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Kidney allograft inflammation, mostly attributed to rejection and infection, is an important cause of graft injury and loss. Standard histopathological assessment of allograft inflammation provides limited insights into biological processes and the immune landscape. Here, using imaging mass cytometry with a panel of 28 validated biomarkers, we explored the single-cell landscape of kidney allograft inflammation in 32 kidney transplant biopsies and 247 high-dimensional histopathology images of various phenotypes of allograft inflammation (antibody-mediated rejection, T cell-mediated rejection, BK nephropathy, and chronic pyelonephritis). Using novel analytical tools, for cell segmentation, we segmented over 900 000 cells and developed a tissue-based classifier using over 3000 manually annotated kidney microstructures (glomeruli, tubules, interstitium, and arteries). Using PhenoGraph, we identified 11 immune and 9 nonimmune clusters and found a high prevalence of memory T cell and macrophage-enriched immune populations across phenotypes. Additionally, we trained a machine learning classifier to identify spatial biomarkers that could discriminate between the different allograft inflammatory phenotypes. Further validation of imaging mass cytometry in larger cohorts and with more biomarkers will likely help interrogate kidney allograft inflammation in more depth than has been possible to date., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background & Aims: Metabolic dysfunction-associated steatohepatitis (MASH) is characterized by excessive circulating toxic lipids, hepatic steatosis, and liver inflammation. Monocyte adhesion to liver sinusoidal endothelial cells (LSECs) and transendothelial migration (TEM) are crucial in the inflammatory process. Under lipotoxic stress, LSECs develop a proinflammatory phenotype known as endotheliopathy. However, mediators of endotheliopathy remain unclear., Methods: Primary mouse LSECs isolated from C57BL/6J mice fed chow or MASH-inducing diets rich in fat, fructose, and cholesterol (FFC) were subjected to multi-omics profiling. Mice with established MASH resulting from a choline-deficient high-fat diet (CDHFD) or FFC diet were also treated with two structurally distinct GSK3 inhibitors (LY2090314 and elraglusib [9-ING-41])., Results: Integrated pathway analysis of the mouse LSEC proteome and transcriptome indicated that leukocyte TEM and focal adhesion were the major pathways altered in MASH. Kinome profiling of the LSEC phosphoproteome identified glycogen synthase kinase (GSK)-3β as the major kinase hub in MASH. GSK3β-activating phosphorylation was increased in primary human LSECs treated with the toxic lipid palmitate and in human MASH. Palmitate upregulated the expression of C-X-C motif chemokine ligand 2, intracellular adhesion molecule 1, and phosphorylated focal adhesion kinase, via a GSK3-dependent mechanism. Congruently, the adhesive and transendothelial migratory capacities of primary human neutrophils and THP-1 monocytes through the LSEC monolayer under lipotoxic stress were reduced by GSK3 inhibition. Treatment with the GSK3 inhibitors LY2090314 and elraglusib ameliorated liver inflammation, injury, and fibrosis in FFC- and CDHFD-fed mice, respectively. Immunophenotyping using cytometry by mass cytometry by time of flight of intrahepatic leukocytes from CDHFD-fed mice treated with elraglusib showed reduced infiltration of proinflammatory monocyte-derived macrophages and monocyte-derived dendritic cells., Conclusion: GSK3 inhibition attenuates lipotoxicity-induced LSEC endotheliopathy and could serve as a potential therapeutic strategy for treating human MASH., Impact and Implications: LSECs under lipotoxic stress in MASH develop a proinflammatory phenotype known as endotheliopathy, with obscure mediators and functional outcomes. The current study identified GSK3 as the major driver of LSEC endotheliopathy, examined its pathogenic role in myeloid cell-associated liver inflammation, and defined the therapeutic efficacy of pharmacological GSK3 inhibitors in murine MASH. This study provides preclinical data for the future investigation of GSK3 pharmacological inhibitors in human MASH. The results of this study are important to hepatologists, vascular biologists, and investigators studying the mechanisms of inflammatory liver disease and MASH, as well as those interested in drug development., Competing Interests: The authors declare no conflicts of interest. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2024 The Author(s).)

Intrahepatic macrophages in nonalcoholic steatohepatitis (NASH) are heterogenous and include proinflammatory recruited monocyte-derived macrophages. The receptor for advanced glycation endproducts (RAGE) is expressed on macrophages and can be activated by damage associated molecular patterns (DAMPs) upregulated in NASH, yet the role of macrophage-specific RAGE signaling in NASH is unclear. Therefore, we hypothesized that RAGE-expressing macrophages are proinflammatory and mediate liver inflammation in NASH. Compared with healthy controls, RAGE expression was increased in liver biopsies from patients with NASH. In a high-fat, -fructose, and -cholesterol-induced (FFC)-induced murine model of NASH, RAGE expression was increased, specifically on recruited macrophages. FFC mice that received a pharmacological inhibitor of RAGE (TTP488), and myeloid-specific RAGE KO mice (RAGE-MKO) had attenuated liver injury associated with a reduced accumulation of RAGE+ recruited macrophages. Transcriptomics analysis suggested that pathways of macrophage and T cell activation were upregulated by FFC diet, inhibited by TTP488 treatment, and reduced in RAGE-MKO mice. Correspondingly, the secretome of ligand-stimulated BM-derived macrophages from RAGE-MKO mice had an attenuated capacity to activate CD8+ T cells. Our data implicate RAGE as what we propose to be a novel and potentially targetable mediator of the proinflammatory signaling of recruited macrophages in NASH.

Ductular reactive (DR) cells exacerbate cholestatic liver injury and fibrosis. Herein, we posit that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) emanates from recruited macrophages and restrains DR cell expansion, thereby limiting cholestatic liver injury. Wild type (WT), Trail fl/fl and myeloid-specific Trail deleted (Trail Δmye ) C57BL/6 mice were exposed to DDC diet-induced cholestatic liver injury, which induced hepatomegaly and liver injury as compared to control diet-fed mice. However, parameters of liver injury, fibrosis, and inflammation were all increased in the Trail Δmye mice as compared to the WT and Trail fl/fl mice. High dimensional mass cytometry indicated that cholestasis resulted in increased hepatic recruitment of subsets of macrophages and neutrophils in the Trail Δmye mice. Spatial transcriptomics analysis revealed that the PanCK + cholangiocytes from Trail Δmye mice had increased expression of the known myeloid attractants S100a8, Cxcl5, Cx3cl1, and Cxcl1. Additionally, in situ hybridization of Cxcl1, a potent neutrophil chemoattractant, demonstrated an increased expression in CK19 + cholangiocytes of Trail Δmye mice. Collectively, these data suggest that TRAIL from myeloid cells, particularly macrophages, restrains a subset of DR cells (i.e., Cxcl1 positive cells), limiting liver inflammation and fibrosis. Reprogramming macrophages to express TRAIL may be salutary in cholestasis., (© 2024. The Author(s).)

T-lymphocytes are prevalent in the tumor microenvironment of follicular lymphoma (FL). However, the phenotype of T-cells may vary, and the prevalence of certain T-cell subsets may influence tumor biology and patient survival. We therefore analyzed a cohort of 82 FL patients using CyTOF to determine whether specific T-cell phenotypes were associated with distinct tumor microenvironments and patient outcome. We identified four immune subgroups with differing T-cell phenotypes and the prevalence of certain T-cell subsets was associated with patient survival. Patients with increased T cells with early differentiation stage tended to have a significantly better survival than patients with increased T-cells of late differentiation stage. Specifically, CD57 + T FH cells, with a late-stage differentiation phenotype, were significantly more abundant in FL patients who had early disease progression and therefore correlated with an inferior survival. Single cell analysis (CITE-seq) revealed that CD57 + T FH cells exhibited a substantially different transcriptome from CD57 - T FH cells with upregulation of inflammatory pathways, evidence of immune exhaustion and susceptibility to apoptosis. Taken together, our results show that different tumor microenvironments among FL patients are associated with variable T-cell phenotypes and an increased prevalence of CD57 + T FH cells is associated with poor patient survival., (© 2023. Springer Nature Limited.)

