Anti-PD-1 and Extended Half-life IL2 Synergize for Treatment of Murine Glioblastoma Independent of Host MHC Class I Expression.

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.

Zika virus spreads through infection of lymph node-resident macrophages.

To disseminate through the body, Zika virus (ZIKV) is thought to exploit the mobility of myeloid cells, in particular monocytes and dendritic cells. However, the timing and mechanisms underlying shuttling of the virus by immune cells remains unclear. To understand the early steps in ZIKV transit from the skin, at different time points, we spatially mapped ZIKV infection in lymph nodes (LNs), an intermediary site en route to the blood. Contrary to prevailing hypotheses, migratory immune cells are not required for the virus to reach the LNs or blood. Instead, ZIKV rapidly infects a subset of sessile CD169+ macrophages in the LNs, which release the virus to infect downstream LNs. Infection of CD169+ macrophages alone is sufficient to initiate viremia. Overall, our experiments indicate that macrophages that reside in the LNs contribute to initial ZIKV spread. These studies enhance our understanding of ZIKV dissemination and identify another anatomical site for potential antiviral intervention.

Rejuvenation of the aged brain immune cell landscape in mice through p16-positive senescent cell clearance.

Cellular senescence is a plausible mediator of inflammation-related tissue dysfunction. In the aged brain, senescent cell identities and the mechanisms by which they exert adverse influence are unclear. Here we used high-dimensional molecular profiling, coupled with mechanistic experiments, to study the properties of senescent cells in the aged mouse brain. We show that senescence and inflammatory expression profiles increase with age and are brain region- and sex-specific. p16-positive myeloid cells exhibiting senescent and disease-associated activation signatures, including upregulation of chemoattractant factors, accumulate in the aged mouse brain. Senescent brain myeloid cells promote peripheral immune cell chemotaxis in vitro. Activated resident and infiltrating immune cells increase in the aged brain and are partially restored to youthful levels through p16-positive senescent cell clearance in female p16-InkAttac mice, which is associated with preservation of cognitive function. Our study reveals dynamic remodeling of the brain immune cell landscape in aging and suggests senescent cell targeting as a strategy to counter inflammatory changes and cognitive decline.

Microglia and Perivascular Macrophages Act as Antigen Presenting Cells to Promote CD8 T Cell Infiltration of the Brain.

CD8 T cell infiltration of the central nervous system (CNS) is necessary for host protection but contributes to neuropathology. Antigen presenting cells (APCs) situated at CNS borders are thought to mediate T cell entry into the parenchyma during neuroinflammation. The identity of the CNS-resident APC that presents antigen via major histocompatibility complex (MHC) class I to CD8 T cells is unknown. Herein, we characterize MHC class I expression in the naïve and virally infected brain and identify microglia and macrophages (CNS-myeloid cells) as APCs that upregulate H-2Kb and H-2Db upon infection. Conditional ablation of H-2Kb and H-2Db from CNS-myeloid cells allowed us to determine that antigen presentation via H-2Db, but not H-2Kb, was required for CNS immune infiltration during Theiler's murine encephalomyelitis virus (TMEV) infection and drives brain atrophy as a consequence of infection. These results demonstrate that CNS-myeloid cells are key APCs mediating CD8 T cell brain infiltration.

Immunomodulation Mediated by Anti-angiogenic Therapy Improves CD8 T Cell Immunity Against Experimental Glioma.

