Pfaller, Christian K.

Abstract Introduction Bone marrow (BM) is the primary immune organ responsible for stem cell maintenance. Neurotropic infections elicit sequestration of T cells within the BM. Changes of the BM niche during brain viral infections are unknown. Intracranial infection with the neurotropic Theiler’s Murine Encephalomyelitis Virus (TMEV) in C57BL/6 mouse is cleared by 30 days due to generation of an antigen-specific CD8 T cell response. Methods We used brain viral infection and injury models to test T cell responses against brain antigens in the bone marrow. Results We determined that 1-10% of CD8 T cells present in the sternal, femoral, and cranial BM were virus antigen-specific 7 days post intracranial infection. Over 70% of these CD8 T cells were tissue resident. We determined antigen-specific CD8 T cells were generated in the BM within 4 days of infection. Continuous treatment with FTY720, which sequesters T cells outside of the blood, did not eliminate antigen-specific CD8 T cells in any BM compartment. CD8 T cells in the BM established durable memory populations and were reactivated upon antigen reencounter. In the memory phase (200 days post brain TMEV infection), bone marrow resident CD8 T cell could be reactivated upon cognate peptide delivery. Such reactivation of antigen specific memory CD8 T cells caused an increase in lineage-, Sca-1+, ckit + (LSK) cells in the BM in a CD8 dependent manner. Similarly, injection of the model antigen AF488-conjugated OVA mixed with Poly IC into the brain recapitulated antigen specific CD8 T cell generation within the bone marrow and further demonstrated the antigen presenting cell involved in the response to be a CD103 expressing myeloid DC. We conclude that viral infections and inflammatory stimulation in the brain induce in situ effector and memory T cell responses within the BM which is associated with significant stem cell dysregulation. Conclusion Our data paves the way for crucial studies of BM resident antigen-specific CD8 T cells in health and disease. Funding Source NIH K99/R00 Topic Categories Viral Immunology (VIR)

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 Bone marrow is the primary immune organ responsible for stem cell maintenance and hematopoiesis. However, the contribution of the bone marrow niche for the generation of adaptive immune responses is less well-understood. We therefore assessed the capacity of virus antigen-specific CD8 T cells to be generated and expanded in bone marrow following acute neurotropic virus infection. Intracranial infection with the neurotropic Theiler’s Murine Encephalomyelitis Virus (TMEV) results in an acute infection in the C57BL/6 mouse which is cleared within 4 weeks post infection due to the generation of an antigen-specific CD8 T cell response against the immunodominant epitope VP2 121-130 . We determined that 1-10% of CD8 T cells present in the femoral, and sternal bone marrow were virus antigen-specific 5-7 days after intracranial TMEV infection. We determined that antigen-specific CD8 T cells were generated in the bone marrow with similar kinetics to that of conventional responses in the secondary lymphoid organs. Importantly, continuous treatment with FTY720, which sequesters T cells outside of the blood, did not eliminate antigen-specific CD8 T cells in the bone marrow compartment indicating generation within the bone marrow niche. Similarly, injection of Poly I:C admixed with fluorescently labeled ovalbumin into the brain generated a similar antigen-specific CD8 T cell response in the bone marrow indicating these responses occur following various brain insults. This model also identified the likely antigen presenting cell (APC) responsible for scavenging antigens from the brain and trafficking into the bone marrow as a migratory myeloid-derived APC. Moreover, following clearance of TMEV infection, CD8 T cells in the bone marrow established durable memory. Antigen-specific memory CD8 T cells in the bone marrow reactivated and expanded upon cognate antigen reencounter. Antigen-specific reactivation of memory CD8 T cells within the bone marrow caused a concurrent increase in Lineage-, Sca-1+, c-Kit+ (LSK) and CD11b+ MHCII+ myeloid cells. This increase in the LSKs and myeloid cells in the bone marrow niche was abrogated with CD8 T cell depletion. We conclude that brain viral infections induce in situ effector and memory T cell responses within the bone marrow compartment. Memory recall CD8 T cell responses induce niche dysregulation by expanding LSK cells and lead to an influx of MHCII+ myeloid cells. Our data pave the way for crucial studies of bone marrow resident antigen-specific CD8 T cells in health and diseases.