Senescence drives organismal aging, yet the deep characterization of senescent cells in vivo remains incomplete. Here, we apply mass cytometry by time-of-flight using carefully validated antibodies to analyze senescent cells at single-cell resolution. We use multiple criteria to identify senescent mesenchymal cells that are growth-arrested and resistant to apoptosis. These p16 + Ki67-BCL-2+ cells are highly enriched for senescence-associated secretory phenotype and DNA damage markers, are strongly associated with age, and their percentages are increased in late osteoblasts/osteocytes and CD24 high osteolineage cells. Moreover, both late osteoblasts/osteocytes and CD24 high osteolineage cells are robustly cleared by genetic and pharmacologic senolytic therapies in aged mice. Following isolation, CD24+ skeletal cells exhibit growth arrest, senescence-associated β-galactosidase positivity, and impaired osteogenesis in vitro. These studies thus provide an approach using multiplexed protein profiling to define senescent mesenchymal cells in vivo and identify specific skeletal cell populations cleared by senolytics., (© 2023. The Author(s).)

The intrinsic and acquired resistance to PD-1/PD-L1 immune checkpoint blockade is an important challenge for patients and clinicians because no reliable tool has been developed to predict individualized response to immunotherapy. In this study, we demonstrate the translational relevance of an ex vivo functional assay that measures the tumor cell killing ability of patient-derived CD8 T and NK cells (referred to as "cytotoxic lymphocytes," or CLs) isolated from the peripheral blood of patients with renal cell carcinoma. Patient-derived PBMCs were isolated before and after nephrectomy from patients with renal cell carcinoma. We compared the efficacy of U.S. Food and Drug Administration (FDA)-approved PD-1/PD-L1 inhibitors (pembrolizumab, nivolumab, atezolizumab) and a newly developed PD-L1 inhibitor (H1A Ab) in eliciting cytotoxic function. CL activity was improved at 3 mo after radical nephrectomy compared with baseline, and it was associated with higher circulating levels of tumor-reactive effector CD8 T cells (CD11ahighCX3CR1+GZMB+). Treatment of PBMCs with FDA-approved PD-1/PD-L1 inhibitors enhanced tumor cell killing activity of CLs, but a differential response was observed at the individual-patient level. H1A demonstrated superior efficacy in promoting CL activity compared with FDA-approved PD-1/PD-L1 inhibitors. PBMC immunophenotyping by mass cytometry revealed enrichment of effector CD8 T and NK cells in H1A-treated PBMCs and immunosuppressive regulatory T cells in atezolizumab-treated samples. Our study lays the ground for future investigation of the therapeutic value of H1A as a next-generation immune checkpoint inhibitor and the potential of measuring CTL activity in PBMCs as a tool to predict individual response to immune checkpoint inhibitors in patients with advanced renal cell carcinoma., (Copyright © 2023 by The American Association of Immunologists, Inc.)

Background: NASH is the progressive form of NAFLD characterized by lipotoxicity, hepatocyte injury, tissue inflammation, and fibrosis. Previously, Rho-associated protein kinase (ROCK) 1 has been implicated in lipotoxic signaling in hepatocytes in vitro and high-fat diet-induced lipogenesis in vivo. However, whether ROCK1 plays a role in liver inflammation and fibrosis during NASH is unclear. Here, we hypothesized that pathogenic activation of ROCK1 promotes murine NASH pathogenesis., Methods and Results: Patients with NASH had increased hepatic ROCK1 expression compared with patients with fatty liver. Similarly, hepatic ROCK1 levels and activity were increased in mice with NASH induced by a western-like diet that is high in fat, fructose, and cholesterol (FFC). Hepatocyte-specific ROCK1 knockout mice on the FFC diet displayed a decrease in liver steatosis, hepatic cell death, liver inflammation, and fibrosis compared with littermate FFC-fed controls. Mechanistically, these effects were associated with a significant attenuation of myeloid cell recruitment. Interestingly, myeloid cell-specific ROCK1 deletion did not affect NASH development in FFC-fed mice. To explore the therapeutic opportunities, mice with established NASH received ROCKi, a novel small molecule kinase inhibitor of ROCK1/2, which preferentially accumulates in liver tissue. ROCK inhibitor treatment ameliorated insulin resistance and decreased liver injury, inflammation, and fibrosis., Conclusions: Genetic or pharmacologic inhibition of ROCK1 activity attenuates murine NASH, suggesting that ROCK1 may be a therapeutic target for treating human NASH., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Association for the Study of Liver Diseases.)

Total joint arthroplasty (TJA) implants are composed of metal components. Although they are regarded safe, the long-term immunological effects of chronic exposure to the specific implant materials are unknown. We recruited 115 hip and/or knee TJA patients (mean age 68 years) who provided a blood draw for measurement of chromium, cobalt, titanium concentrations, inflammatory markers and systemic distribution of immune cells. We examined differences between the immune markers and the systemic concentrations of chromium, cobalt and titanium. CD66-b neutrophils, early natural killer cells (NK), and eosinophils were present in higher percentages in patients with chromium and cobalt concentrations greater than the median. The opposite pattern was observed with titanium where the percentages of CD66-b neutrophils, early NK, and eosinophils were higher in patients with undetectable titanium. Cobalt concentrations were positively correlated with a higher percentage of gamma delta T cells. Both chromium and cobalt concentrations were positively correlated with higher percentages of plasmablasts. Titanium concentrations were positively correlated with higher CD4 effector memory T cells, regulatory T cell count and Th1 CD4 helper cells. In this exploratory study, we observed altered distribution of immune cells in TJA patients with elevated systemic metal concentrations. Although these correlations were not strong, these exploratory findings warrant further investigation into the role of increased metals circulating in blood and its role in immune modulation., Competing Interests: MV received funding in the past from F. Hoffmann-La Roche Ltd. and Biogen and consulted for F. Hoffmann-La Roche Ltd., outside of this study. MV has equity ownership in Abbott Laboratories, Johnson and Johnson, Medtronic, Merck, AbbVie and Amgen and is receiving research funding from NIH. KP has received consulting fees from Akna Health. DL has received research funding from NIH; Royalties and Consulting Fees from Zimmer and Biomet; serves on the American Joint Replacement Registry and Orthopaedic Research and Education Foundation; has stock in Acuitive Technologies and Ketai Medical Devices. HMK reported receiving research funding from NIH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Peterson, O’Byrne, Brennan, Jannetto, Pavelko, Lewallen, Vassilaki and Maradit Kremers.)

Seven different anti-PD-1 and PD-L1 mAbs are now widely used in the United States to treat a variety of cancer types, but no clinical trials have compared them directly. Furthermore, because many of these Abs do not cross-react between mouse and human proteins, no preclinical models exist in which to consider these types of questions. Thus, we produced humanized PD-1 and PD-L1 mice in which the extracellular domains of both mouse PD-1 and PD-L1 were replaced with the corresponding human sequences. Using this new model, we sought to compare the strength of the immune response generated by Food and Drug Administration-approved Abs. To do this, we performed an in vivo T cell priming assay in which anti-PD-1/L1 therapies were given at the time of T cell priming against surrogate tumor Ag (OVA), followed by subsequent B16-OVA tumor challenge. Surprisingly, both control and Ab-treated mice formed an equally robust OVA-specific T cell response at the time of priming. Despite this, anti-PD-1/L1-treated mice exhibited significantly better tumor rejection versus controls, with avelumab generating the best protection. To determine what could be mediating this, we identified the increased production of CX3CR1+PD-1+CD8+ cytotoxic T cells in the avelumab-treated mice, the same phenotype of effector T cells known to increase in clinical responders to PD-1/L1 therapy. Thus, our model permits the direct comparison of Food and Drug Administration-approved anti-PD-1/L1 mAbs and further correlates successful tumor rejection with the level of CX3CR1+PD-1+CD8 + T cells, making this model a critical tool for optimizing and better utilizing anti-PD-1/L1 therapeutics., (Copyright © 2023 The Authors.)