Glioblastoma (GBM) is a lethal cancer of the central nervous system with a median survival rate of 15 months with treatment. Thus, there is a critical need to develop novel therapies for GBM. Immunotherapy is emerging as a promising therapeutic strategy. However, current therapies for GBM, in particular anti-angiogenic therapies that block vascular endothelial growth factor (VEGF), may have undefined consequences on the efficacy of immunotherapy. While this treatment is primarily prescribed to reduce tumor vascularization, multiple immune cell types also express VEGF receptors, including the most potent antigen-presenting cell, the dendritic cell (DC). Therefore, we assessed the role of anti-VEGF therapy in modifying DC function. We found that VEGF blockade results in a more mature DC phenotype in the brain, as demonstrated by an increase in the expression of the co-stimulatory molecules B7-1, B7-2, and MHC II. Furthermore, we observed reduced levels of the exhaustion markers PD-1 and Tim-3 on brain-infiltrating CD8 T cells, indicating improved functionality. Thus, anti-angiogenic therapy has the potential to be used in conjunction with and enhance immunotherapy for GBM.

Non-equivalent antigen presenting capabilities of dendritic cells and macrophages in generating brain-infiltrating CD8 + T cell responses.

The contribution of antigen-presenting cell (APC) types in generating CD8+ T cell responses in the central nervous system (CNS) is not fully defined, limiting the development of vaccines and understanding of immune-mediated neuropathology. Here, we generate a transgenic mouse that enables cell-specific deletion of the H-2Kb MHC class I molecule. By deleting H-2Kb on dendritic cells and macrophages, we compare the effect of each APC in three distinct models of neuroinflammation: picornavirus infection, experimental cerebral malaria, and a syngeneic glioma. Dendritic cells and macrophages both activate CD8+ T cell responses in response to these CNS immunological challenges. However, the extent to which each of these APCs contributes to CD8+ T cell priming varies. These findings reveal distinct functions for dendritic cells and macrophages in generating CD8+ T cell responses to neurological disease.

Immunovirotherapy with measles virus strains in combination with anti-PD-1 antibody blockade enhances antitumor activity in glioblastoma treatment.

Background: Glioblastoma (GBM) is the most common primary malignant brain tumor and has a dismal prognosis. Measles virus (MV) therapy of GBM is a promising strategy due to preclinical efficacy, excellent clinical safety, and its ability to evoke antitumor pro-inflammatory responses. We hypothesized that combining anti- programmed cell death protein 1 (anti-PD-1) blockade and MV therapy can overcome immunosuppression and enhance immune effector cell responses against GBM, thus improving therapeutic outcome. Methods: In vitro assays of MV infection of glioma cells and infected glioma cells with mouse microglia ± aPD-1 blockade were established to assess damage associated molecular pattern (DAMP) molecule production, migration, and pro-inflammatory effects. C57BL/6 or athymic mice bearing syngeneic orthotopic GL261 gliomas were treated with MV, aPD-1, and combination treatment. T2* weighted immune cell-specific MRI and fluorescence activated cell sorting (FACS) analysis of treated mouse brains was used to examine adaptive immune responses following therapy. Results: In vitro, MV infection induced human GBM cell secretion of DAMP (high-mobility group protein 1, heat shock protein 90) and upregulated programmed cell death ligand 1 (PD-L1). MV infection of GL261 murine glioma cells resulted in a pro-inflammatory response and increased migration of BV2 microglia. In vivo, MV+aPD-1 therapy synergistically enhanced survival of C57BL/6 mice bearing syngeneic orthotopic GL261 gliomas. MRI showed increased inflammatory cell influx into the brains of mice treated with MV+aPD-1; FACS analysis confirmed increased T-cell influx predominantly consisting of activated CD8+ T cells. Conclusions: This report demonstrates that oncolytic measles virotherapy in combination with aPD-1 blockade significantly improves survival outcome in a syngeneic GBM model and supports the potential of clinical/translational strategies combining MV with αPD-1 therapy in GBM treatment.

Superior isolation of antigen-specific brain infiltrating T cells using manual homogenization technique.

