Goddery, Emma N.

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.

Abstract Description In the tumor microenvironment of glioblastoma, myeloid cells act as a double-edged sword—they are a major cellular component suppressing the immune response while serving as a promising therapeutic target. Our study sheds light on the voltage-gated proton channel Hv1, predominantly expressed in myeloid cells, as a pivotal regulator of their physiological functions. Elevated Hv1 expression correlates strongly with poor prognosis in glioblastoma patients, underscoring its clinical significance. Using a glioma mouse model, we demonstrated that Hv1 depletion markedly extended survival. To overcome the limitations of existing tools, we engineered novel transgenic mouse lines optimized for Hv1 research. Our findings revealed tumor-induced Hv1 upregulation, with glioma-associated macrophages identified as the principal contributors. Crucially, we identified that Hv1 in infiltrating macrophages as the major driver of survival phenotype differences. Through a multi-faceted approach combining in vivo two-photon imaging, spectral flow cytometry, and spatial transcriptomic sequencing, we discovered that Hv1 depletion limits macrophage infiltration and enhances antigen presentation, ultimately fostering a stronger adaptive immune response. These findings establish the Hv1 channel as a crucial new immune regulator within the brain tumor milieu, offering a promising target for reprogramming macrophage function to combat glioblastoma. Funding Sources Supported by NIH R35NS132326 (LJW); Mayo Clinic Cancer Center and Mayo Clinic Center for Biomedical Discovery. Topic Categories Neuroimmunology (NEUR)

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 Brain atrophy is a common feature of many neurological diseases as diverse as Alzheimer’s disease, cerebral palsy, Huntington’s disease, multiple sclerosis, epilepsy, encephalitis, neurosyphilis, neuroAIDS, and Covid-19 infection. We have developed a murine model of brain atrophy using the Theiler’s murine encephalomyelitis virus (TMEV) infection mouse model of multiple sclerosis. In this study, we investigated the contribution of the MHC class I molecule, H-2Db, in generating an immune response associated with the onset of brain atrophy using mice with a C57BL/6 background. The H-2DbClass I molecule not only contributed to significantly more ventricular enlargement in a T2-weighted MRI at 45 d.p.i., but this ventricular enlargement correlated to CD8 T cell infiltration. To further define the cellular and molecular mechanisms of MHC class I molecules in TMEV induced brain atrophy we investigated the cell specific contribution of brain resident antigen presenting cells (APCs) in ventricular atrophy. We developed novel bi-transgenic mouse lines with tamoxifen induced conditional ablation of the H-2Db class I molecule in Cx3CR1+ brain resident myeloid cells. Cx3CR1cre+/Db Lox P mice presented with significantly less ventricular atrophy at 45 d.p.i., compared to cre- littermates along with a decrease in CD8 T cell infiltration. These results strongly imply that antigen presentation by H-2Db on myeloid cells, at least in part is responsible for the development of brain atrophy and provided insight into the molecular mechanism of this poorly understood neuropathology. NS103212, NS122174

Abstract Glioblastoma (GBM) is the most common malignant brain tumor in adults, responsible for approximately 225,000 deaths per year. Despite preclinical successes, most interventions have failed to extend patient survival by more than a few months. Treatment with anti—programmed cell death protein 1 (anti–PD-1) immune checkpoint blockade (ICB) monotherapy has been beneficial for malignant tumors such as melanoma and lung cancers but has yet to be effectively employed in GBM. This study aimed to determine whether supplementing anti–PD-1 ICB with engineered extended half-life IL2, a potent lymphoproliferative cytokine, could improve outcomes. This combination therapy, subsequently referred to as enhanced checkpoint blockade (ECB), delivered intraperitoneally, reliably cures approximately 50% of C57BL/6 mice bearing orthotopic GL261 gliomas and extends median survival of the treated cohort. In the CT2A model, characterized as being resistant to CBI, ECB caused a decrease in CT2A tumor volume in half of measured animals similar to what was observed in GL261-bearing mice, promoting a trending survival increase. ECB generates robust immunologic responses, features of which include secondary lymphoid organ enlargement and increased activation status of both CD4 and CD8 T cells. This immunity is durable, with long-term ECB survivors able to resist GL261 rechallenge. Through employment of depletion strategies, ECB's efficacy was shown to be independent of host MHC class I–restricted antigen presentation but reliant on CD4 T cells. These results demonstrate ECB is efficacious against the GL261 glioma model through an MHC class I–independent mechanism and supporting further investigation into IL2-supplemented ICB therapies for tumors of the central nervous system.