Introduction: Immune checkpoint inhibitors (ICIs) induce impressive antitumor responses but may lead to acute kidney injury (AKI) associated with ICI therapy (AKI-ICI). Biomarkers distinguishing AKI-ICI from AKI because of other causes (AKI-other) are currently lacking. Because ICIs block immunoregulatory pathways, we hypothesized that biomarkers related to immune cell dysregulation, including tumor necrosis factor alpha (TNF-α) and other markers of B and T cell activation in the systemic circulation and kidney tissue, may aid with the diagnosis of AKI-ICI., Methods: This is a prospective study consisting of 24 participants who presented with AKI during ICI therapy, adjudicated to either have AKI-ICI ( n  = 14) or AKI-other ( n  = 10). We compared markers of kidney inflammation and injury (neutrophil gelatinase-associated lipocalin, kidney injury molecule-1) as well as plasma and urine levels of T cell-associated cytokines (TNF-α, interferon-γ, interleukin (IL)-2, IL-4, IL-6, IL-8, IL-9, and IL-10) between groups. We also compared T-cell responses in the systemic circulation and in kidney tissue across groups, using mass cytometry systems., Results: We observed increase in several specific immune cells, including CD4 memory, T helper cells, and dendritic cells in the kidney tissue, as well as in the urine cytokines IL-2, IL-10, and TNF-α, in patients who developed AKI-ICI compared to patients with AKI-other ( P  < 0.05 for all). The discriminatory ability of TNF-α on AKI cause was strong (area under the curve = 0.814, 95% confidence interval: 0.623-1.00. The CD4+ T cells with memory phenotype formed the dominant subset., Conclusion: These results suggest that specific T-cell responses and their respective cytokines may be indicative of AKI associated with ICI therapy and may help to differentiate AKI-ICI from AKI-other. Urine TNF-α is a promising biomarker for AKI-ICI, which is most often caused by acute interstitial nephritis (AIN), and TNF-α pathway may serve as a potential target for therapeutic intervention., (© 2022 Published by Elsevier Inc. on behalf of the International Society of Nephrology.)

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., (© 2022. The Author(s).)

Neurotropic viruses target the brain and contribute to neurologic diseases. C-type lectin receptors (CLRs) are pattern recognition receptors that recognize carbohydrate structures on endogenous molecules and pathogens. The myeloid CLR dendritic cell immunoreceptor (DCIR) is expressed by antigen presenting cells and mediates inhibitory intracellular signalling. To investigate the effect of DCIR on neurotropic virus infection, mice were infected experimentally with Theiler's murine encephalomyelitis virus (TMEV). Brain tissue of TMEV-infected C57BL/6 mice and DCIR -/- mice were analysed by histology, immunohistochemistry and RT-qPCR, and spleen tissue by flow cytometry. To determine the impact of DCIR deficiency on T cell responses upon TMEV infection in vitro, antigen presentation assays were utilised. Genetic DCIR ablation in C57BL/6 mice was associated with an ameliorated hippocampal integrity together with reduced cerebral cytokine responses and reduced TMEV loads in the brain. Additionally, absence of DCIR favoured increased peripheral cytotoxic CD8 + T cell responses following TMEV infection. Co-culture experiments revealed that DCIR deficiency enhances the activation of antigen-specific CD8 + T cells by virus-exposed dendritic cells (DCs), indicated by increased release of interleukin-2 and interferon-γ. Results suggest that DCIR deficiency has a supportive influence on antiviral immune mechanisms, facilitating virus control in the brain and ameliorates neuropathology during acute neurotropic virus infection., (© 2021. The Author(s).)

Objective: To compare coronavirus disease 2019 (COVID-19) acute kidney injury (AKI) to sepsis-AKI (S-AKI). The morphology and transcriptomic and proteomic characteristics of autopsy kidneys were analyzed., Patients and Methods: Individuals 18 years of age and older who died from COVID-19 and had an autopsy performed at Mayo Clinic between April 2020 to October 2020 were included. Morphological evaluation of the kidneys of 17 individuals with COVID-19 was performed. In a subset of seven COVID-19 cases with postmortem interval of less than or equal to 20 hours, ultrastructural and molecular characteristics (targeted transcriptome and proteomics analyses of tubulointerstitium) were evaluated. Molecular characteristics were compared with archived cases of S-AKI and nonsepsis causes of AKI., Results: The spectrum of COVID-19 renal pathology included macrophage-dominant microvascular inflammation (glomerulitis and peritubular capillaritis), vascular dysfunction (peritubular capillary congestion and endothelial injury), and tubular injury with ultrastructural evidence of mitochondrial damage. Investigation of the spatial architecture using a novel imaging mass cytometry revealed enrichment of CD3 + CD4 + T cells in close proximity to antigen-presenting cells, and macrophage-enriched glomerular and interstitial infiltrates, suggesting an innate and adaptive immune tissue response. Coronavirus disease 2019 AKI and S-AKI, as compared to nonseptic AKI, had an enrichment of transcriptional pathways involved in inflammation (apoptosis, autophagy, major histocompatibility complex class I and II, and type 1 T helper cell differentiation). Proteomic pathway analysis showed that COVID-19 AKI and to a lesser extent S-AKI were enriched in necroptosis and sirtuin-signaling pathways, both involved in regulatory response to inflammation. Upregulation of the ceramide-signaling pathway and downregulation of oxidative phosphorylation in COVID-19 AKI were noted., Conclusion: This data highlights the similarities between S-AKI and COVID-19 AKI and suggests that mitochondrial dysfunction may play a pivotal role in COVID-19 AKI. This data may allow the development of novel diagnostic and therapeutic targets., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The transcription factor TCF-1 is essential for the development and function of regulatory T (T reg ) cells; however, its function is poorly understood. Here, we show that TCF-1 primarily suppresses transcription of genes that are co-bound by Foxp3. Single-cell RNA-sequencing analysis identified effector memory T cells and central memory T reg cells with differential expression of Klf2 and memory and activation markers. TCF-1 deficiency did not change the core T reg cell transcriptional signature, but promoted alternative signaling pathways whereby T reg cells became activated and gained gut-homing properties and characteristics of the T H 17 subset of helper T cells. TCF-1-deficient T reg cells strongly suppressed T cell proliferation and cytotoxicity, but were compromised in controlling CD4 + T cell polarization and inflammation. In mice with polyposis, T reg cell-specific TCF-1 deficiency promoted tumor growth. Consistently, tumor-infiltrating T reg cells of patients with colorectal cancer showed lower TCF-1 expression and increased T H 17 expression signatures compared to adjacent normal tissue and circulating T cells. Thus, T reg cell-specific TCF-1 expression differentially regulates T H 17-mediated inflammation and T cell cytotoxicity, and can determine colorectal cancer outcome., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Neurotropic viruses target the brain and contribute to neurologic diseases. Caspase recruitment domain containing family member 9 (CARD9) controls protective immunity in a variety of infectious disorders. To investigate the effect of CARD9 in neurotropic virus infection, CARD9 -/- and corresponding C57BL/6 wild-type control mice were infected with Theiler's murine encephalomyelitis virus (TMEV). Brain tissue was analyzed by histology, immunohistochemistry and molecular analyses, and spleens by flow cytometry. To determine the impact of CARD9 deficiency on T cell responses in vitro, antigen presentation assays were utilized. Genetic ablation of CARD9 enhanced early pro-inflammatory cytokine responses and accelerated infiltration of T and B cells in the brain, together with a transient increase in TMEV-infected cells in the hippocampus. CARD9 -/- mice showed an increased loss of neuronal nuclear protein + mature neurons and doublecortin + neuronal precursor cells and an increase in β-amyloid precursor protein + damaged axons in the hippocampus. No effect of CARD9 deficiency was found on the initiation of CD8 + T cell responses by flow cytometry and co-culture experiments using virus-exposed dendritic cells or microglia-enriched glial cell mixtures, respectively. The present study indicates that CARD9 is dispensable for the initiation of early antiviral responses and TMEV elimination but may contribute to the modulation of neuroinflammation, thereby reducing hippocampal injury following neurotropic virus infection.