Effective recovery of activated brain infiltrating lymphocytes is critical for investigations involving murine neurological disease models. To optimize lymphocyte recovery, we compared two isolation methods using brains harvested from seven-day Theiler's murine encephalomyelitis virus (TMEV) and TMEV-OVA infected mice. Brains were processed using either a manual dounce based approach or enzymatic digestion using type IV collagenase. The resulting cell suspensions from these two techniques were transferred to a percoll gradient, centrifuged, and lymphocytes were recovered. Flow cytometric analysis of CD45hi cells showed greater percentage of CD44hiCD62lo activated lymphocytes and CD19+ B cells using the dounce method. In addition, we achieved a 3-fold greater recovery of activated virus-specific CD8 T cells specific for the immunodominant Db:VP2121-130 and engineered Kb:OVA257-264 epitopes through manual dounce homogenization approach as compared to collagenase digest. A greater percentage of viable cells was also achieved through dounce homogenization. Therefore, we conclude that manual homogenization is a superior approach to isolate activated T cells from the mouse brain.

The Effect of Vector Silencing during Picornavirus Vaccination against Experimental Melanoma and Glioma.

Virus vector-based vaccination against tumor-specific antigens remains a promising therapeutic approach to overcome the immune suppressive tumor microenvironment. However, the extent that the desired CD8 T cell response against the targeted tumor antigen is impacted by the CD8 T cell response against the virus vector is unclear. To address this question, we used picornavirus vaccination with Theiler's murine encephalomyelitis virus (TMEV) as our vector against tumor-expressed ovalbumin (OVA257-264) antigen in both the B16-OVA murine melanoma and GL261-quad cassette murine glioma models. Prior to vaccination, we employed vector silencing to inhibit the CD8 T cell response against the immunodominant TMEV antigen, VP2121-130. We then monitored the resulting effect on the CD8 T cell response against the targeted tumor-specific antigen, ovalbumin. We demonstrate that employing vector silencing in the context of B16-OVA melanoma does not reduce tumor burden or improve survival, while TMEV-OVA vaccination without vector silencing controls tumor burden. Meanwhile, employing vector silencing during picornavirus vaccination against the GL261-quad cassette glioma resulted in a lower frequency of tumor antigen-specific CD8 T cells. The results of this study are relevant to antigen-specific immunotherapy, in that the virus vector-specific CD8 T cell response is not competing with tumor antigen-specific CD8 T cells. Furthermore, vector silencing may have the adverse consequence of reducing the tumor antigen-specific CD8 T cell response, as demonstrated by our findings in the GL261-quad cassette model.

Improved Treatment Efficacy of Antiangiogenic Therapy when Combined with Picornavirus Vaccination in the GL261 Glioma Model.

The addition of antiangiogenic therapy to the standard-of-care treatment regimen for recurring glioblastoma has provided some clinical benefits while also delineating numerous caveats, prompting evaluation of the elicited alterations to the tumor microenvironment. Of critical importance, given the steadily increasing incorporation of immunotherapeutic approaches clinically, is an enhanced understanding of the interplay between angiogenic and immune response pathways within tumors. In the present study, the GL261 glioma mouse model was used to determine the effects of antiangiogenic treatment in an immune-competent host. Following weekly systemic administration of aflibercept, an inhibitor of vascular endothelial growth factor, tumor volume was assessed by magnetic resonance imaging and changes to the tumor microenvironment were determined. Treatment with aflibercept resulted in reduced tumor burden and increased survival compared with controls. Additionally, decreased vascular permeability and preservation of the integrity of tight junction proteins were observed. Treated tumors also displayed hallmarks of anti-angiogenic evasion, including marked upregulation of vascular endothelial growth factor expression and increased tumor invasiveness. Aflibercept was then administered in combination with a picornavirus-based antitumor vaccine and tumor progression was evaluated. This combination therapy significantly delayed tumor progression and extended survival beyond that observed for either therapy alone. As such, this work demonstrates the efficacy of combined antiangiogenic and immunotherapy approaches for treating established gliomas and provides a foundation for further evaluation of the effects of antiangiogenic therapy in the context of endogenous or vaccine-induced inflammatory responses.