Abstract CD8 T cell responses are essential for clearance of infections in the CNS but contribute to neuropathology. T cell interaction with antigen presenting cells (APCs) at CNS barriers is thought to allow activated antigen-specific T cells to infiltrate the CNS during neuroinflammation. However, the role of CNS-resident APCs during CD8 T cell infiltration of the CNS is unclear. To address this, we utilized Theiler’s murine encephalomyelitis virus (TMEV) and Plasmodium berghei ANKA (PbA) to assess the role of APCs during CD8 T cell responses to CNS infection in mice. During infection, we observed robust upregulation of MHC class I on microglia and brain associated macrophages (BAM). Microglia and BAMs were located in close proximity to inflamed vasculature where antigen presentation to T cells could occur. Thus, we hypothesized that microglia and BAMs are critical APCs for CNS infiltration of antigen specific CD8 T cells. To test this, we generated mice with inducible deletion of MHC class I on microglia and BAMs by crossing CX3CR1creERT2 mice with H-2Db or H-2Kb floxed mice (Db cKO or Kb cKO). We found that brain infiltrating virus-specific CD8 T cells were reduced in Db cKO mice compared to controls during TMEV infection, while peripheral priming of T cells was unaffected. Conversely, antigen-specific CD8 T cell infiltration was not impacted in Kb cKO mice infected with TMEV-OVA257–264. In Db cKO mice, brain-infiltrating CD8 T cells were increased during PbA induced experimental cerebral malaria (ECM), indicating a potential inhibitory role for CNS APCs. These results demonstrate that local antigen presentation by microglia/BAMs can both positively and negatively regulate CD8 T cell infiltration of the brain in discrete neurologic disease models.

Abstract Glioblastoma multiforme (GBM) is a deadly CNS malignancy with an average survival of ~12–18 months post diagnosis. The unique immune environment of the brain and the immunosuppressive features of GBM complicate the use of immunotherapies against these tumors. We aimed to promote the efficacy of αPD-1 against the GL261 murine model of GBM by supplementing this therapy with an engineered IL-2 cytokine. IL-2 therapy has been used clinically for metastatic melanoma and renal cancer, but its short half-life in circulation necessitated high doses to keep the drug concentration sufficiently high. Fusing IL-2 to the mouse serum albumin molecule (MSA-IL-2), extends the cytokine’s half-life. Neither αPD-1 nor MSA-IL-2 are effective monotherapies for GL261 tumors, but the combination provides durable tumor clearance and prevents subsequent rechallenge. Most strikingly, this combination therapy cleared established GL261 tumors even in mice incapable of MHC class I restricted antigen presentation. Instead, therapeutic efficacy was abrogated by the depletion of CD4 T cells. Our combination therapy also reversed multiple key features of GBM-associated peripheral immune suppression, including depressed CD4 T cell counts and dampened MHC class II expression. Our combination therapy’s ability to overcome these immune derangements associated with glioma growth and to function independent of MHC class I restricted antigen presentation enhance the appeal of this therapy for future translational adaptation. This work was supported by the National Institutes of Health (R01 NS103212) and the Mayo Clinic-Koch Institute Cancer Solutions Team Grant