Cellular senescence is characterized by an irreversible cell cycle arrest as well as a pro-inflammatory phenotype, thought to contribute to aging and age-related diseases. Neutrophils have essential roles in inflammatory responses; however, in certain contexts their abundance is associated with a number of age-related diseases, including liver disease. The relationship between neutrophils and cellular senescence is not well understood. Here, we show that telomeres in non-immune cells are highly susceptible to oxidative damage caused by neighboring neutrophils. Neutrophils cause telomere dysfunction both in vitro and ex vivo in a ROS-dependent manner. In a mouse model of acute liver injury, depletion of neutrophils reduces telomere dysfunction and senescence. Finally, we show that senescent cells mediate the recruitment of neutrophils to the aged liver and propose that this may be a mechanism by which senescence spreads to surrounding cells. Our results suggest that interventions that counteract neutrophil-induced senescence may be beneficial during aging and age-related disease., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

Monocyte homing to the liver and adhesion to the liver sinusoidal endothelial cells (LSECs) are key elements in nonalcoholic steatohepatitis (NASH) pathogenesis. We reported previously that VCAM-1 mediates monocyte adhesion to LSECs. However, the pathogenic role of VCAM-1 in NASH is unclear. Herein, we report that VCAM-1 was a top upregulated adhesion molecule in the NASH mouse liver transcriptome. Open chromatin landscape profiling combined with genome-wide transcriptome analysis showed robust transcriptional upregulation of LSEC VCAM-1 in murine NASH. Moreover, LSEC VCAM-1 expression was significantly increased in human NASH. LSEC VCAM-1 expression was upregulated by palmitate treatment in vitro and reduced with inhibition of the mitogen-activated protein 3 kinase (MAP3K) mixed lineage kinase 3 (MLK3). Likewise, LSEC VCAM-1 expression was reduced in the Mlk3-/- mice with diet-induced NASH. Furthermore, VCAM-1 neutralizing Ab or pharmacological inhibition attenuated diet-induced NASH in mice, mainly via reducing the proinflammatory monocyte hepatic population as examined by mass cytometry by time of flight (CyTOF). Moreover, endothelium-specific Vcam1 knockout mice were also protected against NASH. In summary, lipotoxic stress enhances the expression of LSEC VCAM-1, in part, through MLK3 signaling. Inhibition of VCAM-1 was salutary in murine NASH and might serve as a potential therapeutic strategy for human NASH.

Importance: Recognizing the presenting and immunopathological features of Kelch-like protein-11 immunoglobulin G seropositive (KLHL11 IgG+) patients may aid in early diagnosis and management., Objective: To describe expanding neurologic phenotype, cancer associations, outcomes, and immunopathologic features of KLHL11 encephalitis., Design, Setting, and Participants: This retrospective tertiary care center study, conducted from October 15, 1998, to November 1, 2019, prospectively identified 31 KLHL11 IgG+ cases in the neuroimmunology laboratory. Eight were identified by retrospective testing of patients with rhomboencephalitis (confirmed by tissue-based-immunofluorescence and transfected-cell-based assays)., Main Outcomes and Measures: Outcome variables included modified Rankin score and gait aid use., Results: All 39 KLHL11 IgG+ patients were men (median age, 46 years; range, 28-73 years). Initial clinical presentations were ataxia (n = 32; 82%), diplopia (n = 22; 56%), vertigo (n = 21; 54%), hearing loss (n = 15; 39%), tinnitus (n = 14; 36%), dysarthria (n = 11; 28%), and seizures (n = 9; 23%). Atypical neurologic presentations included neuropsychiatric dysfunction, myeloneuropathy, and cervical amyotrophy. Hearing loss or tinnitus preceded other neurologic deficits by 1 to 8 months in 10 patients (26%). Among patients screened for malignancy (n = 36), testicular germ-cell tumors (n = 23; 64%) or testicular microlithiasis and fibrosis concerning for regressed germ cell tumor (n = 7; 19%) were found in 83% of the patients (n = 30). In 2 patients, lymph node biopsy diagnosed metastatic lung adenocarcinoma in one and chronic lymphocytic leukemia in the other. Initial brain magnetic resonance imaging revealed T2 hyperintensities in the temporal lobe (n = 12), cerebellum (n = 9), brainstem (n = 3), or diencephalon (n = 3). Among KLHL11 IgG+ patients who underwent HLA class I and class II genotyping (n = 10), most were found to have HLA-DQB1*02:01 (n = 7; 70%) and HLA-DRB1*03:01 (n = 6; 60%) associations. A biopsied gadolinium-enhancing temporal lobe lesion demonstrated T cell-predominant inflammation and nonnecrotizing granulomas. Cerebellar biopsy (patient with chronic ataxia) and 2 autopsied brains demonstrated Purkinje neuronal loss and Bergmann gliosis, supporting early active inflammation and later extensive neuronal loss. Compared with nonautoimmune control peripheral blood mononuclear cells, cluster of differentiation (CD) 8+ and CD4+ T cells were significantly activated when patient peripheral blood mononuclear cells were cultured with KLHL11 protein. Most patients (58%) benefitted from immunotherapy and/or cancer treatment (neurological disability stabilized [n = 10] or improved [n = 9]). Kaplan-Meier curve demonstrated significantly higher probability of wheelchair dependence among patients without detectable testicular cancer. Long-term outcomes in KLHL11-IgG+ patients were similar to Ma2 encephalitis., Conclusions and Relevance: Kelch-like protein-11 IgG is a biomarker of testicular germ-cell tumor and paraneoplastic neurologic syndrome, often refractory to treatment. Described expanded neurologic phenotype and paraclinical findings may aid in its early diagnosis and treatment.

In ovarian cancer (OC), IL-17-producing T cells (Th17s) predict improved survival, whereas regulatory T cells predict poorer survival. We previously developed a vaccine whereby patient-derived dendritic cells (DCs) are programmed to induce Th17 responses to the OC antigen folate receptor alpha (FRα). Here we report the results of a single-arm open-label phase I clinical trial designed to determine vaccine safety and tolerability (primary outcomes) and recurrence-free survival (secondary outcome). Immunogenicity is also evaluated. Recruitment is complete with a total of 19 Stage IIIC-IV OC patients in first remission after conventional therapy. DCs are generated using our Th17-inducing protocol and are pulsed with HLA class II epitopes from FRα. Mature antigen-loaded DCs are injected intradermally. All patients have completed study-related interventions. No grade 3 or higher adverse events are seen. Vaccination results in the development of Th1, Th17, and antibody responses to FRα in the majority of patients. Th1 and antibody responses are associated with prolonged recurrence-free survival. Antibody-dependent cell-mediated cytotoxic activity against FRα is also associated with prolonged RFS. Of 18 patients evaluable for efficacy, 39% (7/18) remain recurrence-free at the time of data censoring, with a median follow-up of 49.2 months. Thus, vaccination with Th17-inducing FRα-loaded DCs is safe, induces antigen-specific immunity, and is associated with prolonged remission.

