Citations

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)

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.

Aging is a major risk factor for increased morbidity and mortality following acute respiratory virus infections. To elucidate the immune determinants underlying viral pathogenesis and delayed lung repair in the aged lung, a comprehensive time-course study was conducted. Single-cell RNA sequencing (scRNAseq) and high-dimensional flow cytometry were utilized to compare lungs from young and aged mice infected with influenza A virus (IAV). Aged hosts displayed diminished alveolar macrophage (AM) and dendritic cell (DC) but elevated monocyte-derived macrophage (MoM) and interstitial macrophage (IM) presence following infection. Additionally, enhanced accumulation of adaptive immune cells, including CD4+ tissue-resident helper (TRH) cells, CD8+ tissue-resident memory (TRM) cells, and a B cell subset resembling age-associated B cells, was observed in the memory phase. Pathway analysis revealed that elevated type I and II interferon (IFNα/γ) signaling, especially in MoM/IM subsets, distinguished the aged hosts from the young. Inhibition of IFNα/γ signaling after viral clearance improved long-term respiratory outcomes and reduced both IM and TRH populations in aged mice. These findings highlight the pivotal role of IFNα/γ signaling, likely within MoM/IM subsets, in driving the exuberant persistence of adaptive immune cells and chronic immunopathology in the aged lung following acute viral infection.

Aging is a major risk factor for increased morbidity and mortality following acute respiratory virus infections. To elucidate the immune determinants underlying viral pathogenesis and delayed lung repair in the aged lung, a comprehensive time-course study was conducted. Single-cell RNA sequencing (scRNAseq) and high-dimensional flow cytometry were utilized to compare lungs from young and aged mice infected with influenza A virus (IAV). Aged hosts displayed diminished alveolar macrophage (AM) and dendritic cell (DC) but elevated monocyte-derived macrophage (MoM) and interstitial macrophage (IM) presence following infection. Additionally, enhanced accumulation of adaptive immune cells, including CD4+ tissue-resident helper (TRH) cells, CD8+ tissue-resident memory (TRM) cells, and a B cell subset resembling age-associated B cells, was observed in the memory phase. Pathway analysis revealed that elevated type I and II interferon (IFNα/γ) signaling, especially in MoM/IM subsets, distinguished the aged hosts from the young. Inhibition of IFNα/γ signaling after viral clearance improved long-term respiratory outcomes and reduced both IM and TRH populations in aged mice. These findings highlight the pivotal role of IFNα/γ signaling, likely within MoM/IM subsets, in driving the exuberant persistence of adaptive immune cells and chronic immunopathology in the aged lung following acute viral infection.

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 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 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 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 α-PD-1 checkpoint blockade is a promising immunotherapeutic approach for many solid tumors; however, this strategy is minimally effective for GBM patients as a monotherapy. One potential reason why α-PD-1 checkpoint blockade is ineffective is the tumor microenvironment and systemic derangements of the immune system which are prevalent in GBM patients. We have successfully modeled peripheral immune derangements in experimental murine GBM models. These include low peripheral blood CD4 T cell counts, reduced MHC class II expression on monocytes, and atrophy of primary immune organs in animals harboring gliomas. We therefore hypothesized that extended half-life IL-2, a potent cytokine which promotes the proliferation, differentiation, and killing activity of T cells, could overcome immune derangements and could potentially synergize with α-PD-1 checkpoint blockade therapy. In cohorts of C57Bl/6 mice, we supplemented α-PD-1 checkpoint blockade with multiple bio-engineered extended half-life IL-2 molecules to treat GL261 and CT2A orthotopic glioma models which are normally resistant to monotherapy. We observe a statistically significant increase in survival in GL261-bearing animals receiving the combination of extended half-life IL-2 reagents and αPD-1 therapy relative to control-treated mice. Some animals receiving this combination therapy had a reduction in tumor burden to levels below detection. These long-term survivors underwent a rechallenge experiment with a second inoculation of GL261-Luc and effectively cleared tumor without further therapeutic intervention. This intervention causes a significant shift in the immune profile, including the formation of mature T cell responses, as well as restoring the diminished peripheral immune cell counts in GL261-bearing animals. Finally, combining extended half-life IL-2 with α-PD-1 checkpoint blockade is effective independently of the CD8 T cell response. Together, these data suggest that combination immunotherapies employing extended half-life IL-2 fusion proteins are translational to the clinic and do not require the identification of tumor specific antigens for efficacy

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.