Abstract The contribution of antiviral resident memory T cells (TRM) to blood-brain barrier disruption has not been defined despite the implication of this lymphocyte in clinical encephalitis. We therefore evaluated the capacity of TRM cells to induce CNS vascular permeability in mice which resolved Theiler’s murine encephalomyelitis (TMEV) infection. Following TMEV infection of the brain, persistent populations of virus antigen specific resident memory T cells are induced within the brain. This population of resident memory T cells can be further reactivated at 60 dpi through administration of the cognate immunodominant virus peptide antigen, VP2, inducing blood brain barrier disruption in the process. We determined that brain resident memory T cells expand following reactivation with administered VP2 peptide. This reactivation is associated with infiltration of T cells, and inflammatory myeloid cells into the brain. Importantly, using depletion and T cell sequestrating strategies with low dose anti CD8 antibodies and FTY720, we determined that TRM induced blood-brain barrier disruption is independent of peripheral T cells. We conclude that reactivation of brain TRM cells during peptide administration occurs without dependency on peripheral responses, highlighting the importance of this cell type in inducing neuroinflammation and underlying neuropathology.

Abstract Brain atrophy is a common feature of many neurological diseases as diverse as Alzheimer’s disease, cerebral palsy, Huntington’s disease, Krabbe disease, multiple sclerosis, Pick’s disease, epilepsy, encephalitis, neurosyphilis, neuroAIDS, and Covid-19 infection. We have developed a murine model of brain atrophy using the Theiler’s murine encephalomyelitis virus (TMEV) infection mouse model of multiple sclerosis. In this study, we investigated the contribution of the MHC class I molecule, H-2Db, in generating an immune response associated with the onset of brain atrophy. To define the roll of the MHC class I molecule in the cellular and molecular mechanisms of brain atrophy, we developed a novel bi-transgenic mouse lines with tamoxifen induced conditional ablation of the H-2Db class I molecule in Cx3CR1+ brain resident myeloid cells on the C57BL/6 background. Interestingly, both CX3CR1cre/Db LoxP mice and their cre- littermate controls experienced short-term memory loss using novel object recognition tests. However, Cx3CR1cre+/Db LoxP mice presented with significantly less brain atrophy as assessed by analysis of lateral ventricular volume at 45 d.p.i. using T2-weighted MRI, compared to cre- littermates. This change in ventricular volume was sustained at 6 months post infection. These results strongly imply a requirement for antigen presentation by H-2Db on myeloid cells for the development of brain atrophy and provided insight into the molecular mechanism of this poorly understood neuropathology. Supported by NIH (RFI NS122174)

Abstract Myeloid cells comprising up to 50% of total tumor mass in glioblastoma (GBM) contribute to tumor progression and immunosuppression. Restraining glioma-induced myeloid cell infiltration increases functional T cells within the tumors, improves the efficacy of checkpoint blockade, and significantly extends mouse survival. Here, we focus on an ion channel, voltage-gated proton channel Hv1, which is mainly expressed in myeloid cells and shapes the physiological functions of myeloid cells. Our bioinformatics analysis demonstrated that elevated Hv1 expression in the tumor mass correlates with poor disease prognosis in patients. Using the leading immunocompetent model of GBM in mice, we showed that Hv1 knockout mice (Hv1−/−) exhibit slower glioma progression and prolonged survival. Using in vivo two-photon imaging, we found that myeloid cells infiltrate into tumor mass since early time points and have intimate interaction with tumor cells. Notably, by full-spectrum flow cytometry, we found that Hv1−/− mice have reduced monocyte/macrophage infiltration, and increased PD-1+ CD4+ T cells in the tumor-burdened hemisphere. As a result, we combined anti-PD-1 treatment with Hv1 knockout in the middle stage of glioma, and found that approximately 30% of Hv1−/− mice were cured. Together, our results demonstrated that Hv1 controls myeloid infiltration and could be a novel therapeutic target for the treatment of GBM. Supported by R01NS110825, R01NS088627