Background & Aims: Endoplasmic reticulum to nucleus signaling 1 (ERN1, also called IRE1A) is a sensor of the unfolded protein response that is activated in the livers of patients with nonalcoholic steatohepatitis (NASH). Hepatocytes release ceramide-enriched inflammatory extracellular vesicles (EVs) after activation of IRE1A. We studied the effects of inhibiting IRE1A on release of inflammatory EVs in mice with diet-induced steatohepatitis., Methods: C57BL/6J mice and mice with hepatocyte-specific disruption of Ire1a (IRE1α Δhep ) were fed a diet high in fat, fructose, and cholesterol to induce development of steatohepatitis or a standard chow diet (controls). Some mice were given intraperitoneal injections of the IRE1A inhibitor 4μ8C. Mouse liver and primary hepatocytes were transduced with adenovirus or adeno-associated virus that expressed IRE1A. Livers were collected from mice and analyzed by quantitative polymerase chain reaction and chromatin immunoprecipitation assays; plasma samples were analyzed by enzyme-linked immunosorbent assay. EVs were derived from hepatocytes and injected intravenously into mice. Plasma EVs were characterized by nanoparticle-tracking analysis, electron microscopy, immunoblots, and nanoscale flow cytometry; we used a membrane-tagged reporter mouse to detect hepatocyte-derived EVs. Plasma and liver tissues from patients with NASH and without NASH (controls) were analyzed for EV concentration and by RNAscope and gene expression analyses., Results: Disruption of Ire1a in hepatocytes or inhibition of IRE1A reduced the release of EVs and liver injury, inflammation, and accumulation of macrophages in mice on the diet high in fat, fructose, and cholesterol. Activation of IRE1A, in the livers of mice, stimulated release of hepatocyte-derived EVs, and also from cultured primary hepatocytes. Mice given intravenous injections of IRE1A-stimulated, hepatocyte-derived EVs accumulated monocyte-derived macrophages in the liver. IRE1A-stimulated EVs were enriched in ceramides. Chromatin immunoprecipitation showed that IRE1A activated X-box binding protein 1 (XBP1) to increase transcription of serine palmitoyltransferase genes, which encode the rate-limiting enzyme for ceramide biosynthesis. Administration of a pharmacologic inhibitor of serine palmitoyltransferase to mice reduced the release of EVs. Levels of XBP1 and serine palmitoyltransferase were increased in liver tissues, and numbers of EVs were increased in plasma, from patients with NASH compared with control samples and correlated with the histologic features of inflammation., Conclusions: In mouse hepatocytes, activated IRE1A promotes transcription of serine palmitoyltransferase genes via XBP1, resulting in ceramide biosynthesis and release of EVs. The EVs recruit monocyte-derived macrophages to the liver, resulting in inflammation and injury in mice with diet-induced steatohepatitis. Levels of XBP1, serine palmitoyltransferase, and EVs are all increased in liver tissues from patients with NASH. Strategies to block this pathway might be developed to reduce liver inflammation in patients with NASH., (Copyright © 2020 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Immune checkpoint blockade (ICB) has revolutionized cancer therapeutics. Desmoplastic malignancies, such as cholangiocarcinoma (CCA), have an abundant tumor immune microenvironment (TIME). However, to date, ICB monotherapy in such malignancies has been ineffective. Herein, we identify tumor-associated macrophages (TAMs) as the primary source of programmed death-ligand 1 (PD-L1) in human and murine CCA. In a murine model of CCA, recruited PD-L1+ TAMs facilitated CCA progression. However, TAM blockade failed to decrease tumor progression due to a compensatory emergence of granulocytic myeloid-derived suppressor cells (G-MDSCs) that mediated immune escape by impairing T cell response. Single-cell RNA sequencing (scRNA-Seq) of murine tumor G-MDSCs highlighted a unique ApoE G-MDSC subset enriched with TAM blockade; further analysis of a human scRNA-Seq data set demonstrated the presence of a similar G-MDSC subset in human CCA. Finally, dual inhibition of TAMs and G-MDSCs potentiated ICB. In summary, our findings highlight the therapeutic potential of coupling ICB with immunotherapies targeting immunosuppressive myeloid cells in CCA.

Theiler's murine encephalomyelitis virus (TMEV) infection of the CNS is cleared in C57BL/6 mice by a CD8 T cell response restricted by the MHC class I molecule H-2D b The identity and function of the APC(s) involved in the priming of this T cell response is (are) poorly defined. To address this gap in knowledge, we developed an H-2D b LoxP-transgenic mouse system using otherwise MHC class I-deficient C57BL/6 mice, thereby conditionally ablating MHC class I-restricted Ag presentation in targeted APC subpopulations. We observed that CD11c + APCs are critical for early priming of CD8 T cells against the immunodominant TMEV peptide VP2 121-130 Loss of H-2D b on CD11c + APCs mitigates the CD8 T cell response, preventing early viral clearance and immunopathology associated with CD8 T cell activity in the CNS. In contrast, animals with H-2D b -deficient LysM + APCs retained early priming of D b :VP2 121-130 epitope-specific CD8 T cells, although a modest reduction in immune cell entry into the CNS was observed. This work establishes a model enabling the critical dissection of H-2D b -restricted Ag presentation to CD8 T cells, revealing cell-specific and temporal features involved in the generation of CD8 T cell responses. Employing this novel system, we establish CD11c + cells as pivotal to the establishment of acute antiviral CD8 T cell responses against the TMEV immunodominant epitope VP2 121-130 , with functional implications both for T cell-mediated viral control and immunopathology., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Background & Aims: Hepatic recruitment of monocyte-derived macrophages (MoMFs) contributes to the inflammatory response in non-alcoholic steatohepatitis (NASH). However, how hepatocyte lipotoxicity promotes MoMF inflammation is unclear. Here we demonstrate that lipotoxic hepatocyte-derived extracellular vesicles (LPC-EVs) are enriched with active integrin β 1 (ITGβ 1 ), which promotes monocyte adhesion and liver inflammation in murine NASH., Methods: Hepatocytes were treated with either vehicle or the toxic lipid mediator lysophosphatidylcholine (LPC); EVs were isolated from the conditioned media and subjected to proteomic analysis. C57BL/6J mice were fed a diet rich in fat, fructose, and cholesterol (FFC) to induce NASH. Mice were treated with anti-ITGβ 1 neutralizing antibody (ITGβ 1 Ab) or control IgG isotype., Results: Ingenuity® Pathway Analysis of the LPC-EV proteome indicated that ITG signaling is an overrepresented canonical pathway. Immunogold electron microscopy and nanoscale flow cytometry confirmed that LPC-EVs were enriched with activated ITGβ 1 . Furthermore, we showed that LPC treatment in hepatocytes activates ITGβ 1 and mediates its endocytic trafficking and sorting into EVs. LPC-EVs enhanced monocyte adhesion to liver sinusoidal cells, as observed by shear stress adhesion assay. This adhesion was attenuated in the presence of ITGβ 1 Ab. FFC-fed, ITGβ 1 Ab-treated mice displayed reduced inflammation, defined by decreased hepatic infiltration and activation of proinflammatory MoMFs, as assessed by immunohistochemistry, mRNA expression, and flow cytometry. Likewise, mass cytometry by time-of-flight on intrahepatic leukocytes showed that ITGβ 1 Ab reduced levels of infiltrating proinflammatory monocytes. Furthermore, ITGβ 1 Ab treatment significantly ameliorated liver injury and fibrosis., Conclusions: Lipotoxic EVs mediate monocyte adhesion to LSECs mainly through an ITGβ 1 -dependent mechanism. ITGβ 1 Ab ameliorates diet-induced NASH in mice by reducing MoMF-driven inflammation, suggesting that blocking ITGβ 1 is a potential anti-inflammatory therapeutic strategy in human NASH., Lay Summary: Herein, we report that a cell adhesion molecule termed integrin β 1 (ITGβ 1 ) plays a key role in the progression of non-alcoholic steatohepatitis (NASH). ITGβ 1 is released from hepatocytes under lipotoxic stress as a cargo of extracellular vesicles, and mediates monocyte adhesion to liver sinusoidal endothelial cells, which is an essential step in hepatic inflammation. In a mouse model of NASH, blocking ITGβ 1 reduces liver inflammation, injury and fibrosis. Hence, ITGβ 1 inhibition may serve as a new therapeutic strategy for NASH., (Copyright © 2019 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Successful efforts to activate T cells capable of recognizing weak cancer-associated self-antigens have employed altered peptide antigens to activate T cell responses capable of cross-reacting on native tumor-associated self. A limitation of this approach is the requirement for detailed knowledge about the altered self-peptide ligands used in these vaccines. In the current study we considered allorecognition as an approach for activating CTL capable of recognizing weak or self-antigens in the context of self-MHC. Nonself antigen-presenting molecules typically contain polymorphisms that influence interactions with the bound peptide and TCR interface. Recognition of these nonself structures results in peptide-dependent alloimmunity. Alloreactive T cells target their inducing alloantigens as well as third-party alloantigens but generally fail to target self-antigens. Certain residues located on the alpha-1/2 domains of class I antigen-presenting molecules primarily interface with TCR. These residues are more conserved within and across species than are residues that determine peptide antigen binding properties. Class I variants designed with amino acid substitutions at key positions within the conserved helical structures are shown to provide strong activating signals to alloreactive CD8 T cells while avoiding changes in naturally bound peptide ligands. Importantly, CTL activated in this manner can break self-tolerance by reacting to self-peptides presented by native MHC. The ability to activate self-tolerant T cells capable of cross-reacting on self-peptide-MHC in vivo represents an approach for inducing autoimmunity, with possible application in cancer vaccines., Competing Interests: Conflict of interest statement: The authors have a pending US patent application, US20160361402A1, relevant to the delivery of MHC class I heavy chain mutant genes using adenoviral transduction in humans.

