations indicates the potential involvement of IL34 in neuronal development in the thymus. Conclusion We describe a novel population of neuronal cells within the thymus that interact with developing thymocytes to establish optimal thymic architecture. Funding Source NIH K99/R00 Topic Categories Neuroimmunology (NEUR)

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 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 Introduction The anatomical locations of generating adaptive immune responses to neurotropic viruses are still being investigated. Skull bone marrow (BM) has recently been identified as a pivotal immune site that can sense and respond to a local insult in the brain. Yet, there is a lack of understanding of the role of skull BM during viral encephalitis. We hypothesized that the skull BM is an important niche for antiviral CD8 T cells priming, expansion, and maintenance during Theiler’s murine encephalomyelitis virus infection. Methods Using peptide:MHC tetramer analysis, we determined that antiviral CD8 T cells are primed in the skull BM in early infection, at time points comparable to cervical lymph nodes. Employing MHC class I conditional knockout mice generated by our research group, we inactivated H-2Kb and H-2Db class I molecules on discrete antigen-presenting cell types. Results We determined using these transgenic mice that viral antigen presentation on dendritic cells (DCs) and endothelial cells is essential for the generation of virus-specific T cells in skull BM during acute viral encephalitis. However, antigen presentation by DCs is not required for long-lived antiviral CD8 T cells that reside in skull BM. Moreover, the use of FTY720 to block lymphocyte migration diminished skull antiviral CD8 T cell numbers, suggesting that peripheral trafficking is required for generating skull antiviral CD8 T cells. Conclusion We present compelling evidence for the importance of skull BM as a responsive site that is highly suitable for rapid generation of antiviral CD8 T cells during acute viral encephalitis. Skull BM also serves as a reservoir for memory T cells, long after the resolution of a neurotropic virus infection. This work sheds light on the skull as a potential target for further modulation of the immune response to viral infections in the brain. Funding Source NIH grants R01 NS5103212 and RF1 NS122174 Topic Categories Viral Immunology (VIR)

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.

Primary and metastatic brain tumors exhibit resistance to immunotherapies that demonstrate efficacy in peripheral cancer settings. While many immunotherapies aim to enhance CD8 + T cell infiltration and functionality in established tumors, identification of neoantigens support emerging immunopreventative tactics against brain cancer. Functionally potent tissue-resident memory CD8 + T cells (T RM ) can be generated in the brain following peripheral infection or vaccination. However, the ability of brain T RM to prevent intracranial malignancy remains unknown. Here, mice were seeded with tumor-specific or bystander brain T RM via peripheral infection prior to depletion of circulating memory T cells (T CIRCM ) and subsequent brain tumor challenge. Tumor-specific brain T RM durably protected mice against intracranial malignancy even in the absence T CIRCM . These brain T RM persisted in tumor-surviving mice and protected against a second antigen-matched challenge. Importantly, a translationally-relevant mRNA-lipid nanoparticle (LNP) vaccine phenocopied peripheral infection-induced outcomes, generating functional brain T RM that controlled tumor growth. Altogether, this work points to the utility of brain T RM in cancer immunoprevention, supporting the development of antitumor mRNA-LNP vaccines to bolster brain immunity.

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 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 Cerebral malaria is the deadliest complication that can arise from Plasmodium infection. CD8 T-cell engagement of brain vasculature is a putative mechanism of neuropathology in cerebral malaria. To define contributions of brain endothelial cell major histocompatibility complex (MHC) class I antigen-presentation to CD8 T cells in establishing cerebral malaria pathology, we developed novel H-2Kb LoxP and H-2Db LoxP mice crossed with Cdh5-Cre mice to achieve targeted deletion of discrete class I molecules, specifically from brain endothelium. This strategy allowed us to avoid off-target effects on iron homeostasis and class I-like molecules, which are known to perturb Plasmodium infection. This is the first endothelial-specific ablation of individual class-I molecules enabling us to interrogate these molecular interactions. In these studies, we interrogated human and mouse transcriptomics data to compare antigen presentation capacity during cerebral malaria. Using the Plasmodium berghei ANKA model of experimental cerebral malaria (ECM), we observed that H-2Kb and H-2Db class I molecules regulate distinct patterns of disease onset, CD8 T-cell infiltration, targeted cell death and regional blood–brain barrier disruption. Strikingly, ablation of either molecule from brain endothelial cells resulted in reduced CD8 T-cell activation, attenuated T-cell interaction with brain vasculature, lessened targeted cell death, preserved blood–brain barrier integrity and prevention of ECM and the death of the animal. We were able to show that these events were brain-specific through the use of parabiosis and created the novel technique of dual small animal MRI to simultaneously scan conjoined parabionts during infection. These data demonstrate that interactions of CD8 T cells with discrete MHC class I molecules on brain endothelium differentially regulate development of ECM neuropathology. Therefore, targeting MHC class I interactions therapeutically may hold potential for treatment of cases of severe malaria.

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)