Johnson, Aaron J.

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)

Hypoxia is a prevalent characteristic of neurological diseases, including ischemic injury, neurodegeneration and infectious disease complications. Concurrently, hypoxia shapes both protective and pathological responses within the central nervous system (CNS). Central to this process is hypoxia-inducible factor 1α (HIF1α), a transcription factor that regulates cellular adaptation to reduced oxygen availability through coordinated glycolytic, inflammatory and cell survival pathways. Under hypoxic conditions, HIF1α transcriptional activity influences microglial activation, mitochondrial quality control, and cytokine production, thereby modulating neuroinflammation and neuroprotection. Preclinical evidence points toward hypoxia preconditioning being neuroprotective through HIF1α-dependent mechanisms in a context-dependent matter. This review synthesizes the current understanding of the role of HIF1α across neurological disease contexts, highlighting the intersection of hypoxia, neuroinflammation and neuronal survival. Ultimately, defining the cell-specific and context-dependent involvement of HIF1α will be critical for targeted therapeutic approaches to alleviate neuronal death and slow disease progression.

Abstract The intestinal epithelium harbors a large population of microbiota-dependent CD8αβ + T cells whose antigen specificity and regulation are ill-defined. By identifying MHCIa-restricted TCRs and generating tetramers against the gut commensal Segmented Filamentous Bacteria, we demonstrate that a single commensal species drives a clonally expanded, antigen-specific CD8αβ + T cell within the intraepithelial lymphocyte compartment. Mechanistically, the intestinal epithelium coordinates coincident signals governing this population: peptide:MHC-dependent TCR engagement drives pIEL accumulation, while αvβ6-mediated TGFβ activation restraints effector cell differentiation. Perturbation of epithelial cell-mediated TGFβ activation diverts commensal-specific CD8 + T cells toward inflammatory differentiation states transcriptionally convergent with those observed in ulcerative colitis. The intestinal epithelium thus functions as a dual-signal organizer of commensal-specific CD8 + T cell responses, coupling differentiation to restraint through spatially coincident molecular cues.

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.

Blood-brain barrier (BBB) disruption is a potentially deadly complication of numerous neurological diseases as diverse as cerebral malaria, dengue hemorrhagic fever, tauopathies, multiple sclerosis, schizophrenia, Parkinson’s disease, narcolepsy, and COVID-19. Neuroinflammation and immune cell infiltration are both drivers and consequences of BBB disruption. In this review, we will provide an overview of how brain-infiltrating T-cell responses engage the cellular components of the neurovascular unit (NVU), which regulates the BBB in preclinical models. This will provide context on how the immune system can contribute to vascular leakage and neuropathology in neurological diseases. We will discuss how discrete major histocompatibility complex (MHC) class I and class II molecules on NVU cell types govern T-cell entry, retention, and barrier disruption in neuroinflammatory diseases. Finally, we will summarize our current understanding of how discrete human leukocyte antigen (HLA) genes associate with specific neurological diseases characterized by neuroinflammation and BBB disruption.

Abstract In many cancers, a major barrier to effective immunotherapy is the dominance of tumor-associated myeloid cells (TAMs), which establish a profoundly immunosuppressive tumor microenvironment (TME). This challenge is particularly pronounced in glioblastoma (GBM), where myeloid cells represent the predominant immune population and actively suppress antitumor immunity. Identifying targets that can reprogram TAMs from an immunosuppressive state toward a pro-inflammatory, antigen-presenting phenotype remains a critical unmet need. Here, we identify the voltage-gated proton channel Hv1 as an immune-restricted regulator of glioma-associated macrophage function. Hv1 is notable not only for its immune-specific expression but also for its established roles in central nervous system (CNS) pathologies, where Hv1 depletion alters microglial and macrophage polarization, reduces myeloid accumulation, dampens inflammatory cytokine production, and limits secondary tissue damage. However, Hv1’s role in shaping the GBM immune landscape—particularly in vivo—has remained poorly understood due to low Hvcn1 transcript abundance and the lack of reliable antibodies and genetic tools. Given the dominance of myeloid cells in GBM and their central role in tumor immune regulation, we sought to define Hv1 function within this highly immunosuppressive brain TME. Analysis of patient datasets revealed that elevated Hv1 expression correlates with poor clinical prognosis. Consistently, Hv1 loss significantly prolonged survival in multiple murine glioma models. Using in vivo two-photon imaging and spectral flow cytometry, we show that Hv1 depletion reprograms TAMs toward an anti-tumor phenotype characterized by enhanced tumor cell phagocytosis, improved antigen presentation, and increased T cell infiltration. By generating new Hv1 reporter and conditional mouse lines, we further demonstrate that tumor-associated microglia and infiltrating macrophages are the principal sources of Hv1 and markedly upregulate its expression in response to glioma. Notably, selective deletion of Hv1 in infiltrating macrophages phenocopied the survival benefit observed with global Hv1 loss. Mechanistically, using mass spectrometry and genetic models, we identify the Stimulator of Interferon Genes (STING) as a previously unrecognized binding partner of Hv1, placing Hv1 within the STING signaling pathway, a central regulator of innate and adaptive immune responses. Together, these findings fill a critical gap in understanding myeloid Hv1 function in GBM and identify Hv1 as a promising target for reprogramming the tumor immune microenvironment. Citation Format: Jiaying Zheng, Lingxiao Wang, Shunyi Zhao, Katayoun Ayasoufi, Meijie Wang, Praveen N. Pallegar, Peter R. Strege, Langni Liu, Yushan Wan, Wenjing Zhang, Emma N. Goddery, Manling Xie, Arthur Beyder, Aaron J. Johnson, Long-Jun Wu. Myeloid Hv1-STING axis tunes glioma immunity [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Brain Cancer; 2026 Mar 23-25; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(6_Suppl):Abstract nr A016.

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 Therapeutic in situ chimeric antigen receptor (isCAR) T cells can be generated by targeting activated T cells within a lymph node (LN) with retroviruses encoding CARs. While isCAR T cells are successfully generated by intradermal LN priming using adenovirus encoding allogeneic MHC in all mouse lines tested, differences in emerging therapeutic peripheral isCAR T cells are found in a strain dependent manner. Potentially harmful off target retroviral transduction of B cells with CARs is observed within the targeted LN. Here we report the blockade of B cell transduction using anti-CD20 antibody B cell depletion with the introduction of anti-CD19 chimeric antigen receptors into the activated CD8+ T cells. Lymphocytes were analyzed in blood and LN following isCAR T cell generation. When B cells were depleted by systemic administration of anti-CD20 antibodies during isCAR T cell induction, >97.5% systemic depletion of B cells was achieved. Whereas very small numbers of CAR transduced B cells were found in the blood of mice not treated with anti-CD20, none were observed in animals systemically depleted of B cells. In contrast, CAR B cells persisted in the LN of local anti-CD20 treated animals. However, animals expressing dominant CD8+ isCAR T cell populations eliminated LN B cells expressing CARs by week two. Furthermore, systemic treatment of lymphoma in animals with anti-CD20 antibody complemented anti-CD19 isCAR T therapy while blocking off target B cell transduction. Funding Sources Mayo Clinic Ventures Mayo Clinic Graduate School of Biomedical Sciences Mayo Clinic Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)