During αβ T cell development, T cell antigen receptor (TCR) engagement transduces biochemical signals through a protein-protein interaction (PPI) network that dictates dichotomous cell fate decisions. It remains unclear how signal specificity is communicated, instructing either positive selection to advance cell differentiation or death by negative selection. Early signal discrimination might occur by PPI signatures differing qualitatively (customized, unique PPI combinations for each signal), quantitatively (graded amounts of a single PPI series), or kinetically (speed of PPI pathway progression). Using a novel PPI network analysis, we found that early TCR-proximal signals distinguishing positive from negative selection appeared to be primarily quantitative in nature. Furthermore, the signal intensity of this PPI network was used to find an antigen dose that caused a classic negative selection ligand to induce positive selection of conventional αβ T cells, suggesting that the quantity of TCR triggering was sufficient to program selection outcome. Because previous work had suggested that positive selection might involve a qualitatively unique signal through CD3δ, we reexamined the block in positive selection observed in CD3δ 0 mice. We found that CD3δ 0 thymocytes were inhibited but capable of signaling positive selection, generating low numbers of MHC-dependent αβ T cells that expressed diverse TCR repertoires and participated in immune responses against infection. We conclude that the major role for CD3δ in positive selection is to quantitatively boost the signal for maximal generation of αβ T cells. Together, these data indicate that a quantitative network signaling mechanism through the early proximal TCR signalosome determines thymic selection outcome., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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.

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-2K b 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.

Therapies that target the PD-1/B7-H1 axis have revolutionized cancer treatment, yet precise knowledge of how this pathway provides benefit continues to evolve. Here, we report a novel role for the immune checkpoint ligand B7-H1 in the accumulation of tissue-resident memory CD8 + T-cells (T RM ). After intracranial infection, Theiler's murine encephalomyelitis virus (TMEV) generates T RM that are maintained in the central nervous system (CNS) tissues of B7-H1 WT animals. Although no differences in acute T-cell responses between B7-H1 WT and B7-H1 KO are observed, at long-term periods post-infection the maintenance of CD8 + T RM is diminished in B7-H1 KO animals. This is accompanied by redistribution of the resident CD8 + population from primarily CD103 + T RM to a diminished population of T RM and a preponderance of non-specified PD-1 + CD103 - CD8 + T-cells. T-cell transfer studies demonstrate that host B7-H1 is necessary for maintaining T RM and limiting accumulation of PD-1 + CD103 - CD8 + T-cells. The lack of host B7-H1 results in compromised control of a heterologous virus re-challenge demonstrating a functional defect in T RM mediated virus control. This study reveals a new role for B7-H1 in T RM and pro-inflammatory PD-1 + CD103 - CD8 + T-cell accumulation in the CNS and gives insight for using B7-H1/PD-1 blockade in modulating long-term T-cell protection.

Brain atrophy is a common feature of numerous neurologic diseases in which the role of neuroinflammation remains ill-defined. In this study, we evaluated the contribution of major histocompatibility complex class I molecules to brain atrophy in Theiler's murine encephalomyelitis virus (TMEV)-infected transgenic FVB mice that express the D b class I molecule. FVB/D b and wild-type FVB mice were evaluated for changes in neuroinflammation, virus clearance, neuropathology, and development of brain atrophy via T2-weighted MRI and subsequent 3-dimensional volumetric analysis. Significant brain atrophy and hippocampal neuronal loss were observed in TMEV-infected FVB/D b mice, but not in wild-type FVB mice. Brain atrophy was observed at 1 mo postinfection and persisted through the 4-mo observation period. Of importance, virus-infected FVB/D b mice elicited a strong CD8 T-cell response toward the immunodominant D b -restricted TMEV-derived peptide, VP2 121-130 , and cleared TMEV from the CNS. In addition, immunofluorescence revealed CD8 T cells near virus-infected neurons; therefore, we hypothesize that class I restricted CD8 T-cell responses promote development of brain atrophy. This model provides an opportunity to analyze the contribution of immune cells to brain atrophy in a system where persistent virus infection and demyelination are not factors in long-term neuropathology.-Huseby Kelcher, A. M., Atanga, P. A., Gamez, J. D., Cumba Garcia, L. M., Teclaw, S. J., Pavelko, K. D., Macura, S. I., Johnson. A. J. Brain atrophy in picornavirus-infected FVB mice is dependent on the H-2D b class I molecule., (© FASEB.)

Background: CD8 T cell-mediated blood-brain barrier (BBB) disruption is dependent on the effector molecule perforin. Human perforin has extensive single nucleotide variants (SNVs), the significance of which is not fully understood. These SNVs can result in reduced, but not ablated, perforin activity or expression. However, complete loss of perforin expression or activity results in the lethal disease familial hemophagocytic lymphohistiocytosis type 2 (FHL 2). In this study, we address the hypothesis that a single perforin allele can alter the severity of BBB disruption in vivo using a well-established model of CNS vascular permeability in C57Bl/6 mice. The results of this study provide insight into the significance of perforin SNVs in the human population., Methods: We isolated the effect a single perforin allele has on CNS vascular permeability through the use of perforin-heterozygous (perforin+/-) C57BL/6 mice in the peptide-induced fatal syndrome (PIFS) model of immune-mediated BBB disruption. Seven days following Theiler's murine encephalomyelitis virus (TMEV) CNS infection, neuroinflammation and TMEV viral control were assessed through flow cytometric analysis and quantitative real-time PCR of the viral genome, respectively. Following immune-mediated BBB disruption, gadolinium-enhanced T1-weighted MRI, with 3D volumetric analysis, and confocal microscopy were used to define CNS vascular permeability. Finally, the open field behavior test was used to assess locomotor activity of mice following immune-mediated BBB disruption., Results: Perforin-null mice had negligible CNS vascular permeability. Perforin-WT mice have extensive CNS vascular permeability. Interestingly, perforin-heterozygous mice had an intermediate level of CNS vascular permeability as measured by both gadolinium-enhanced T1-weighted MRI and fibrinogen leakage in the brain parenchyma. Differences in BBB disruption were not a result of increased CNS immune infiltrate. Additionally, TMEV was controlled in a perforin dose-dependent manner. Furthermore, a single perforin allele is sufficient to induce locomotor deficit during immune-mediated BBB disruption., Conclusions: Perforin modulates BBB disruption in a dose-dependent manner. This study demonstrates a potentially advantageous role for decreased perforin expression in reducing BBB disruption. This study also provides insight into the effect SNVs in a single perforin allele could have on functional deficit in neurological disease.