Abstract Description Peripheral nervous system innervation of various organs has been associated with the development and maintenance of the immune system. Various neurological insults including brain tumors induce thymic involution. Using flow cytometry and confocal microscopy, we identified a population of cells within the thymus that contained neuronal markers and morphology. Subsets expressed tyrosine hydroxylase, BIII tubulin and/or NeuN, distinguishing them from thymic epithelial cells. These cells do not express AIRE. Neuronal cell bodies and/or axonal processes were identified within the thymus. Using retrograde rabies vector neurotracers, we confirmed axonal connections and the identity of thymic neurons. To determine developmental T cell stages that support neuronal development, we evaluated RAG1/2 KO, TCRβ KO, TCRα KO, TCRγδ KO, CD4 KO, CD8 KO and OT-I and -II transgenics. RAG1/2 and TCRβ deficiency resulted in complete loss of thymic neurons. This is while TCRα KO, TCRγδ KO, CD4/8 KO and OT-II transgenics had normal thymic neuron development. Importantly, OT-I transgenics had a significant loss of neuron-like cells, indicating developing CD8 T cells pre-alpha arrangement control thymic innervation. Finally, introduction of healthy bone marrow into irradiated RAG KO mice can restore neuron development. To summarize, we describe a novel population of neuronal cells within the thymus that interact with developing thymocytes to establish optimal thymic innervation. Funding Sources R00NS117799 Topic Categories Neuroimmunology (NEUR)

Abstract Description Glioblastoma (GBM) is an aggressive brain cancer with poor survival. GBM patients have severe peripheral immunosuppression, including defective T cell responses. We showed that serum-derived factors were sufficient to inhibit T cell proliferation. This factor was nonsteroidal and larger than 100kDa. Mass-spectrometry and pathway analysis of suppressive serum implicated proteins involved in DNA response elements. We hypothesized that the suppressive factor is cell-free DNA (cfDNA). In support of this, serum levels of cfDNA are increased in glioma-bearing mice. Exposure to cfDNA from serum of glioma-bearing mice was sufficient to directly and potently inhibit T cell proliferation. 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 was sufficient to partially restore proliferation capacity. We determined that the T cell proliferation defects were independent of AIM2 DNA sensing mechanisms, implicating a novel pathway of DNA sensing in T cells. Finally, to determine the origin of the cfDNA, we used methylation-seq and found that the majority of cfDNA originated from neutrophils, implicating NETosis. Together, we contend that cfDNA from neutrophils induces immunosuppressive effects in GBM. Our work highlights a potential role for targeting cfDNA, neutrophils, and/or NETs in order to reverse T cell dysfunction and improve patient outcomes in GBM. Funding Sources R00NS117799 Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Abstract Description The live-attenuated measles virus (MeV) vaccine is one of the most efficacious vaccines ever developed and has the potential to eradicate MeV from the human population. While having been administered to millions of children over the past 60 years, the molecular mechanisms of its attenuation remain elusive. To determine these mechanisms, we have performed a comparative study of wild type (WT) and vaccine strain MeV infections in peripheral blood mononuclear cells (PBMCs) from human donors, the natural target cells of MeV. We performed in vitro growth kinetics, high-parameter spectral flow cytometry, next generation RNA-seq, and protein analyses. We found that the vaccine strain specifically activated cell intrinsic innate immunity signaling cascades causing a strong type-I interferon (IFN) signature. This resulted in 100-fold reduced titers of vaccine compared to WT virus. Surprisingly, while MeV vaccine infected B and T cells expressing the natural MeV receptor CD150 with similar efficiency as the WT virus, we found a subset of CD150-negative, CD11b+CD11c+CD123+ myeloid cells to become infected with the vaccine strain. We propose that these innate immune cells are highly specialized in pathogen sensing and prime the IFN response preventing vaccine from productive replication and generation of progeny virus in PBMCs. We currently investigate whether altered cell entry or innate immune control mechanisms are the underlying cause of attenuation. Funding Sources Supported by the German Research Foundation (DFG) Collaborative Research Center (SFB) 1021 (Project number 197785619/B12), internal funding of the German Ministry of Health, and by Mayo Clinic. Topic Categories Viral Immunology (VIR)