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., Competing Interests: The authors have declared that no competing interests exist.

Picornaviruses have emerged as promising cancer therapies due to their ability to drive cytotoxic cellular immune responses and for promoting oncolysis. These properties include preferential replication in tumor cells, the induction of strong innate and adaptive immune responses, and the ease with which their genomes can be manipulated. We have developed Theiler's murine encephalomyelitis virus (TMEV) as an immunotherapy vector that promotes strong adaptive immune responses to tumor antigens embedded within its genome. To further explore its usefulness as cancer therapy, we investigated whether direct intratumoral delivery of TMEV could promote tumor regression. We generated several picornavirus hybrids using substrains of TMEV that have unique immunopathologic characteristics, despite their extensive sequence homology. These hybrids exhibit a unique propensity to infect and replicate in melanoma. We have identified GD7-KS1, a virus that is particularly effective at replicating and infecting B16 melanoma in vitro and provides benefit as an oncolytic therapy in vivo after intratumoral injection. In addition, this virus promotes the mobilization and accumulation of CD8(+) T cells within treated tumors. Altogether, these findings demonstrate that picornavirus substrains can be used to rationally design virus hybrids that promote antitumor responses and add to the known strategies identified by us and others to further enhance the therapeutic potential of vectors used to treat cancer., (©2016 American Association for Cancer Research.)

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., Competing Interests: Compliance with Ethical Standards Required Author Forms Disclosure forms provided by the authors are available with the online version of this article.

Antigen-specific T cell responses can be visualized using MHC:peptide multimers. In cases where robust T cell controls are not readily available to assess the integrity of multimer reagents prior to analyzing limited sample, the ability to assess the structural integrity of MHC multimers before their use in critical experiments would be useful. We present a method to probe the structural integrity of MHC multimers using antibodies specific for conformational determinants. Beads coated with anti-mouse Ig are incubated with conformation-specific mouse monoclonal antibody and then with fluorescently tagged MHC multimer. The ability of the bead to capture the labeled multimer can be measured semi-quantitatively by flow cytometry. In this manner, the correct folding of MHC multimers can be visualized and batches of multimer can be compared for quality control. Because there are multiple conformational epitopes formed by various molecular interactions among heavy chain, peptide, and β2M, this capture assay can assess the fidelity of each aspect of multimer structure, depending on the availability of antibodies. The described approach could be particularly useful for studies using irreplaceable samples, including patient samples collected in clinical trials.

Glioblastoma (GBM) is among the most invasive and lethal of cancers, frequently infiltrating surrounding healthy tissue and giving rise to rapid recurrence. It is therefore critical to establish experimental model systems and develop therapeutic approaches that enhance anti-tumor immunity. In the current study, we have employed a newly developed murine glioma model to assess the efficacy of a novel picornavirus vaccination approach for the treatment of established tumors. The GL261-Quad system is a variation of the GL261 syngeneic glioma that has been engineered to expresses model T cell epitopes including OVA257-264. MRI revealed that both GL261 and GL261-Quad tumors display characteristic features of human gliomas such as heterogeneous gadolinium leakage and larger T2 weighted volumes. Analysis of brain-infiltrating immune cells demonstrated that GL261-Quad gliomas generate detectable CD8+ T cell responses toward the tumor-specific Kb:OVA257-264 antigen. Enhancing this response via a single intracranial or peripheral vaccination with picornavirus expressing the OVA257-264 antigen increased anti-tumor CD8+ T cells infiltrating the brain, attenuated progression of established tumors, and extended survival of treated mice. Importantly, the efficacy of the picornavirus vaccination is dependent on functional cytotoxic activity of CD8+ T cells, as the beneficial response was completely abrogated in mice lacking perforin expression. Therefore, we have developed a novel system for evaluating mechanisms of anti-tumor immunity in vivo, incorporating the GL261-Quad model, 3D volumetric MRI, and picornavirus vaccination to enhance tumor-specific cytotoxic CD8+ T cell responses and track their effectiveness at eradicating established gliomas in vivo.

Recombinant virus vaccines are often less effective due to immunodominant responses against endogenous vector antigens. However, the use of small RNA virus vectors provides an opportunity to limit host exposure to endogenous virus antigens and focus immune responses on the desired vaccine antigen. Using the Daniel's strain of Theiler's murine encephalomyelitis virus, we have identified strategies to modulate responses to endogenous viral proteins by manipulating the host CD8+ T-cell repertoire prior to infection or through the use of mutations introduced into the virus genome. Both of these approaches enhance responses to vaccine antigens introduced into the picornavirus. However, the use of mutant immunodominant epitopes provides an opportunity for enhancing vaccine responses without further manipulation of the host. Using this strategy, we demonstrate that modification of the consensus MHC class I anchor residue within the virus genome can promote enhanced immunity to foreign antigens and self-antigens embedded in the virus genome.

The emergence of novel viral pathogens can lead to devastating consequences in the infected population. However, on occasion, rare hyper-responsive elite controllers are able to mount a protective primary response to infection and clear the new pathogen. Factors distinguishing elite controllers from other members of the population are not completely understood. We have been using Theiler's murine encephalomyelitis as a model of primary infection in mice and clearance of the virus is limited to one MHC genotype capable of generating a protective response to a single viral peptide VP2121-130. The genetics of host susceptibility to TMEV, a natural mouse pathogen, has been studied extensively and non-protective CD8 responses to other peptides have been documented, however, little is known why the protective response to infection focuses on the VP2121-130 peptide. To study this question, we have generated TMEV mutants that encode for mutations within the VP2121-130 peptide. We find that very few of mutants are able to assemble and infect in vitro. These mutations are not related to virus RNA structure since non-coding mutations do not interfere with assembly. In the rare event when functional VP2121-130 mutant viruses did emerge, they were attenuated to some level or retained the ability to develop an immune response to the wild-type VP2121-130 sequence, demonstrating that the virus is incapable of escaping the protective response. These findings advance our understanding of how characteristics of the host immune response and an infectious agent can interact to lead to the appearance of rare super controllers in a population. Furthermore, the immutable nature of the viral antigen highlights the importance of choosing appropriate vaccine antigens and has implications for the development of agents that are able to generate protective CD8 T-cell responses.

Picornaviruses have been developed as potential therapies for gene delivery and vaccination. One drawback to their use is the potential for recombination and viral persistence. Therefore, the engineering strategies used must take into account the possibility for virus escape. We have developed Theiler's murine encephalomyelitis virus (TMEV) as a potential vaccine vector for use in immunotherapy. This study shows that insertion of a vaccine epitope at a unique site within the TMEV leader protein can dramatically increase the type I interferon (IFN) response to infection and promote rapid viral clearance. This live virus vaccine maintains its ability to drive antigen-specific CD8(+) T-cell responses to a model antigen as well as to the weakly immunogenic tumor antigen Her2/neu. Furthermore, the epitope integration site does not affect the efficacy of this vaccine as cancer immunotherapy for treating models of melanoma and breast cancer as demonstrated by delayed tumor outgrowth and increased survival in animals implanted with these tumors. These findings show that an attenuated virus retaining limited ability to replicate nonetheless can effectively mobilize CD8(+) cellular immunity and will be important for the design of picornavirus vectors used as immunotherapy in clinical settings.

Background: Altered permeability of the blood-brain barrier (BBB) is a feature of numerous neurological conditions including multiple sclerosis, cerebral malaria, viral hemorrhagic fevers and acute hemorrhagic leukoencephalitis. Our laboratory has developed a murine model of CD8 T cell-initiated central nervous system (CNS) vascular permeability in which vascular endothelial growth factor (VEGF) signaling plays a prominent role in BBB disruption., Findings: In this study, we addressed the hypothesis that in vivo blockade of VEGF signal transduction through administration of peptide (ATWLPPR) to inhibit neuropilin-1 (NRP-1) would have a therapeutic effect following induction of CD8 T cell-initiated BBB disruption. We report that inhibition of NRP-1, a co-receptor that enhances VEGFR2 (flk-1) receptor activation, decreases vascular permeability, brain hemorrhage, and mortality in this model of CD8 T cell-initiated BBB disruption. We also examine the expression pattern of VEGFR2 (flk-1) and VEGFR1 (flt-1) mRNA expression during a time course of this condition. We find that viral infection of the brain leads to increased expression of flk-1 mRNA. In addition, flk-1 and flt-1 expression levels decrease in the striatum and hippocampus in later time points following induction of CD8 T cell-mediated BBB disruption., Conclusion: This study demonstrates that NRP-1 is a potential therapeutic target in neuro-inflammatory diseases involving BBB disruption and brain hemorrhage. Additionally, the reduction in VEGF receptors subsequent to BBB disruption could be involved in compensatory negative feedback as an attempt to reduce vascular permeability.

Structural diversity in the peptide binding sites of the redundant classical MHC antigen presenting molecules is strongly selected in humans and mice. Although the encoded antigen presenting molecules overlap in antigen presenting function, differences in polymorphism at the MHC I A, B and C loci in humans and higher primates indicate these loci are not functionally equivalent. The structural basis of these differences is not known. We hypothesize that classical class I loci differ in their ability to direct effective immunity against intracellular pathogens. Using a picornavirus infection model and chimeric H-2 transgenes, we examined locus specific functional determinants distinguishing the ability of class I sister genes to direct effective anti viral immunity. Whereas, parental FVB and transgenic FVB mice expressing the H-2K(b) gene are highly susceptible to persisting Theiler's virus infection within the CNS and subsequent demyelination, mice expressing the D(b) transgene clear the virus and are protected from demyelination. Remarkably, animals expressing a chimeric transgene, comprised primarily of K(b) but encoding the peptide binding domain of D(b), develop a robust anti viral CTL response yet fail to clear virus and develop significant demyelination. Differences in expression of the chimeric K(b)α1α2D(b) gene (low) and D(b) (high) in the CNS of infected mice mirror expression levels of their endogenous H-2(q) counterparts in FVB mice. These findings demonstrate that locus specific elements other than those specifying peptide binding and T cell receptor interaction can determine ability to clear virus infection. This finding provides a basis for understanding locus-specific differences in MHC polymorphism, characterized best in human populations.

MRI is sensitive to tissue pathology in multiple sclerosis (MS); however, most lesional MRI findings have limited correlation with disability. Chronic T1 hypointense lesions or "T1 black holes" (T1BH), observed in a subset of MS patients and thought to represent axonal damage, show moderate to strong correlation with disability. The pathogenesis of T1BH remains unclear. We previously reported the first and as of yet only model of T1BH formation in the Theiler's murine encephalitis virus induced model of acute CNS neuroinflammation induced injury, where CD8 T-cells are critical mediators of axonal damage and related T1BH formation. The purpose of this study was to further analyze the role of CD8 and CD4 T-cells through adoptive transfer experiments and to determine if the relevant CD8 T-cells are classic epitope specific lymphocytes or different subsets. C57BL/6 mice were used as donors and RAG-1 deficient mice as hosts in our adoptive transfer experiments. In vivo 3-dimensional MRI images were acquired using a 7 Tesla small animal MRI system. For image analysis, we used semi-automated methods in Analyze 9.1; transfer efficiency was monitored using FACS of brain infiltrating lymphocytes. Using a peptide depletion method, we demonstrated that the majority of CD8 T-cells are classic epitope specific cytotoxic cells. CD8 T-cell transfer successfully restored the immune system's capability to mediate T1BH formation in animals that lack adaptive immune system, whereas CD4 T-cell transfer results in an attenuated phenotype with significantly less T1BH formation. These findings demonstrate contrasting roles for these cell types, with additional evidence for a direct pathogenic role of CD8 T-cells in our model of T1 black hole formation.

The induction of sterilizing T-cell responses to tumors is a major goal in the development of T-cell vaccines for treating cancer. Although specific components of anti-viral CD8+ immunity are well characterized, we still lack the ability to mimic viral CD8+ T-cell responses in therapeutic settings for treating cancers. Infection with the picornavirus Theiler's murine encephalomyelitis virus (TMEV) induces a strong sterilizing CD8+ T-cell response. In the absence of sterilizing immunity, the virus causes a persistent infection. We capitalized on the ability of TMEV to induce strong cellular immunity even under conditions of immune deficiency by modifying the virus to evaluate its potential as a T-cell vaccine. The introduction of defined CD8+ T-cell epitopes into the leader sequence of the TMEV genome generates an attenuated vaccine strain that can efficiently drive CD8+ T-cell responses to the targeted antigen. This virus activates T-cells in a manner that is capable of inducing targeted tissue damage and glucose dysregulation in an adoptive T-cell transfer model of diabetes mellitus. As a therapeutic vaccine for the treatment of established melanoma, epitope-modified TMEV can induce strong cytotoxic T-cell responses and promote infiltration of the T-cells into established tumors, ultimately leading to a delay in tumor growth and improved survival of vaccinated animals. We propose that epitope-modified TMEV is an excellent candidate for further development as a human T-cell vaccine for use in immunotherapy.

Vaccine strategies for cancer immunotherapy have focused on peptide ligands with high affinity for MHC class I. Largely, these vaccines have not been therapeutic. We have examined the peptide specificity of a strongly protective T-cell response that eradicates established B16 melanoma and find that the recognized epitope is generated by a low-affinity MHC class I ligand from tyrosinase-related protein 1 (TRP1). Cytotoxic T-cell responses are induced against TRP1(222-229) by several vaccination schemes using a Toll-like receptor agonist, T regulatory cell depletion, or the immune modulator B7-DCXAb to drive immunity. TRP1(222) CTL are generated from multiple antigen sources, including antigens expressed by tumors growing in situ, tumor cell lysates, and peptide vaccines. The key finding in this study is that protection from freshly implanted or established B16 tumors is primarily mediated by TRP1(222)-specific CTL and not by CTL specific for more traditional melanoma antigens such as TRP2 or gp100. This finding challenges the assumption that the optimal peptide antigens for cancer vaccines are high-affinity MHC ligands. We propose that when administered appropriately, native low-affinity MHC ligands are optimal inducers of immunotherapeutic CTL.

We evaluated the participatory role of human HLA-DR molecules in control of virus from the central nervous system and in the development of subsequent spinal cord demyelination. The experiments utilized intracranial infection with Theiler's murine encephalomyelitis virus (TMEV), a picornavirus that, in some strains of mice, results in primary demyelination. We studied DR2 and DR3 transgenic mice that were bred onto a combined class I-deficient mouse (beta-2 microglobulin deficient; beta2m(0)) and class II-deficient mouse (Abeta(0)) of the H-2(b) background. Abeta(0).beta2m(0) mice infected with TMEV died within 18 days of infection. These mice showed severe encephalomyelitis due to rapid replication of virus genome. In contrast, transgenic mice with insertion of a single human class II major histocompatibility complex (MHC) gene (DR2 or DR3) survived the acute infection. DR2 and DR3 mice controlled virus infection by 45 days and did not develop spinal cord demyelination. Levels of virus RNA were reduced in HLA-DR transgenic mice compared to Abeta(0).beta2m(0) mice. Virus-neutralizing antibody responses did not explain why DR mice survived the infection and controlled virus replication. However, DR mice showed an increase in gamma interferon and interleukin-2 transcripts in the brain, which were associated with protection. The findings support the hypothesis that the expression of a single human class II MHC molecule can, by itself, influence the control of an intracerebral pathogen in a host without a competent class I MHC immune response. The mechanism of protection appears to be the result of cytokines released by CD4(+) T cells.