Abstract Description Bone marrow (BM) is the primary immune organ responsible for stem cell maintenance. Neurotropic infections elicit sequestration of T cells within the BM. Changes of the BM niche during brain viral infections are unknown. Intracranial infection with the neurotropic Theiler’s Murine Encephalomyelitis Virus (TMEV) in C57BL/6 mouse is cleared by 30 days due to generation of an antigen-specific CD8 T cell response. We determined that 1-10% of CD8 T cells present in the sternal, femoral, and cranial BM were virus antigen-specific 7 days post intracranial infection. Over 70% of these CD8 T cells were tissue resident. We determined antigen-specific CD8 T cells were generated in the BM within 4 days of infection, a timeline similar to lymph nodes. Continuous treatment with FTY720, which sequesters T cells outside of the blood, did not eliminate antigen-specific CD8 T cells in any BM compartment. CD8 T cells in the BM established durable memory populations and were reactivated upon antigen reencounter. Both acute infection and reactivation caused an increase in lineage-, Sca-1+, ckit + (LSK) cells in the BM. This LSK dysregulation was abrogated upon CD8 T cell depletion and was limited to the intracranial routes of infection. We conclude that brain viral infections induce in situ effector and memory T cell responses within the BM and cause stem cell dysregulation. Our data paves the way for crucial studies of BM resident antigen-specific CD8 T cells in health and disease. Topic Categories Viral Immunology (VIR)

Abstract Description Innate immune sensing of viral double-stranded (ds) RNA induces the transcription of hundreds of interferon stimulated genes (ISGs) that shift the cell into an antiviral state. We have shown that measles virus (MeV) utilizes the 150 kDa isoform of adenosine deaminase acting on RNA 1 (ADAR1-p150) to prevent activation of protein kinase R (PKR), thereby subverting a type-I interferon response. Hypothesizing that cytoplasmic localization of ADAR1-p150 is required for this pro-viral activity, we generated cell lines expressing single isoforms and localization mutants of ADAR1. Based on MeV replication studies in these cells, we concluded that both nuclear and cytoplasmic ADAR1-p150 prevents PKR activation. However, only cytoplasmic but not nuclear ADAR1-p110 can efficiently prevent PKR activation. These observations suggest a bimodal activation of IFN responses. Initial sensing of viral dsRNA leads to upregulation of a subset of ISGs forming p150-specific self-RNA substrates driving PKR activation. Through ADAR editome analyses, we have identified candidate p150- and p110-specific self-RNA substrates that may be the drivers of this positive feedback loop. RNA immunoprecipitation studies to identify the specific substrates of ADAR1-p150 and PKR during MeV infection are underway to strengthen this novel model of a positive feedback loop driven by self-RNAs. Altogether, our findings shift the paradigm on the regulatory properties of self-RNA on amplifying antiviral innate immunity. Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)

Abstract Measles virus (MeV) is a highly contagious pathogen capable of infecting immune cells and thereby causing immunosuppression and immune amnesia. A live-attenuated MeV vaccine is highly efficacious and has been safely administered for over 60 years. However, a detailed mechanistic understanding of the mode of its attenuation remains elusive. Here, we determined the molecular mechanisms underlying MeV vaccine attenuation in primary human immune cells. Enhanced infection of innate immune cells resulting in uncontrolled viral RNA synthesis and reduced ability to evade host antiviral responses induced a strong type-I interferon signature that arrested replication of the vaccine strain. In contrast, the wild type virus replicated slowly but steadily, avoiding detection by innate immunity.

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.